WO1998001155A1

WO1998001155A1 - Compositions and methods for regulating hiv gene expression

Info

Publication number: WO1998001155A1
Application number: PCT/US1997/012756
Authority: WO
Inventors: Richard B. Gaynor; Wu-Baer Foon
Original assignee: Board Of Regents, The University Of Texas System
Priority date: 1995-07-31
Filing date: 1997-06-30
Publication date: 1998-01-15

Abstract

The invention relates to compositions and methods for inhibition of expression from the long terminal repeat region of Lentiviruses, in particular, from HIV-1. The compositions and methods derive from the discovery that RNA polymerase II has unusually high affinity and specificity for a TAR RNA region of the long terminal repeat. A Tat peptide having binding affinity for RNA polymerase II, an RNA polymerase II peptide having binding affinity for Tat, or an RNA polymerase II peptide having binding affinity for TAR RNA in the presence of a cofactor fraction are provided for inhibition of expression from the TAR region of the long terminal repeat and, therefore, for inhibition of replication of the virus.

Description

COMPOSITIONS AND METHODS FOR REGULATING HIV GENE

EXPRESSION

The present application claims priority to SN 60/001.686 filed July 31. 1995 and SN 08/610,959 filed June 28, 1996.

FIELD OF THE INVENTION

The invention relates to the fields of proteins and oligonucleotides, most particularly cellular and viral proteins and viral oligonucleotide capable of regulating gene expression. The invention also relates to the field of reagents useful in the regulation of viral gene expression. In addition, the invention relates to the field of therapeutic methods and reagents for the treatment of viral diseases, such as AIDS and HIV-related pathologies.

BACKGROUND OF THE INVENTION

Human immunodeficiency virus type 1 (HIV-1) gene expression is regulated by the interaction of cellular factors with distinct regulatory elements in the HIV-1 long terminal repeat (LTR) (Gaynor, 1992). One of these regulatory regions, designated the transactivating region (TAR) (Rosen et al., 1985), is important for transcriptional activation by the transactivator protein Tat (Dayton, et al., 1986; Fisher, et al.. 1986). TAR was defined as extending from -17 to +80 in the HIV LTR and the structural integrity of TAR RNA is a key element for tat activation. TAR forms a stable stem loop RNA structure extending between +1 and +60 that contains several important regulatory elements. These include a three nucleotide bulge located between +23 and -^L25 (Berkhout and Jeang, 1989) and a six nucleotide loop between +30 and +35 (Feng and Holland, 1988: Garcia, et al, 1989). In addition, the preservation of stem base-pairing in TAR RNA between +18 and +44 is also important for Tat activation (Feng and Holland. 1988; Garcia. et al. 1989). A number of studies have indicated that Tat is capable of specific binding to the HIV-1 TAR RNA bulge sequences (Dingwall. C. et al. 1990: Calnan. B.J.. et a/..1991 : Roy. S.. et al.. 1990: Weeks. K. . & Crothers. D.M. 1991 ) while a cellular factor. TRP- 185. binds specifically to the HIV-1 TAR RNA loop sequences (Wu. F.. et al.. 1991 : Sheιinc. C.T.. e«- α/.. 1991). In contrast to the single TAR element that is found in the HIV-1 LTR. HIV-2 contains a duplicated TAR RNA structure whose sequences have diverged from that of HIV-1 TAR RNA. However, the bulge and the loop sequences in the HIV-1 and HIV-2 TAR elements are highly conserved (Emerman et al. 1987). The HIV-2 TAR element is also critical for activation of gene expression in response to the HIV-2 Tat protein. The HIV-1 and HIV-2 Tat proteins have a similar domain structure containing a cysteine. core, and basic domains, each of which is critical for Tat function (Emerman et al. 1987). The HIV-1 Tat protein will activate gene expression of the HIV-1 and HIV-2 LTRs to similar levels while the HIV-2 Tat protein activates the HIV-1 LTR to a much lesser degree than the HIV-1 Tat protein (Emerman et al, 1987). However, a single amino acid change between the basic and core domains of Tat2, which converts glutamine to glycine at amino acid residue 77, results in an HIV-2 Tat protein which is able to activate the HIV-1 LTR to similar levels as the HIV-1 Tat (Elangovan et al, 1992).

In the absence of Tat, transcripts synthesized from the HIV-1 promoter reportedly pause at approximately +60 (Kao, S.-Y., et al, 1987). These short or nonprocessive transcripts are thought to arise from a poorly processive RNA polymerase II that is unable to function efficiently in transcriptional elongation from the HIV-1 promoter (Laspia, M., et al. 1989; Ratnasabapathy, R., et al, 1990; Feinberg, M.B., et al, 1991 ; Marciniak, R.A.

& Sharp, P. A. 1991; Kato, H. et al, 1992). The generation of these short transcripts is dependent on TAR (Ratnasabapathy, R., et al, 1990). However, in the presence of Tat, there is reportedly a marked decrease in the number of short transcripts synthesized and a subsequent increase in the number of full-length transcripts that may be capable of extending the 9.0 kb length of the HIV-1 genome (Feinberg, M.B.. et al, 1991). The role of cellular and viral TAR RNA "binding" proteins on the regulation of H1V-1 gene expression has not been fully characterized.

Several viruses, such as those of the HIV and the HTLV (human leukemia lymphoma virus) type, have within their gene structure a downstream regulatory region, which binds cellular proteins that are required for transactivation (a protein that acts in conjunction with a viral protein as a transactivating factor). A more complete understanding of the mechanisms which govern viral gene expression and, in particular, the role of TAR RNA binding proteins would provide methods for selectively "turning on" and "turning off viral genes. Therapeutic agents that selectively "turn off viral expression in individuals infected with a variety of viruses including HIV, HTLV, herpes virus, hepatitis B. and adenovirus could also be developed using this selective mechanism. HIV is recognized as the causative agent of Acquired Immunodeficiency Syndrome (AIDS). Therapeutic agents which have been used in the treatment of AIDS include AZT (azidothymidine) and DDI (dideoxyinosine) (Broder et al, 1994). Both of these agents are ^c. nucleotide analogs that target the viral enzyme, reverse transcriptase. While these agents have been used with varying degrees of success, they are also unfortunately associated with a variety of severe side effects. Some of these side effects include peripheral neuropathy (DDI), pancreatitis, granulocytopenia, anemia, severe headache, nausea, insomnia, neurotoxicity, and seizure. These agents have also been associated with a potential

10 carcinogenicity and teratogenicity.

Other molecular targets under investigation as anti-viral targets include an HIV- gene encoded protease (Broder, et al; 1994). The protease is encoded on the polygene of HIV-1. The polygene encodes three proteins - a reverse transcriptase, a self-cleaving protease (that is required for processing the reverse transcriptase) and a nuclease that is lb essential for integration of viral DNA into the genome of a host cell. Inhibitors of the HIV protease have been developed using the crystal structure of the protein.

Other potential molecular targets for affecting viral gene expression include the glycosylated envelope protein of HIV and the receptor protein CD4 (Broder, et a 1994). CD4 is a T cell co-receptor glycoprotein on the surface of lymphocytes to which the virus

20 binds. A soluble form of CD4 can bind to the viral envelope protein and prevent the virus from entering cells. Alternatively, a conjugate of CD4 and a toxin might be used to attack HIV-infected cells, since such cells express the envelope protein on their surfaces. Another drug, dextran sulfate, has also been used in the treatment of AIDS. This drug blocks the binding of HIV to target cells.

25 None of these molecular targets for anti-viral therapy relates to an agent of cellular origin capable of specifically affecting viral gene expression. An enhanced understanding of the particular role of cellular proteins in the molecular events of both cellular and viral (HIV) gene expression would provide a new avenue for the development of effective antiviral agents. Such information would further provide for the development of a new genus

30 of drugs based on the regulation of host proteins for the treatment of diseases such as AIDS and AIDS-related diseases in addition to a variety of other pathogenic viruses that infect humans.

It is also important to identify factors that bind to TAR RNA and determine how they modulate Tat-mediated effects on HIV-1 transcriptional elongation via effects on RNA polymerase II. Methods for the specific inhibition of viruses, such as HIV and HTLV may be developed from this information.

It is an object of the invention to provide a tool which is useful in the inhibition of viral gene expression. It is still another object of the invention to provide a reagent which is useful in the study of viral gene regulation. It is further an object of the present invention to provide a method for treating HIV-disease. How these and other objects of this invention are achieved will become apparent in light of the accompanying disclosure.

SUMMARY OF THE INVENTION

The present invention addresses one or more of the problems in the art relating to the characterization and control of gene expression, particularly viral HIV gene expression.

The present invention also addresses the need for highly specific alternative AIDS and AIDS-related disease therapeutic agents.

In particular aspects, the agents of the present invention target molecular events of HIV gene expression by affecting the interaction of specific proteins, such as RNA polymerase II, TRP-185 or Tat with each other, with TAR RNA, or both.

The present invention provides a method for inhibiting expression from the TAR region of HIV- 1, and stems from the present inventors' identification and characterization of unusual and unexpected activities of a cellular RNA polymerase. More specifically, a cellular RNA polymerase is observed to have an unusual and unexpected binding affinity and specificity for the TAR region of the LTR of HI V-l . In some embodiments, the method comprises administering a composition comprising a pharmacologically active amount of a Tat peptide having binding affinity for RNA polymerase II. an RNA polymerase II peptide having binding affinity for a Tat peptide, or an RNA polymerase II peptide having binding affinity for TAR RNA in the presence of a cofactor fraction, or a mixture thereof.

In the present invention, Tat is representative of a number of transactivator proteins required for viral replication. The present inventors envision use of the methods herein for obtaining compositions useful for inhibition of other viruses of the Lentivirus family that have TAR RNA elements. Furthermore, similar types of interaction between viral transactivator and RNA polymerase II may be inhibited by defining interactions between RNA polymerase II and the respective viral transactivator. Similarly, RNA polymerase II is representative of a polymerase that interacts with a transactivator protein. Inhibition of other polymerases apart from or in addition to RNA polymerase II is therefore envisioned by the present inventors, including the HIV-1 polymerase. reverse transcriptase.

In some aspects of the invention, the Tat peptide has a sequence of amino acids corresponding to a basic region of Tat from about amino acid 49 to about 57. That sequence is Arg Lys Lys Arg Arg Gin Aig Arg Arg (SEQ ID NO:l). The amino acid sequence of Tat (SEQ ID NO: 3) is described in Modesti et al. (1991), which is specifically incorporated herein by reference for this purpose. In some embodiments, the peptide is capable of inhibiting HIV-1 expression in vivo, such provides an approach for inhibiting HIV-1 gene expression in a subject having HIV-1 or other viral infection.

The cofactor fraction may be even further defined as comprising elongation factor- 1 alpha, polypyrimidine tract-binding protein, and a peptide stimulator of TAR RNA binding proteins (SRB peptide).

The stimulator peptide of TAR RNA binding proteins is a novel peptide provided by the present disclosure. For purposes of describing the present invention, it is referenced as SRB peptide. In some embodiments, the SRB peptide is substantially purified, that is. it is present in a preparation substantially free of other proteins or peptides that do not stimulate binding of proteins to TAR RNA. The SRB peptide may be further defined as capable of enhancing the binding of both TRP-185 and RNA polymerase II to TAR RNA in the presence of elongation factor- 1 alpha and polypyrimidine tract-binding protein. The SRB peptide in some embodiments may be even further defined as having an amino acid sequence substantially as shown in SEQ ID No:5.

The SRB peptide may be further defined as a peptide obtained by a process comprising the steps of obtaining a nucleic acid encoding an SRB peptide. and expressing the nucleic acid to obtain an SRB peptide. Methods for obtaining said nucleic acid and expressing the nucleic acid to obtain SRB peptide are found in Examples 18 and 19 of the present disclosure. In an embodiment of the present invention, the nucleic acid has a nucleotide sequence as defined by SEQ ID NO:4.

Any of a variety of mammalian cells may be used as a source to prepare SRB or TRP-185. Preferably, mammalian cells used to prepare the nuclear extract are cells that are susceptible to HIV infection or related viruses. By way of example, particularly useful mammalian cell lines include VERO (ATCC CCL 81), HeLa cells (ATCC CCL 2.1. ATCC CCL 2.2), W138, COS, Jurkat. CEM, 293 (human embryonic kidney cell line ATCC CRL 1573) and MDCK cell lines. Most preferably, the mammalian cell line employed to prepare a mammalian cell nuclear extract for purposes of isolating the herein described binding proteins, TRP-185 or SRB. are HeLa cells or HeLa cell lines.

In some embodiments of the invention, the RNA polymerase II peptide having binding affinity for TAR RNA may be further defined as being from the largest subunit of RNA polymerase II having a molecular weight of about 210 kDa, or the largest subunit in addition to other subunits.

A method for inhibiting expression from a TAR region of HIV-1 is a further aspect of the invention. In some embodiments, the method comprises administering a pharmacologically active amount of an oligonucleotide having a nucleotide sequence corresponding to a TAR region of HIV-1 LTR, and even further as having binding affinity for RNA polymerase II or Tat. The oligonucleotide may comprise about 25 nucleotides from an about +18 to an about +44 region of TAR (SEQ ID NO: 6). The oligonucleotide may be a deoxyribonucleotide or a ribonucleotide.

A further embodiment of the present invention is an oligonucleotide having binding affinity for R A polymerase II and capable of inhibiting expression from a TAR region of

HIV-1. Preferably, the oligonucleotide has a nucleotide sequence corresponding to a position +18 to a position +44 of an HIV-1 TAR RNA region (SEQ ID NO: 6).

A method of screening for a candidate substance that is capable of inhibiting the binding of RNA polymerase II to TAR RNA in the presence of a cofactor fraction is a further aspect of the present invention. The method comprises obtaining an RNA polymerase II protein and a cofactor fraction; admixing a candidate substance with the

RNA polymerase II protein and the cofactor fraction in the presence of a nucleic acid sequence including a TAR region; and selecting a candidate substance that inhibits the binding of RNA polymerase II to TAR RNA in the presence of the cofactor fraction. The cofactors may be defined as herein described above. "Selecting a candidate substance that inhibits the binding of RNA polymerase II to TAR RNA" means identifying a candidate substance that has some inhibitory effect compared to the amount of binding observed in the absence of the candidate substance.

Following long-standing patent law convention, the terms "a" and "an" mean "one or more" when used in this application, including the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. FIG. 1. Purification Scheme for TRP-185. The protocol for the fractionation of

HeLa cell nuclear extract to purify TRP-185 is shown with the molarity of the buffers used in the column elution indicated.

FIG. 2: The HPLC profile of peptides released after Lys C protease digestion of TRP-185 is shown and the position where the peptides which generated the 14-mer and 24- mer TRP-185 peptides is marked with * and a diamond respectively.

FIG. 3. Purification scheme of cellular cofactors from HeLa cells. The protocol for the fractionation of HeLa cell nuclear extract to purify the cellular cofactors which stimulate TRP-185 binding is shown. The numbers in the figure indicate the concentration of KC1 used to elute each column with the exception of the hydroxyapatite Bio Gel column in which the concentration of potassium phosphate is indicated.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention results from the discovery that cellular RNA polymerase II binds with unusually and unexpectedly high specificity and affinity to the TAR RNA region of the long terminal repeat (LTR) of HIV-1. This discovery allows for the provision of compositions and methods for the inhibition of expression from the TAR RNA region of HIV and, therefore, for inhibition of replication of the virus.

For therapeutic applications, the present inventors have found that Tat can bind to the largest (210 kDa) subunit of RNA polymerase II. The interaction is mediated by a group of amino acids extending from about amino acid 49-57 of Tat. Tat is described in detail in U.S. patent application SN 07/910,867, incorporated by reference herein. The corresponding binding site on RNA polymerase II will be identified as described in Example 23. Peptides corresponding to either this region of Tat or an interaction domain of the polymerase may be used to inhibit this binding with subsequent prevention of Tat activation and HIV replication. Similar studies will be performed to identify binding sites for TRP-185 with either Tat or RNA polymerase II and peptides inhibition technology will be developed. Other viral regulatory proteins in addition to Tat such as contained in adenovirus. CMV. herpes simplex, hepatitis B, or human T cell leukemia virus may also bind a polymerase and, therefore, similar technology based on defining interacting domains may be used to identify peptides that inhibit viral replication.

Eukaryotic RNA polymerase II contains 10 to 12 subunits with two large subunits of molecular weights of approximately 220 and 140 kDa respectively (Young, R.A.. 1991).

The largest subunit of RNA polymerase II contains 52 repeats of the amino acid sequence

Tyr-Ser-Pro-Thr-Ser-Pro-Ser (SEQ ID NO:28) that is referred to as the C-terminal domain or CTD. The CTD is highly phosphorylated in a substantial portion of the RNA polymerase II molecules in the cell. The regulation of CTD phosphorylation appears to be a key mechanism controlling the transition between transcriptional initiation and elongation. One model that the present inventors have tested was whether TRP-185 or its cofactors could modify the transcriptional elongation properties of RNA polymerase II in either a positive or negative manner. The results presented herein suggest that TAR RNA. in conjunction with a multiprotein complex that includes RNA polymerase II, is the ultimate target for Tat-mediated transcriptional activation of the HIV-1 promoter. While not wanting to be bound by theory, passage of the transcriptional complex through TAR may lead to the transfer of TAT from the preinitiation complex to the transcriptional elongation complex.

The present invention also provides for oligonucleotide reagents that may be administered to bind RNA polymerase II and thereby prevent the polymerase from binding to TAR RNA. In general, there are three commonly used solid phase-based approaches to the synthesis of oligonucleotide containing conventional 5'-3' linkages. These are the phosphoramidite method, the phosphonate method, and the triester method.

A brief description of a current method used commercially to synthesize oligomeric DNA is as follows: Oligomers up to ca. 100 residues in length are prepared on a commercial synthesizer, eg., Applied Biosystems Inc. (ABI) model 392, that uses phosphoramidite chemistry. DNA is synthesized from the 3' to the 5' direction through the sequential addition of highly reactive phosphorous(III) reagents called phosphoramidites. The initial 3' residue is covalently attached to a controlled porosity silica solid support, which greatly facilitates manipulation of the polymer. After each residue is coupled to the growing polymer chain, the phosphorus(III) is oxidized to the more stable phosphorus(V) state by a short treatment with iodine solution. Unreacted residues are capped with acetic anhydride, the 5'- protective group is removed with weak acid, and the cycle may be repeated to add a further residue until the desired DNA polymer is synthesized. The full length polymer is released from the solid support, with concomitant removal of remaining protective groups, by exposure to base. A common protocol uses saturated ethanolic ammonia.

The phosphonate based synthesis is conducted by the reaction of a suitably protected nucleotide containing a phosphonate moiety at a position to be coupled with a solid phase-derivatized nucleotide chain having a free hydroxyl group, in the presence of a suitable activator to obtain a phosphonate ester linkage, which is stable to acid. Thus, the oxidation to the phosphate or thiophosphate can be conducted at any point during synthesis of the oligonucleotide or after synthesis of the oligonucleotide is complete. The phosphonates can also be converted to phosphoramidate derivatives by reaction with a primary or secondary amine in the presence of carbon tetrachloride.

In the triester synthesis, a protected phosphodiester nucleotide is condensed with the free hydroxyl of a growing nucleotide chain derivatized to a solid support in the presence of coupling agent. The reaction yields a protected phosphate linkage which may be treated with an oximate solution to form unprotected oligonucleotide.

Preferred oligonucleotide resistant to in vivo hydrolysis may contain a phosphorothioate substitution at each base (J. Org. Chem., 55 :4693-4699, ( 1990). Oligodeoxynucleotides or their phosphorothioate analogues may be synthesized using an Applied Biosystem 380B DNA synthesizer (Applied Biosystems, Inc., Foster City, CA). Standard methods for Southern, Northern and Western analysis were carried out as follows. For Southern analysis (Sambrook et al, 1989), 6 μg of human and mouse genomic DNA isolated from human and mouse lymphocytes (ClonTech, Palo Alto. CA) was digested with either BamHI, PstI, or EcoRI and then subject to electrophoresis on a 1 % agarose gel in 1 x Tris-acetate EDTA (TAE) buffer at 150V. The gel was then treated. blotted and the DNA was fixed to Hybon-N membrane as described in the manufactures protocol (Amersham). The membrane was then pre-hybridized at 65°C for 3 hours in 5 X SSPE, l Ox Denhardt's, 100 μg/ml salmon sperm DNA, and 2% SDS and hybridized overnight in the same buffer containing 1 x 10⁶ cpm/ml of labeled DNA probe. The probes were made from nick translation of portions of the TRP- 185 cDNA encoding amino acids position 392 to 817, 817 to 1 162 and 1 162 to 1572, respectively. The same blots were also probed with a portion of the largest subunit of an RNA polymerase II cDNA encoding amino acids position of 1290 to 1640. Southern blot analysis of the zoo blot used in this study (Clontech) and the somatic cell hybrid panel used for chromosome mapping was purchased from Oncor Inc., and probed with the same portions of the TRP-185 cDNA used above as described in the manufacture's protocols. Northern analysis of TRP-185 on poly A selected RNA isolated from HeLa cells was performed as described in the rapid hybridization protocol from Amersham (Sambrook et al, 1989). Northern analysis of the human multiple tissue (MTN) blot from Clontech were done as described in the product protocol. The probe used in these analysis was a portion of the TRP-185 cDN encoding amino acids between 392-817 prepared by nick translation. A full length GAPDH probe was used as a control for these Northern blot.

Western analysis were performed using either 12CA5 monoclonal antibody (Field et. al., 1988) which is directed against the twelve amino acid influenza virus hemagglutinin sequence (HA1) or a monoclonal antibody (NK 5.18) raised against a portion of the TRP- 185 amino acids extending from position 1409-1541. ECL reagents and protocols from Amersham were used in this analysis.

Table 1 lists the identity of sequences of the present disclosure having sequence identifiers.

TABLE 1

11

12

Biologically Functional Equivalent Amino Acids. Modifications and changes may be made in the sequence of the transactivator or polymerase peptides of the present invention and still obtain a peptide having like or otherwise desirable characteristics. For example, certain amino acids may be substituted for other amino acids in a peptide without appreciable loss of interactive binding capacity. Since it is the interactive capacity and nature of an amino acid sequence that defines the peptide's functional activity, certain amino acid sequences may be chosen (or, of course, its underlying DNA coding sequence) and nevertheless obtain a peptide with like properties. It is thus contemplated by the inventors that certain changes may be made in the sequence of a peptide (or underlying DNA) without appreciable loss of its ability to function.

Substitution of like amino acids can be made on the basis of hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanme (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent peptide. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those which are within ± 1 are more preferred, and those within ±0.5 are most preferred.

As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. Two designations for amino acids are used interchangeably throughout this application, as is common practice in the art. Alanine = Ala (A); Arginine = Arg (R);

Aspartate = Asp (D); Asparagine = Asn (N); Cysteine = Cys (C); Glutamate = Glu (E);

Glutamine = Gin (Q); Glycine = Gly (G); Histidine = His (H); Isoleucine = He (I); Leucine = Leu (L); Lysine = Lys (K); Methionine = Met (M); Phenylalanine = Phe (F); Proline=

Pro (P): Serine = Ser (S); Threonine^ Thr (T); Tryptophan = Trp (W); Tyrosine = Tyr (Y);

Valine= Val (V).

While discussion has focused on functionally equivalent polypeptides arising from amino acid changes, it will be appreciated that these changes may be effected by alteration of the encoding DNA. taking into consideration also that the genetic code is degenerate and that two or more codons may code for the same amino acid.

The synthesis of peptides is readily achieved using conventional peptide synthetic techniques such as the solid phase method (e.g., through the use of commercially available peptide synthesizer such as an Applied Biosystems Model 430A Peptide Synthesizer. Foster City, California). It is desirable for the amino terminal end of synthetic peptides to be protected from degradation, for example, by using acetic anhydride to acetylate the N- terminal end. Similarly, protection for the carboxyl end may be achieved by forming an amide linkage. These protecting groups will prevent synthetic peptides from being degraded by proteolytic enzymes once they are introduced into a cell. Peptides synthesized in this manner may then be aliquoted in predetermined amounts and stored in conventional manners, such as in aqueous solutions or, even more preferably, in a powder or lyophilized state pending use.

In general, due to the relative stability of peptides, they may be readily stored in sterile aqueous solutions for fairly long periods of time if desired, e.g., up to six months or more, in virtually any aqueous solution without appreciable degradation or loss of activity.

However, where extended aqueous storage is contemplated, it will generally be desirable to include agents including buffers such as Tris-HCl or phosphate buffers to maintain a pH of 7.0 to 7.5. Moreover, it may be desirable to include agents which will inhibit microbial growth, such as sodium azide or merthiolate. For extended storage in an aqueous state, it will be desirable to store the solutions at 4°C, or more preferably, frozen. Of course, where the peptide(s) are stored in a lyophilized or powdered state, they may be stored virtually indefinitely, e.g., in metered aliquots that may be rehydrated with a predetermined amount of water (preferably distilled) or buffer prior to use. The transactivator or polymerase peptides of the present invention may have 3-4 amino acids or may be 5. 6, 7, 8, 9, 10, 1 1 , 12. 13, 14. 15, 16, 17, 18. 19 or 20 ammo acids long. Longer peptides are also contemplated. Peptides less than about 45 amino acids are synthesized chemically whereas longer peptides are preferably provided by a plasmid or viral expression system. The administration of peptides to HIV- 1 -positive cells is contemplated to be a repetitive or continuous supply of peptides either directly administered or administered as liposomes or other delivery systems known to one of skill in this art in light of the present disclosure.

Nucleic Acid Hybridization. The nucleic acid sequences disclosed herein will find utility as probes and primers in nucleic acid hybridization embodiments. As such, it is contemplated that oligonucleotide fragments corresponding to a sequence of SEQ ID NOS: 4, 6, 8-17, 20 and 21 for stretches of between about 10 nucleotides to about 20 or to about 30 nucleotides will find particular utility, with even longer sequences, e.g., 40. 50. even up to full length, being more preferred for certain embodiments. The ability of such nucleic acid probes to specifically hybridize to TAR nucleic acid sequences will enable them to be of use in a variety of embodiments. For example, the probes can be used in a variety of assays for detecting the presence of complementary sequences in a given sample. However, other uses are envisioned, including the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions.

These probes will be useful in hybridization embodiments, such as Southern and Northern blotting. The total size of fragment, as well as the size of the complementary stretch(es), will ultimately depend on the intended use or application of the particular nucleic acid segment. Smaller fragments will generally find use in hybridization embodiments, wherein the length of the complementary region may be varied, such as between about 20 and about 40 nucleotides, or even up to the full length of the nucleic acid as shown in SEQ ID NOS: 4, 6, 8-17, 20 and 21 according to the complementary sequences one wishes to detect. The use of a hybridization probe of about 10 nucleotides in length allows the formation of a duplex molecule that is both stable and selective. Molecules having complementary sequences over stretches greater than 10 bases in length are preferred. though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having gene-complementary stretches of 15 to 20 nucleotides, or even longer where desired. Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCR technology of U.S. Patent 4,603,102 (herein incorporated by reference) or by introducing selected sequences into recombinant vectors for recombinant production.

Depending on the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, e.g., one will select relatively low salt and\or high temperature conditions, such as provided by 0.02M-0.15M NaCl at temperatures of 50°C to 70°C. Such selective conditions tolerate little, if any. mismatch between the probe and the template or target strand.

Where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template or where one seeks to isolate sequences from related species, functional equivalents, or the like, less stringent hybridization conditions will typically be needed in order to allow formation of the heteroduplex. In these circumstances, one may desire to employ conditions such as 0.15M-0.9M salt, at temperatures ranging from 20°C to 55 °C. Cross-hybridizing species can thereby be readily identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results. In certain embodiments, it will be advantageous to employ nucleic acid sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin biotin, which are capable of giving a detectable signal. In preferred embodiments, one will likely desire to employ a fluorescent label or an enzyme tag, such as ureasc. alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are known which can be employed to provide a means visible to the human eye or spectrophotometrically, to identify' specific hybridization with complementary nucleic acid-containing samples.

In general, it is envisioned that the hybridization probes described herein will be useful both as reagents in solution hybridization as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C contents, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Following washing of the hybridized surface so as to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantified, by means of the label.

Longer DNA segments will often find particular utility in the recombinant production of peptides or proteins. DNA segments which encode peptide antigens from about 15 to about 50 amino acids in length, or more preferably, from about 15 to about 30 amino acids in length are contemplated to be particularly useful. DNA segments encoding peptides will generally have a minimum coding length in the order of about 45 to about 150. or to about 90 nucleotides. DNA segments encoding full length proteins may have a minimum coding length in the order of about 2000 nucleotides for a protein or otherwise biologically active equivalent peptide having at least a sufficient portion of the sequence in accordance with SEQ ID NO: 4 capable of providing said SRB-biological activity. The nucleic acid segments of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. For example, nucleic acid fragments may be prepared in accordance with the present invention which are up to 10,000 base pairs in length, with segments of 5,000 or 3,000 being preferred and segments of about 1,000 base pairs in length being particularly preferred.

It will be understood that this invention is not limited to the particular nucleic acid and amino acid sequences having sequence identifiers as listed in Table 1. Therefore, DNA segments prepared in accordance with the present invention may also encode biologically functional equivalent proteins or peptides which have variant amino acid sequences. Such sequences may arise as a consequence of codon redundancy and functional equivalency which are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged.

Pharmaceutical Preparations. Another aspect of the present invention provides a therapeutic agent for the treatment of HIV or HTLV infection in an animal. The therapeutic agent comprises an admixture of an inhibitor of expression from a TAR RNA region in a pharmaceutically acceptable excipient. Most preferably, the therapeutic agent will be formulated so as to be suitable for administration as a parental formulation or as a capsule (for oral administration). An inhibitor of expression from a TAR RNA region can be an RNA or a DNA that preferentially binds RNA polymerase II, a TRP- 185, or a cofactor with higher or similar affinity to TAR RNA. An inhibitor could also be a peptide having related or higher affinity than that of Tat, TRP- 185, RNA polymerase II, or cellular cofactors that disrupt interaction between either these proteins and/or TAR RNA. An inhibitor can also be defined as a chemical agent that prevents any of the interactions elucidated hereinabove. The active compounds may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form of ingestible tablets. buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of the unit. The amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup of elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.

The active compounds may also be administered parenterally or intraperitoneally. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols. and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol. and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial ad antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. In still another aspect of the present invention, an RNA binding complex is provided. The RNA binding complex may be particularly useful in characterizing the molecular events of gene expression. In one particular embodiment, the RNA binding complex comprises a TRP- 185 cellular protein capable of binding a TAR RNA region of HIV, three cofactors capable of facilitating the binding of the TRP- 185 to a TAR RNA consisting of EF-1 alpha, PTB and SRB, and a volume of TAR RNA sufficient to bind the

TRP-185. In another embodiment, the RNA binding complex comprises an RNA polymerase II capable of binding a TAR RNA region of HIV, three cofactors capable of facilitating the binding of the RNA polymerase II to a TAR RNA consisting of EF-1 alpha, PTB and SRB, and a volume of TAR RNA sufficient to bind the RNA polymerase II.

Most preferably, the cofactors and the cellular proteins are isolated from a HeLa cell nuclear cell extract as described hereinbelow. It is envisioned that the described RNA binding complex may be used as a laboratory and candidate substance screening reagent. most particularly in the characterization of viral and cellular gene expression, and inhibitors thereof.

In an alternative embodiment the RNA binding complex may be used to screen compounds that are able to inhibit the TRP- 185 to TAR RNA interaction or the RNA polymerase II to TAR RNA interaction. It is also envisioned that compounds that alter the effect of the co-factors described herein may be useful for inhibiting these interactions. Assays for Candidate Substances. In still further embodiments, the present invention concerns a method for identifying polymerase-TAR RNA inhibitory compounds, which may be termed as "candidate substances." It is contemplated that this screening technique will prove useful in the general identification of any compound that will serve the purpose of inhibiting the interaction of RNA polymerase II with the TAR region of HIV, HTLV and other Lentivirus family members. It is further contemplated that useful compounds in this regard will in no way be limited to proteinaceous or peptidyl compounds, since the candidate substances may also affect the role of the co-factors described herein. In fact, it may prove to be the case that the most useful pharmacological compounds for identification through application of the screening assay will be non- peptidyl in nature and serve to inactivate the polymerase to TAR interaction through a tight binding or other chemical interaction. Accordingly, in screening assays to identify pharmaceutical agents which disrupt

RNA complex formation, it is proposed that compounds isolated from natural sources such as plants, animals or even sources such as marine, forest or soil samples, may be assayed for the presence of potentially useful pharmaceutical agents. It will be understood that the pharmaceutical agents to be screened could also be derived from chemical compositions or man-made compounds. In important aspects, the candidate substances may be anti-RNA polymerase antibodies, including polyclonal and monoclonal antibodies. The suspected agents could also include proteins and peptides, such as those derived from recombinant

DNA technology or by other means, including peptide synthesis. The active compounds may include fragments or parts of naturally-occurring compounds or may be only found as active combinations of known compounds which are otherwise inactive.

In these embodiments, the present invention is directed to a method for determining the ability of a candidate substance to inhibit RNA polymerase II-TAR sequence interaction, the method including generally the steps of: (a) obtaining an RNA binding complex comprising an RNA polymerase II protein and co-factors capable of binding to TAR nucleic acid;

(b) admixing a candidate substance with the RNA binding complex in the presence of target TAR nucleic acid; and

(c) selecting a candidate substance having the ability to interfere with RNA polymerase II-cofactor binding to TAR nucleic acid.

After obtaining a relatively purified preparation of RNA polymerase II. one will desire to simply admix a candidate substance with the RNA polymerase II and TAR RNA or DNA sequence containing preparation, preferably under conditions which would allow the RNA polymerase to perform its binding function but for inclusion of a inhibitory substance. Thus, for example, one will typically desire to include within the admixture an amount of the known cofactor. In this fashion, one can measure the ability of the candidate substance to reduce binding activity relatively in the presence of the candidate substance. Any method may generally be employed to determine RNA polymerase II binding to TAR nucleic acid sequences. A preferred method is by gel retardation as demonstrated in the following examples. Further methods will be those in which the target TAR nucleic acid incorporates, or is conjugated to, a label, such as an enzymatic, chemical or radiolabel. or incorporates one of the ligands of a two ligand-based detection system such as the avidin/biotin system. For ease and safety, the use of enzymatic labels, such as, for example, horse radish peroxidase, urease or alkaline phosphatase is preferred. In such cases, a colorimetric indicator substrate would be employed to provide a means visible to the human eye, or spectrophotometrically, to identify specific hybridization with labelled target sequences.

In still further embodiments, the present invention is concerned with a method of inhibiting RNA polymerase II-TAR sequence binding which includes subjecting an RNA binding complex to an effective concentration of a candidate inhibitor such as one of the family of protein or non-proteinaceous compounds discussed above, or with a candidate substance identified in accordance with the candidate screening assay embodiments. This is, of course, an important aspect of the invention in that it is believed that by inhibiting the binding of RNA polymerase II to TAR nucleic acid sequences, one will be enabled to treat various aspects of retroviral infection, including the HIV virus and related members of the Lentivirus family. It is believed that the use of such inhibitors to block TAR region activation will serve to treat cells that can be, or have already been infected with a retrovirus. such as HIV, and may be useful by themselves or in conjunction with other therapies, including the use of nucleic acid homologs and the like.

Antibodies. In another aspect, the present invention contemplates an antibody that is immunoreactive with an SRB polypeptide, a Tat peptide having affinity for RNA polymerase II, or a polymerase peptide having affinity for Tat, as described for the invention. An antibody can be a polyclonal or a monoclonal antibody. In a preferred embodiment, an antibody is a monoclonal antibody. Means for preparing and characterizing antibodies are well known in the art (See, e.g., Antibodies "A Laboratory Manual. E. Howell and D. Lane, Cold Spring Harbor Laboratory, 1988). Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide of the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically an animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster or a guinea pig. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.

Antibodies, both polyclonal and monoclonal, specific for the peptides of the present invention may be prepared using conventional immunization techniques, as will be generally known to those of skill in the art. A composition containing antigenic epitopes of the peptide sequences, isolated peptides, or fragments thereof can be used to immunize one or more experimental animals, such as a rabbit or mouse, which will then proceed to produce specific antibodies against TRP- 185. Polyclonal antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and preparing serum samples from the whole blood. To obtain monoclonal antibodies, one would also initially immunize an experimental animal, often preferably a mouse, with a purified peptide composition. One would then, after a period of time sufficient to allow antibody generation, obtain a population of spleen or lymph cells from the animal. The spleen or lymph cells can then be fused with cell lines, such as human or mouse myeloma strains, to produce antibody- secreting hybridomas. These hybridomas may be isolated to obtain individual clones which can then be screened for production of antibody to the desired peptide.

Following immunization, spleen cells are removed and fused, using a standard fusion protocol (see, e.g., The Cold Spring Harbor Manual for Hybridoma Development, incorporated herein by reference) with plasmacytoma cells to produce hybridomas secreting monoclonal antibodies against the desired peptide. Hybridomas which produce monoclonal antibodies to the selected antigens are identified using standard techniques, such as ELISA and Western blot methods.

Hybridoma clones can then be cultured in liquid media and the culture supernatants purified to provide the peptide-specific monoclonal antibodies. In general, monoclonal antibodies to the peptide antigen can be used in the treatment of HIV infections. It is proposed that the monoclonal antibodies of the present invention will find useful application in standard immunochemical procedures, such as ELISA and Western blot methods, as well as other procedures which may utilize antibody specific to common or allelically distinct peptide epitopes. Monoclonal and polyclonal antibodies raised against proteins of the present examples are useful for (1) screening a cDNA expression library in the process of cloning the gene that encodes a particular protein (for example, the SUPERSCREEN® immunoscreening system from AMERSHAM^®), (2) facilitating the purification of a particular protein by using column chromatography to which the monoclonal antibody is bound, and (3) providing reagents necessary for a diagnostic immunoassay for screening biological samples.

Monoclonal antibodies are obtained using the following procedure: Immunization Schedule for Raising Monoclonal Antibodies 1. For each mouse, mix 250 μl of antigen solution containing 10 μg of antigen with

250 μl of complete Freund's adjuvant. Inject six BALB/c female mice ip

(intraperitoneal injection).

2. After 14 days, repeat the injections of antigen and incomplete Freund's adjuvant.

3. Collect tail bleeds from immunized mice on day 24. Do 1 in 5 dilutions in phosphate buffered saline (PBS) and test all samples by comparison with similar dilutions of normal mouse serum in a dot blot.

4. On day 35, inject all animals ip with antigen and incomplete Freund's.

5. Day 45, do tail bleeds and test by dot blot. All serum samples checked by immunoprecipitation against in vivo radiolabeled antigen preparation. 6. Day 56, inject best responder, 100 μl iv and 100 μl ip. All others get ip injection with incomplete Freund's. 7. Day 59. fuse splenocytes from best responder.

The resultant hybridoma tissue culture supernatants are screened for monoclonal antibodies as follows: 1. A protein solution of at least 1 μg/ml of antigen is added to a nitrocellulose sheet at 0.1 ml/cm². Allow the protein to bind to the paper for 1 hr. Higher concentrations of proteins will increase the signal and make screening faster and easier. If the amount of protein is not limiting, concentrations of 10-50 μg/ml should be used. Nitrocellulose can bind approximately 100 μg of protein per cm². 2. Wash the nitrocellulose sheet three times in PBS.

3. Place the sheet in a solution of 3% BSA in PBS with 0.02% sodium azide for 2 hr to overnight. To store the sheet, wash twice in PBS and place at 4°C with 0.02% sodium azide. For long-term storage, shake off excessive moisture from the sheet, cover in plastic wrap, and store at -70°C. 4. Place the wet sheet on a piece of parafilm. and rule with a soft lead pencil in 3-mm squares. Cut off enough paper for the number of assays.

5. Apply 1 μl of the hybridoma tissue culture supernatant to each square. Incubate the nitrocellulose sheet on the parafilm at room temperature in a humid atmosphere for 30 min.

Along with dilutions of normal mouse serum, include dilutions of the mouse serum from the last test bleed as controls. Dilutions of the test sera are essential to control correctly for the strength of the positive signals. Mouse sera will often contain numerous antibodies to different regions of the antigen and therefore will give a stronger signal than a monoclonal antibody. Therefore, dilutions need to be used to lower the signal. Good monoclonal antibodies will appear 10-fold less potent than good polyclonal sera.

6. Quickly wash the sheet three times with PBS, then wash two times for 5 min each with PBS. 7. Add 50,000 cpm of ¹² I-labeled rabbit anti-mouse immunoglobulin per 3-mm square in 3% BSA/PBS with 0.02% sodium azide (about 2.0 ml/cm²).

8. After 30-60 min of incubation with shaking at room temperature, wash extensively with PBS until counts in the wash buffer approach background levels.

9. Cover in plastic wrap and expose to X-ray film with a screen at -70^°C. The hybridoma identified as producing antibody to the protein of interest is passaged as follows:

1 . Inject 10⁷ (or less) cells into female mice that have been injected ip about 1 week earlier with 0.5 ml of pristane or incomplete Freund's adjuvant. These types of injections are also used to prime mice for ascites production, and this may serve as a convenient source of appropriate hosts. If no mice are available, inject mice with incomplete Freund's adjuvant and wait 4 hr to 1 day before injecting the hybridoma cells. The animals must be of the same genetic background as the cell line.

2. If an ascites develops, tap the fluid and transfer into a sterile centrifuge tube.

3. Spin the ascites at 400g for 5 min at room temperature.

4. Remove the supernatant. Resuspend the cell pellet in 10 ml of medium supplemented with 10% fetal bovine serum and transfer to a tissue culture plate. The supernatant can be checked for the presence of the antibody and used for further work if needed. 5. Handle as for normal hybridomas, except keep the cells separate from the other cultures until there is little chance of the contamination reappearing.

Even though the invention has been described with a certain degree of particularity. it is evident that many alternatives, modifications, and variations will be apparent to those

5 skilled in the art in light of the foregoing disclosure. Accordingly, it is intended that all such alternatives, modifications, and variations which fall within the spirit and the scope of the invention be embraced by the defined claims.

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed 0 in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and 5 scope o f the invention .

EXAMPLE 1

RNA POLYMERASE II BINDS TO TAR RNA

IN THE PRESENCE OF CELLULAR COFACTORS 0

The present example demonstrates the interaction of cellular cofactor proteins with RNA polymerase II, and the effect of this interaction on the modulation of transcriptional activity by RNA polymerase II. Purification of HIV- 1 TAR RNA binding proteins.

RNA polymerase II was prepared from HeLa nuclear pellets as described (Reinberg and Roeder, 1987). The preparation of RNA polymerase II involved the high salt extraction of HeLa nuclei followed by multiple chromatographic steps resulting in RNA polymerase II of 80 to 90% purity that was capable of transcribing the HIV-1 promoter in a reconstituted in vitro transcription system in both the presence and absence of Tat. Using 2x10¹⁰ HeLa cells, approximately 0.5 mg of RNA polymerase II was obtained. Calf thymus DNA at a concentration of lOOug/ml was used as the template for measuring the RNA polymerase II activity. The amount of RNA polymerase II required to obtain detectable binding to TAR RNA is the amount of polymerase II activity present in 1.5 μl of nuclear extract. The nuclear extract was tested in vitro transcription analysis with the HIV-1 LTR and was sensitive to 2μg/ml of O-amanitin. TRP-185, cofactors and Tat were purified as described

J O in Wu et al, (1991); which reference is specifically incorporated herein by reference for this purpose.

Gel Retardation Assay for RNA Polymerase II and Tat Binding. Wild-type and mutant HIV mRNAs were constructed by fusing a synthetic linker containing a T7 RNA polymerase promoter to DNA fragments of the indicated TAR constructs from +1 to +80 and RNA probes were made as described in Wu et al, (1991 ). The transcribed RNA was gel isolated, eluted, and used for binding with the modifications that 1.5 mM Pefobloc SC

(AEBSF) and lxTIB buffer (50 mM Hepes, pH 7.9/10 mM ascorbic acid 50 mM mannitol/10% glycerol/0.1% Nonidet P-40/0.1 mM EDTA/5 mM DTT/150 mM NaCl) per

30 μl binding reaction were included. UV cross-linking was performed under identical conditions as used for the gel retardation assays.

Gel retardation analysis was performed with RNA polymerase II and a labeled HIV-1 TAR RNA probe. When RNA polymerase II was tested for its ability to bind to TAR RNA using amounts of this protein (45 ng to 225 ng) that were necessary for the stimulation of in vitro transcription from the HIV-1 LTR, there was only minimal binding to TAR RNA. However, the addition of a similar quantity of cellular cofactors (0.4 μg), required for the binding of TRP- 185 to HIV-1 TAR RNA, resulted in a marked stimulation of RNA polymerase II binding to TAR RNA. The cellular cofactor fraction used was prepared as described in Wu et al, (1991).

No binding to TAR RNA was found using the cofactor fraction alone. Equivalent quantities of other proteins, such as albumin or glutathione S -transferase, did not result in any significant stimulation of RNA polymerase II binding to TAR RNA. It was also found that the addition of increasing amounts of cofactors (0.15 ug - 1.0 ug) further stimulated the binding of RNA polymerase II to TAR RNA while a quantity of bovine serum albumin (BS A) equal to the highest concentration of added cofactors had no effect on the binding of RNA polymerase II to TAR RNA.

These results indicate a highly specific effect of the cofactors on increasing the binding of RNA polymerase II to TAR RNA. Thus, the cellular cofactors that regulate the binding of TRP-185 to the HIV-1 TAR RNA loop sequences also regulate the binding of RNA polymerase II to TAR RNA.

EXAMPLE 2 ANTIBODY TO THE C-TERMINAL DOMAIN OF RNA POLYMERASE IIPREVENTS BINDING OF RNA POLYMERASE II TO TAR RNA The present example provides data that demonstrate that the binding of RNA polymerase II to HIV-1 TAR RNA was specific. For these experiments, a monoclonal antibody 8WG16 directed against the C-terminal domain (CTD) of RNA polymerase II

(Thompson et al, 1989) was used to determine whether the addition of this antibody to a gel retardation assay prevented RNA polymerase II binding to TAR RNA.

The addition of the anti-CTD monoclonal antibody (1 μg) inhibited the binding of

RNA polymerase II (40 ng in the presence of 0.4 μg cofactor fraction) to TAR RNA, while monoclonal antibodies directed against either TRP- 185 or beta-galactosidase did not alter the binding of RNA polymerase II to TAR RNA. To further analyze the specificity of these antibodies, their effects on the binding of TRP- 185 were tested. TRP-185 bound to TAR RNA was supershifted by both polyclonal and monoclonal antibodies directed against TRP-185. Antibodies to the RNA polymerase II CTD or beta-galactosidase did not alter the binding of TRP-185 to TAR RNA. These results indicate that the binding of RNA polymerase II to TAR was not due to the presence of TRP-185. To further elucidate the binding properties of RNA polymerase II, UV cross-linking was performed with the labeled wild-type TAR RNA probe. Amounts of RNA polymerase II were used in these studies (200 ng) that allowed for its binding to TAR RNA in the absence of added cofactor fraction. The RNA polymerase II, either alone (200 ng) or in the presence of the cofactor fraction (0.4 μg), resulted in an approximately 220-240 kDa UV cross-linked species. These results are consistent with the binding of the largest subunit of RNA polymerase II.

EXAMPLE 3 RNA POLYMERASE II BINDING TO TAR RNA IS DEPENDENT ON AN INTACT TAR STRUCTURE

The present example provides studies that demonstrate specificity of RNA polymerase II binding to HIV-1 wild-type TAR RNA.

Competition analysis was performed with a 50-fold molar excess of various unlabeled TAR RNAs, including wild-type as well as TAR RNAs having mutations of the loop, bulge, and the stem. TAR RNA containing mutations at the loop, bulge or stem region have been shown to be extremely defective for /αt-activation in vivo (Wu et al. 1991). These studies demonstrate a correlation between the binding of RNA poiymerase II to TAR RNA and the role of these TAR RNA structures on in vivo rαr-activation. The addition of a 50-fold molar excess of unlabeled wild-type RNA resulted in an approximately 20-fold decrease in the binding of RNA polymerase II (120 ng in the presence of 0.4 μg cofactor) to TAR RNA (SEQ ID NO:6).

Mutation of the HIV-1 TAR RNA loop sequences alone (+31/+34) or both the loop sequences and a portion of the primary sequence of TAR that maintained stem base pairing (TAR-sense)/(+31/+34) were much poorer competitors for RNA polymerase II binding, resulting in decreases of RNA polymerase II binding to the wild-type TAR RNA ranging from two to four-fold. Other TAR RNA mutants which have been demonstrated to be defective in in vivo tarr-activation (Wu et al, 1991) include mutants with changes in the TAR RNA primary sequence (TAR-sense), mutations of the bulge (+23) (deletion of +23/+25). and a TAR stem disruption mutation (+19/+22). These mutants were much more defective in competition for RNA polymerase II binding than the unlabeled wild-type TAR RNA. These mutant TAR sequences are provided as SEQ ID NOS.8-17.

In contrast, a TAR RNA stem restoration mutant, that has been demonstrated to have nearly wild-type levels of gene expression in vivo in response to tat, (+19/+22/+40/+43), resulted in approximately 10-fold competition for the binding of RNA polymerase II to TAR RNA.

These results indicated that the binding of the RNA polymerase II to TAR RNA mutants correlated extremely well with the ability of these mutants to function in in vivo assays for ta -activation of the HIV-1 LTR.

EXAMPLE 4

ALTERATION OF THE PHOSPHORYLATION STATE OF RNA POLYMERASE

II DOES NOT PREVENT BINDING TO TAR RNA

The present examples examines the activity of RNA polymerase II in both its hyperphosphorylated and the hypophosphorylated forms, particularly the ability of the different forms of the RNA polymerase II to bind to HIV-1 TAR RNA.

The RNA polymerase II preparation used in these studies contained a mixture of both phosphorylated (Ho) and nonphosphorylated (lla) forms of RNA polymerase II as judged by Western analysis with the monoclonal antibody 8WG16 (Thompson et al , 1989) which is directed against the RNA polymerase II CTD. Alkaline phosphatase or cdc2 kinase treatment was used to convert the RNA polymerase II largest subunit to primarily either the 240 kDa hyperphosphorylated (IIo) or the 220 kDa hypophosphorylated (lla) form as follows. The hypophosphorylated form of RNA polymerase II (lla) was prepared by using 20 units of alkaline phosphatase (Boehringer-Mannheim. Indianapolis. IN) per 200 ng of

RNA polymerase II in binding buffer in the absence of TAR RNA. The phosphorylated form of RNA polymerase II (Ho) was prepared by using 75 ng of cdc2 kinase (Upstate

Biotechnology, Lake Placid, NY) with 1.4 ug of RNA polymerase II and 1 mM ATP in buffer containing lOmM Tris (pH 7.9), 50 mM KC1, 0.1 mM EDTA, 10 mM MgCL, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonylfluoride and 10 % glycerol.

Gel retardation analysis was then performed with either the predominantly hypophosphorylated or phosphorylated forms of RNA polymerase II. Treatment with alkaline phosphatase did not prevent RNA polymerase II from binding to TAR RNA in the presence of cellular cofactors. Whether alkaline phosphatase treatment altered only the phosphorylation state of the RNA polymerase II or potentially also modified the cofactors could not be addressed. Treatment of the RNA polymerase II with cdc2 kinase resulted in the generation of a slightly slower mobility gel retarded complex. A nonspecific faster mobility species was detected in preparations of the cdc2 kinase. Thus, the ability of RNA polymerase II to bind to TAR RNA does not depend absolutely on the degree of CTD phosphorylation.

EXAMPLE 5

TAT DECREASES BINDING OF RNA POLYMERASE II TO TAR RNA

The present example provides data that test a model consistent with TAR RNA serving as a specific attenuator element that causes RNA polymerase II to pause and Tat functioning to release the bound RNA polymerase II. The amino acid sequence of Tat and the nucleotide sequence encoding Tat, (Modesti et al, 1991), are provided as SEQ ID NOS: 3 and 2, respectively.

Gel retardation analysis was performed with RNA polymerase II and cofactors, followed by the addition of either bacterially produced wild-type Tat or a Tat mutant having substituted amino acids in its basic domain. This Tat mutant had previously been demonstrated to be unable to bind to TAR RNA (Wu et al, 1991). Wild-type TAR RNA or TAR RNA mutated at +23 in the TAR RNA bulge were used in these gel retardation studies. Wild-type Tat has been demonstrated to be unable to bind to this latter mutant in the TAR RNA bulge (Wu et al, 1991). Wild type Tat, but not the Tat mutant, was able to bind specifically to wild-type TAR RNA. The addition of wild type Tat to TAR RNA containing RNA polymerase II (45 ng) and cofactor fraction (0.4 μg) resulted in a marked decrease in the binding of RNA polymerase II to TAR RNA. This was not seen following the addition of the Tat basic mutant. Surprisingly, the addition of wild-type Tat but not mutant Tat was also capable of preventing the binding of RNA polymerase 11 to TAR RNA which contained a mutation at +23 in the TAR RNA bulge. Neither wild-type nor mutant

Tat protein bound to this mutant TAR RNA. These results demonstrate that Tat was capable of interfering with the binding of

RNA polymerase II to TAR RNA even when Tat was unable to bind to TAR RNA.

EXAMPLE 6 TAT INTERACTS WITH THE LARGEST SUBUNIT OF RNA POLYMERASE II

The present example provides studies that examine whether Tat directly interacts with RNA polymerase II. It was first necessary to obtain purified preparations of RNA polymerase II. RNA polymerase II contains ten subunits with the largest subunit being comprised of three species of 240 (Ho), 210 (Ha), and 180 (lib) kDa, respectively, which are due to differences in the phosphorylation state (Ho vs lla) or proteolysis (lla vs lib) (Kim and Dahmus. 1986; Lu et al, 1991). The second largest subunit (He) of RNA polymerase II is 140 kDa while other subunits of 34, 25, 22, 18, 19, 16, 14 and 13 kDa have been characterized (Kim and Dahmus, 1986; Lu et al , 1991). The holo-RNA polymerase II complex migrates at approximately 550 kDa and this complex is critical for transcriptional activity.

Plasmid constructs and antibodies. The HIV-1 Tat (Tatl) from amino acids 1 to 72 was expressed as a fusion protein with glutathione S-transferase (GST; Stratagene. La

Jolla, CA) (Wu et al, 1991). A thrombin recognition site (LVPRGS; SEQ ID NO:29) followed by the GST coding sequence was inserted C-terminal to the Tat coding sequence.

A second version of this construct contained a kinase recognition motif RRASV (SEQ ID

NO:30) inserted at the C-terminus of GST. A Tat mutant in the basic domain of Tat between residues 52 and 57 contains a substitution of Arg-Arg-Gln-Arg-Arg-Arg (SEQ ID

NO:31) to Gly-Gly-Ala-Gly-Gly-Gly (SEQ ID NO:32). (Wu et al. (1991). The Tatl mutant contains a serine residue in place of the first cysteine residue in each of four Cys-X-

X-Cys (SEQ ID NO:33) motifs while the K41 mutant is a substitution of lysine residue 41 to alanine in the Tat protein.

The wild-type and mutant HIV-2 Tat clones, GST-Tat2 and GST-Tat2Δ 84, were obtained from the NIH AIDS Research and Reference Reagent Program and contain the GST moiety followed by a thrombin cleavage site and either residues 1 through 99 or 1 through 83 (Rhim et al, 1993). The Tat2 CL mutant contains a substitution of cysteine residue 50 with serine and lysine residue 70 with alanine and is not able to activate HIV-2 gene expression in either in vitro or in vivo assays. A second version of the above clones with a kinase recognition motif RRASV (SEQ ID NO:30) at the C-terminus of Tat was constructed using PCR.

A portion of the largest subunit of RNA polymerase II extending from amino acids

1325 to 1630 (Wintzerith et al, 1992) and a portion of the second largest subunit of RNA polymerase II extending from amino acids 1 to 550 (Acker et al, 1992) were fused to glutathione S -transferase. The full-length CREB cDNA extending from amino acids 1-341 and either wild-type RAP30 extending from amino acid residues 1 to 249 or a RAP30 truncation mutant extending from amino acids 1 to 101 were constructed by PCR and inserted into pGEX 2T for expression in bacteria. Fusion proteins including GST, GST- Tatl, GST-Tat2, GST-pl 80 and GST-pl40 were affinity purified following E. coli expression as described (Wu et al, 1991) and were used as antigens to obtain rabbit polyclonal antisera. The monoclonal antibody 8WG16 (Thompson et al, 1989) directed against the C-terminal domain of RNA polymerase II was purchased from Promega.

Purification of RNA Polymerase II. RNA polymerase II was isolated from 1000 grams of calf thymus. The peak of α-amanitin sensitive activity from the phosphocellulose column was pooled, precipitated with 0.55 gram of ammonium sulfate/ml, the precipitate was resuspended, and dialyzed as described (Hodo and Blatti, 1977). The yield was approximately 5mg of RNA polymerase II which was active in in vitro transcription assays and whose activity was inhibited by 1 mg/ml of α-amanitin in in vitro transcription assays and α³²P [UTP] incorporation. Nuclear extract was prepared from HeLa cells by the method of Dignam et al. (1983). The C fraction was obtained by collecting the 0.3 to 0.5M KC1 eluate of HeLa nuclear extract following phosphocellulose chromatography as described (Reinberg and Roeder, 1987).

The 210 kDa species in addition to the predominant 180 kDa form of the largest subunit were present in SDS-PAGE gels. The 180 kDa species, which is generated by proteolysis of the C-terminal domain (CTD) of the 210 kDa subunit. has been demonstrated to be the predominant species isolated from calf thymus during purification of RNA polymerase II (Corden et al , 1985). No detectable Ho form of RNA polymerase II is found in calf thymus preparations (Hodo and Blatti, 1977: Kim and Dahmus. 1986). However, RNA polymerase II preparations containing the 180 kDa form of the largest subunit are transcriptionally active in reconstituted in vitro transcription assays (Hodo and Blatti, 1977; Kim and Dahmus. 1986: Corden et al. 1985). The 140 KDa second largest subunit of RNA polymerase II (Acker et al, 1992; Hodo and Blatti, 1977; Kim and

Dahmus. 1986; Lee et al, 1991) in addition to the 34 and 25 kDa polymerase subunits were also detected. The 68 kDa species was due to a small amount of bovine serum

5 albumin which was added to the RNA polymerase II preparations to maintain activity while the 43 kDa species is a contaminant. This calf thymus preparation of RNA polymerase II was active in both α³²P [UTP] incorporation into calf thymus DNA and in vitro transcription assays with the HIV-1 LTR.

Western blot analysis was next performed with antibodies directed against the i o largest and second largest subunits of RNA polymerase II to demonstrate its immunologic properties. Monoclonal antibodies directed against the CTD in the largest subunit of RNA polymerase II (Thompson et al, 1989) in addition to rabbit polyclonal antibodies directed against domains in either the largest subunit or the second largest subunit of RNA polymerase II were tested in Western blot analysis. The monoclonal antibody directed

15 against the CTD reacted with the 210kDa species, the rabbit polyclonal antibody directed against the largest subunit reacted with the 180kDa species, while the rabbit polyclonal antibody directed against the second largest subunit reacted with the 140 kDa species. The failure of the rabbit polyclonal antibody which was directed against the largest subunit of the RNA polymerase II to react with 210 kD species was due to the fact that this species

20 was present at only 10 percent of the level as the 180 kDa species and the decreased sensitivity of the rabbit polyclonal antibody as compared to the monoclonal antibody directed against the CTD (Kim and Dahmus, 1986). However, with 10-fold more of the calf thymus RNA polymerase II, the 210 kDa species could be detected with the rabbit polyclonal antibody directed against the largest subunit of the polymerase.

25 The observations of Example 5, using gel retardation assays with TAR RNA and

RNA polymerase II, demonstrated that wild-type Tat, but not a Tat basic mutant, prevented stable binding of RNA polymerase II to TAR R A. These results suggested that Tat could potentially interact with RNA polymerase II. Wild-type and Tatl basic mutant proteins were constructed that contained a cyclic AMP dependent protein kinase A recognition site

30 in their carboxy-termini to facilitate ³²P labeling. To determine whether either of these Tat proteins interacted directly with the RNA polymerase II, far Western analysis was performed (Feaver et al, 1994). In this assay, the calf thymus RNA polymerase II was first subjected to SDS-PAGE. transferred to nitrocellulose, and probed with ³²P labeled wild-type Tatl or a Tatl basic mutant. Wild-type Tatl bound predominantly to 180kDa form of RNA polymerase II, though a slight degree of binding to the 210 kDa form was noted, while the Tatl basic mutant did not bind to these species. No binding was detected to the 140 kDa second largest subunit or other smaller RNA polymerase II subunits. Thus, the wild-type HIV-1 Tat protein was able to specifically interact with the largest subunit of

RNA polymerase II.

EXAMPLE 7

HIV-1 AND HIV-2 TAT PROTEINS SPECIFICALLY

INTERACT WITH RNA POLYMERASE II

The specificity of the interaction between HIV-2 Tat protein (Tat2) and HIV-1 Tat protein (Tatl), and RNA polymerase II is provided in this example. The example also investigates which domains in the Tat protein were critical for this interaction. A variety of mutants in the cysteine, core, or basic domains of Tat that were defective for activation of HIV-1 and HIV-2 gene expression (Gaynor, R., 1992) were used in these studies to determine the domains which interacted with RNA polymerase II.

Affinity binding of RNA polymerase II and Tat. Glutathione agarose beads containing the fusion proteins (lOμg) previously described were incubated with 5μg of calf thymus RNAP II or 30μg of HeLa nuclear extract fraction C at 4° C for 12 hr. The matrix was pelleted, washed three times with 500μg of binding buffer, and the proteins remaining on the matrix were solubilized. The proteins were resolved on SDS PAGE and transferred to nitrocellulose for Western analysis with the antibodies described. To assay Tat binding to immobilized RNA polymerase II, approximately 5μg of immobilized RNA polymerase II (Sopta et al. 1985), was incubated with 500ng of the HIV-2 Tat proteins or 3μg of the HIV-1 Tat proteins in 200μl of binding buffer. Western blot analysis of the bound proteins was performed using the ECL-protein detection system (Amersham Life Sciences) following the manufacturers protocol.

Purification and labeling of HIV- 1 and HIV-2 Tat. Tat proteins were isolated on glutathione agarose matrix, the matrix was equilibrated in thrombin cleavage buffer (40 mM Hepes-KOH, pH 8.3, 30 mM KC1, 2.5 mM CaCl₂, 0.5% glycerol, 0.01% NP-40, 1 mM DTT, 10 μM heparin) and 5 units of thrombin (Sigma) at 25° C for 1 hr was added. To remove the thrombin, the native Tat proteins were applied to a heparin-agarose column. the column was washed with buffer containing 200 mM NaCl. and Tat was eluted with buffer containing 800 mM NaCl and dialyzed. Native Tat was of greater than 95% purity as judged by Coomassie staining of an SDS PAGE gel.

To prepare ³²P labeled Tatl, glutathione agarose containing glutathione S- transferase fusions with either wild-type or the Tatl basic mutant was incubated in 500μl of buffer supplemented with 2.5 mM MgC , 200 units of the cAMP-dependent protein kinase catalytic subunit (Promega Madison. WI) and 0.33 mCi (7000 Ci/mmol) of gamma

³²P-ATP (ICN Biomedicals) for 1 hr. at 25° C. The matrix was washed free of unincorporated label, and the labeled-Tat proteins were eluted with 60 mM glutathione.

The specific activity was normalized by the addition of unlabeled protein to a specific activity of 1 x 10⁶cpm/μg of protein and 300,000 cpm ml of the Tat protein was used in far Western analysis (Feaver et al, 1994).

Glutathione S-transferase (GST) fusions containing either wild-type Tatl, or Tat2, or mutants in different domains of these proteins were coupled to glutathione agarose beads. Other controls such as glutathione agarose beads alone or these beads coupled to either GST or GST-CREB were used to further demonstrate the specificity of Tat interaction with RNA polymerase II. Similar quantities of each of the GST fusion proteins were bound to glutathione agarose beads and incubated with RNA polymerase II purified from calf thymus. Following this incubation, the beads were extensively washed, subjected to SDS-PAGE, and Western blot analysis was performed with antibodies directed against the largest or the second largest subunits of RNA polymerase II or the GST moiety.

Wild-type HIV-1 and HIV -2 Tat proteins were each able to specifically interact with RNA polymerase II as reflected by the presence of the largest and second largest subunits of RNA polymerase II which remained bound to the Tat beads after extensive washing. Mutants in the cysteine and core domains of both HIV-1 and HIV-2 Tat were also able to interact with RNA polymerase II. However, substitutions or deletions in the basic domains of Tatl and Tat2 were unable to bind to RNA polymerase II as reflected in the fact that neither the largest nor the second largest subunits of RNA polymerase II remained bound to these mutant Tat proteins. GST-CREB, which contains a region of 12 basic amino acids that facilitates its DNA binding properties to cyclic AMP-responsive promoter elements, was unable to bind to RNA polymerase II, indicating that the polymerase did not bind nonspecifically to any basic amino acid domain. There was no interaction of RNA polymerase II with either glutathione agarose beads or these beads coupled with GST alone. Western blot analysis confirmed that relatively equal amounts of the GST fusion proteins were coupled to the glutathione agarose beads. These results indicated that both the HIV-1 and HIV-2 Tat proteins were able to specifically interact with

RNA polymerase II as reflected in the binding of the largest and second largest subunits of

RNA polymerase II to Tat. These results, in conjunction with the far Western analysis, indicate that Tat interacts with the multi-subunit RNA polymerase II complex upon its direct binding to the largest subunit of the RNA polymerase.

EXAMPLE 8 HIV-1 AND HIV-2 TAT PROTEINS INTERACT WITH IMMOBILIZED RNA POLYMERASE II AND WITH RNA POLYMERASE II

PRESENT IN HELA NUCLEAR EXTRACT

The present example provides data that examine whether native HIV-1 and H1V-2 Tat proteins cleaved from the glutathione S-transferase moiety by treatment with thrombin could interact with RNA polymerase II purified from calf thymus and immobilized on an Affi-Gel 10 resin (Sopta et al, 1985).

Preparation of immobilized Tat and RNA polymerase II. Cultures of the E. coli strain BL21 DE3 pLysE, bearing plasmids encoding the GST-fusion proteins previously described were washed with PBS containing ImM DTT and 1 mM ascorbate and resuspended into 3ml of buffer/ 100ml of culture with TIB (20 mM Hepes-KOH. pH 8.3, 400 mM NaCl, 10% glycerol, 50 mM mannitol, 10 mM ascorbate, 5 mM DTT. 0.05% NP- 40, 0.1 mM EDTA, and 1 mM PMSF). Cleared lysate was bound to 0.5 ml glutathione- agarose/400 ml culture, washed extensively with TIB, and the amount of bound protein was determined using the Biorad protein assay and Western blot analysis with an anti-GST polyclonal antibody.

To prepare beads containing RNA polymerase II, 225 μg of RNA polymerase II was dialyzed against coupling buffer (50 mM Hepes-KOH, pH 7.9, 100 mM NaCl, 10% glycerol, 0.1 mM EDTA, 1 mM DTT, 1 mM PMSF). The RNA polymerase II was coupled to 80 μl of Affi-Gel 10 (Biorad Laboratories, Melville, NY) following the manufacturers protocol (Sheline et al, 1991).

The activity of the native Tat proteins was confirmed by in vitro transcription analysis with the HIV-1 and HIV-2 LTRs and RNA gel retardation studies with HIV-1 and HIV-2 TAR RNAs. As a control, this same resin was also coupled to lysozyme and tested for interactions with HIV-1 and HIV-2 Tat. Wild-type Tatl and Tat2 proteins were able to bind to RNA polymerase II immobilized on Affi-Gel 10 as determined by Western blot analysis using specific antibodies directed against either Tatl or Tat2. However, there was no binding of wild-type HIV-1 and HIV-2 Tat proteins to resin coupled to lysozyme, nor did basic domain Tat mutants bind to the immobilized RNA polymerase II. These results demonstrated that native Tatl and Tat2 proteins were able to interact with RNA polymerase II. Furthermore, the basic domain of Tat mediates HIV-1 and HIV-2 Tat interactions with RNA polymerase II.

Further studies addressed whether Tat was able to interact with RNA polymerase II present in HeLa nuclear extract. In contrast to the presence of the 180 kDa form of the largest subunit of RNA polymerase II that is found in preparations isolated from calf thymus (Hodo and Blatti, 1977; Kim and Dahmus, 1986), HeLa nuclear extract contains predominantly the 210 kDa form of the largest subunit which has an intact CTD (Lu et al,

1991). HeLa nuclear extract was fractionated using phosphocellulose chromatography followed by step elution with either (A) 0.1 , (B) 0.35, (C) 0.50, or (D) 1.0M KC1 concentrations (Reinberg and Roeder, 1987b) The present studies indicate that RNA polymerase II is present predominantly in the C fraction of HeLa nuclear extract when eluted with KC1 by phosphocellulose chromatography. The HeLa fraction C was incubated with glutathione beads containing either wild-type or basic mutants of Tatl and Tat2 in addition to wild-type or mutant forms of RAP30. RAP30, along with its dimeric partner RAP74, comprises the transcription factor TFIIF that functions in both transcriptional initiation and elongation by facilitating the binding of RNA polymerase II to the transcriptional preinitiation complex and also by inhibiting the pausing of the elongating

RNA polymerase II. RAP30 has been demonstrated to directly associate with RNA polymerase II and the domains that facilitate this interaction have been defined (Horikoshi et al. 1991 ; Sopta et al, 1985). Thus, the binding of RNA polymerase II could be compared to both wild-type and mutant RAP30, and Tat proteins.

Western blot analysis was performed with monoclonal antibody directed against the

RNA polymerase II CTD. Wild-type Tatl, Tat2 and RAP30 were each able to interact with RNA polymerase II. In contrast, there was no binding of RNA polymerase II detected with the Tatl and Tat2 basic mutants nor a RAP30 mutant that was deleted in its binding site for RNA polymerase II. The second largest subunit of RNA polymerase II also bound to the immobilized Tat and RAP30 proteins. Coomassie staining of an SDS polyacrylamide gel indicated that the amounts of wild-type and mutant Tat and RAP30 proteins coupled to glutathione S-transferase beads were relatively similar. Thus Tat. like the well characterized transcription factor RAP30. binds specifically to RNA polymerase

II.

The magnitude of interactions between Tat and RNA polymerase II were similar to those detected between RNA polymerase II and the cellular transcription factor RAP30 b which stably associates with RNA polymerase II during transcriptional elongation. These studies are consistent with the model that RNA polymerase II is a cellular target for Tat resulting in Tat-mediated increases in transcriptional elongation from the HIV LTR.

EXAMPLE 9 ₀ PURIFICATION OF TRP-185

A cellular factor TRP-185 that bound specifically to the HIV-1 TAR RNA loop sequences has been described (Wu et al., 1991). TRP- 185 binding to TAR RNA required the presence of both the TRP- 185 protein and a separate set of factors designated the 5 cofactor fraction. The mechanism by which these cofactors stimulate TRP- 185 binding to TAR RNA had not before been elucidated. While not intending to be limited to any particular mode of action, it is contemplated that either direct binding to TAR RNA with subsequent dissociation of these factors during gel electrophoresis or post-translational modification of TRP- 185 are possible mechanisms. To further characterize the potential 0 role of TRP-185 on regulating HIV-1 gene expression, the present inventors developed a purification scheme for this factor from HeLa nuclear extract depicted in FIG. 1.

All protein purification procedures were performed at 4°C. Nuclear extract was prepared from 60 liters of HeLa cells as described (Dignam et al, 1983) and applied to a heparin agarose column (2.5 x 9 cm) equilibrated with buffer A (20mM Tris-Cl. pH 7.9, 5 20% glycerol (v/v), 0.2 mM EDTA) containing 0.1 M KCl, 0.5 mM PMSF and 0.5 mM DTT. The column was washed with the same buffer until A₂₈₀ was almost zero and the bound proteins were eluted with buffer A containing 0.4 M KCl, 0.5 mM PMSF and 0.5 mM DTT. The buffer A fractions were pooled and dialyzed against buffer with 0.1 M KCl. 0.5 mM PMSF and 0.5 mM DTT. The dialyzed fraction was then applied to HTP Bio Gel 0 (2.5 x 7 cm) column equilibrated with the same buffer. The column was washed with the same buffer above and eluted with buffer containing 0.1 M potassium phosphate (pH 7.0). 0.5 mM PMSF and 0.5 mM DTT. The active fractions were pooled and precipitated with 70% ammonium sulfate for 20 minutes at 4°C and then centrifuged at 12.000 rpm for 20 minutes. The pellet was resuspended in 6 ml of buffer with 0.1 M KCl with 1 mM DTT 5 and then applied to a Superdex 200 FPLC (HiLoad 26/60 prep grade) column equilibrated with this buffer. The active fractions were pooled and applied to a Bio Rex 70 (1.5 x 3 cm) column. The active flow-through fractions were pooled and applied to Dextran Blue

Sepharose (1 x 2.5 cm) column equilibrated in the same buffer. The column was washed and eluted with buffer containing 0.4 M KCl and 1 mM DTT. The active fractions were pooled and dialyzed against buffer containing 0.1 M KCl and 1 mM DTT. The pooled and dialyzed fraction was then applied to a 1 ml Mono Q FPLC column equilibrated in the same buffer. The column was washed and then eluted with buffer containing 0.4 M KCl and 1 mM DTT. The active fraction was dialyzed against 20 mM Tris-Cl, pH 7.9, 5% glycerol and 0.2 mM EDTA, 0.1 M KCl and 1 mM DTT and loaded onto centrifuge tubes (1.4 x 8.9 cm) containing 10 ml of a 5% to 25% continuous sucrose gradient. A preparative sucrose gradient was then performed using a Beckman SW40 Ti rotor centrifuged at 30,000 rpm for 40 hours to 4°C. The sucrose gradient was fractionated from the boUom of the tube and assayed. The active fractions were pooled, diluted 1 : 1 (v/v) with buffer containing 0.1 M KCl and 1 mM DTT and loaded onto a 1 ml Mono Q FPLC column. The column was washed with the same buffer and eluted with buffer containing 0.2 M KCl and 1 mM DTT. The active fractions were stored at -70°C. This purification scheme yielded TRP-185 with greater than 95% purity as visualized by silver staining of a polyacrylamide gel.

Protein fractions isolated from different column chromatographic steps were assayed for TRP-185 binding using gel retardation analysis with wild-type and mutant TAR RNAs. Cofactors purified from HeLa cells as described in Wu et al, 1991. which by themselves exhibited no binding to wild-type TAR RNA, were added to these assays to stimulate TRP- 185 binding. HeLa nuclear extract prepared from 60 liters of cells was applied sequentially to columns containing heparin agarose, hydroxy apatite. Superdex 200 FPLC. Bio-Rex, dextran blue, and mono Q. Following these chromatographic steps, the fractions containing TRP-185 were applied to a preparative sucrose gradient and concentrated on a mono Q FPLC. A silver-stained polyacrylamide gel of the chromatographic fractions containing TRP- 185 eluted from the final mono Q column and a corresponding gel retardation analysis of these fractions with wild-type TAR RNA indicate that the presence of a 185 kDa species correlates with binding activity to TAR RNA.

EXAMPLE 10 CLONING OF THE GENE ENCODING TRP-185

The gene encoding TRP- 185 was cloned as follows. Active fractions of TRP- 185 as judged by their ability to bind to TAR RNA were isolated from twenty-six 60 liter HeLa cell preparations and purified as described. These fractions were pooled and concentrated on a Centricon 30 membrane which was previously blocked with BSA and washed. The concentrated sample (150 μl) was loaded into three wells of a 8% polyacrylamide. 0.1%

SDS protein gel and was subjected to electrophoresis in Tris-glycine buffer. The gel was then blotted overnight onto a nitrocellulose membrane (0.45μm) and the protein bands were visualized by Ponceaus stain and excised as described (Aebersold et al, 1987). The

NaOH destaining step was omitted. The amount of TRP-185 used for digestion with endoprotease Lys C for this analysis was approximately 50 μg and this was followed by separation by HPLC and N-terminal sequence of the peptides by automated Edman degradation on an ABI model 477A protein sequence (Ha et al, 1991 ; Lane et al, 1991). Two peptides with the amino acid sequence of LKPGDWSQQDIGTNLVEADNQAEW (SEQ ID NO: 18) and TEGYTIIGVEQTAK (SEQ ID NO: 19) were obtained.

Degenerate oligonucleotide primers were made to the 5' and 3' end of the 24-mer peptide sequence obtained from Lys C digestion of TRP-185 and PCR analysis was performed to generate a 72 base pair fragment of the TRP-185 cDNA. PCR primers were then made according to the actual nucleic sequences in this fragment and PCR analysis was performed with the degenerate oligonucleotide primers which corresponded to the 14 amino acid peptide obtained from amino acid sequence analysis. A 435 bp fragment of cDNA encoding TRP-185 was obtained (SEQ ID NO: 21) (from nucleotides 4210 to 4644 of SEQ. ID. NO. 20). This fragment was then used as the probe to screen a HeLa cDNA library in lambda zap (ClonTech). A 5.6kb cDNA (SEQ ID NO:20) encoding a 1621 amino acid open reading frame encoding the full length TRP- 185 protein was obtained.

The full-length 1621 amino acid sequence of the TRP- 185 protein is shown as SEQ ID NO:7. TRP- 185 is a protein lacking classical RNA binding motifs such as zinc fingers or ribonucleoprotein binding domains. However, a leucine zipper domain consisting of leucine residues at every seventh position between amino acids 535 to 556 was identified. In addition, a number of so called lysine-helix repeats, which are a novel structural element consisting of lysine or arginine residues spaced at seven amino acid intervals, were noted throughout the TRP- 185. These repeats were previously noted in the transcription factor nuclear factor 1 and they have been postulated to be involved in its DNA binding properties. Finally, a variety of potential nucleotide binding sites were noted in the C- terminal portion of the TRP- 185 protein as were potential sites for post-translational modification such as phosphorylation and glycosylation.

TRP- 185 messenger RNA has two different forms, one form is represented by SEQ ID NO:20. with the amino acid sequence represented by SEQ ID NO:7: the other form is identical to the TRP-185 sequence up through base number 3792 of SEQ ID NO:20 (amino acid 1264 (lysine) of SEQ ID NO:7) and with an added 3' sequence of bases that encodes 7 carboxy terminal amino acids (GTA AGT TTG TTT GTA AGA ATT (SEQ ID NO:34) encodes the seven amino acids, Val Ser Leu Phe Val Arg He (SEQ ID NO:35). The total length of this alternative message is 3813 nucleotides up to an ochre stop codon (SEQ ID

NO:36) and 1271 amino acids (SEQ ID NO:37). The protein encoded by this alternative message is designated TRP-140.

EXAMPLE 11

EXPRESSION AND PURIFICATION OF RECOMBINANT TRP- 185

The present example provides for the expression, purification, and immunoreactivity of cloned and purified TRP-185. PCR primers were used to modify the 5' ATG and 3' end of the TRP-185 cDNA into Ncol and BamHI restriction sites, respectively. The TRP- 185 full-length cDNA was then cloned downstream of the T7 promoter in a modified pTMl vaccinia expression vector (Elroy-Stein et al, 1989) with codons for the twelve amino acid influenza hemagglutinin amino acid sequence (Field et al, 1988) and codons for six histidine residues placed at the 3' end (Tanog et al. 1995). The histidine residues facilitate purification of the TRP-185 protein. The hemagglutinin sequences facilitate detection of the protein with monoclonal antibody 12CA5. This construct was transfected by phosphate precipitation onto HeLa cells infected with a vaccinia recombinant virus that produced T7 polymerase (Janknecht et al. 1991 ). The cells were harvested at 48 hours post-transfection and nuclear extract was prepared as described previously except 1 μg/ml of leupeptin and aprotinin were included in the buffers. The nuclear extract prepared from 30 plates (150mM) of HeLa cells and then subjected to chromatography on a 2 ml Q-Sepharose column (1.5 x 2 cm) equilibrated with buffer A containing 0.1 M KCl, 0.5 MM PMSF. 1 μg/ml of leupeptin and aprotinin and 10 mM β mercaptoethanol. The column was washed and eluted with this same buffer containing 0.3 M KCl. The eluted fractions were pooled and then loaded onto a 1 ml Ni- NTA agarose (Qiagen) column equilibrated with this same buffer. The flow through was reloaded onto this column a second time and the column was washed with (1) 20 ml of the same buffer (2) 20 ml of this buffer containing 1.0 M KCl and (3) 20 ml of this buffer containing 0.1 M KCl. The column was then eluted with this same buffer containing 0.1 M KCl and 60 mM imidazole. The eluted fractions were then dialyzed against buffer containing 0.1 M KCl and 1 mM DTT and stored at -70°C. A typical yield of recombinant

TRP-185 from 30 plates of HeLa cells is 60-70 μg with greater than 90% purity as judged by silver staining following polyacrylamide gel electrophoresis.

The recombinant TRP-185 was purified by finding and elution of the histidine tagged TRP-185 protein from agarose nickel beads using increasing concentrations of imidazole as described (Tanog et al, 1995).

Western blot analysis was performed with equivalent amounts of both vaccinia expressed TRP- 185 and native TRP- 185 purified from HeLa nuclear extract using a monoclonal antibody raised to a portion of TRP- 185 extending from amino acids 1409 to 1541. This analysis demonstrated that both the recombinant and the native TRP- 185 proteins had the same molecular weight.

To determine if both the recombinant and the native TRP- 185 proteins were present in a gel-retarded complex bound to TAR RNA, gel retardation assays were performed with TRP- 185 in the presence of either monoclonal or polyclonal antibodies directed against TRP- 185. There was no binding of either native or recombinant preparations of TRP- 185 to wild-type TAR RNA in the absence of added cofactors. However, in the presence of the cofactor fraction, there was binding of both of these TRP-185 preparations to the wild-type TAR RNA. There was no binding of the cofactors to the wild-type TAR RNA alone. Addition of pre-immune rabbit sera did not alter the mobility of the TRP- 185 gel retarded complex, however, the addition of either polyclonal or monoclonal antibody directed against TRP- 185 resulted in a supershifted complex. Thus, both the recombinant and the endogenous TRP-185 proteins require the presence of cofactors for binding to TAR RNA and the TRP- 185 protein was present in the gel-retarded complex bound to TAR RNA.

EXAMPLE 12

RECOMBINANT TRP-185 BINDS TO TAR RNA LOOP SEQUENCES

The present example provides studies that analyze the binding properties of recombinant TRP- 185 to wild-type and mutant TAR RNAs (SEQ ID NOS 6. 8-17). The HIV-1 TAR RNA constructs included wild-type (SEQ ID NO: 6), (+31/+34) (SEQ ID NO: 8). TAR-sense (SEQ ID NO: 9), TAR-sense/(+31/+34) (SEQ ID NO: 10). +30 (SEQ ID NO: 1 1 ), +32 (SEQ ID NO: 12), +34 (SEQ ID NO: 13), +23 (SEQ ID NO: 14). a deletion of bulge nucleotides (+23/+25) (SEQ ID NO: 15), (+19/+22)/(+40/+43) (SEQ ID NO: 16), and (+19/+22) (SEQ ID NO: 17). Recombinant TRP- 185 in the presence of cellular cofactors bound to wild-type TAR RNA. There was a 20-fold reduction in the amount of the TRP-185 gel retarded complex using a 50-fold molar excess of unlabeled wild-type

TAR RNA competitor, but little competition with a similar excess of two TAR RNAs. that contained mutations of the loop sequences between +31 and +34. A TAR RNA containing mutations in its primary sequence and that maintained both stem base pairing and the bulge and loop sequences competed as well as wild-type TAR RNA for TRP- 185 binding.

Competition with TAR RNAs containing point mutations in the loop sequences was performed. TAR RNA containing mutations of individual nucleotides in the loop sequences +30 and +34 were very defective for competition of TRP-185 binding. Though a TAR RNA mutant at nucleotide +32 was not defective for competition of TRP-185 binding, this mutant was still defective for competition of TRP- 185 relative to wild-type TAR RNA using a 5-fold lower molar excess of competitor RNA. The results with both single and multiple mutations in the TAR RNA loop indicate that the primary sequence of the loop is critical for TRP- 185 binding.

The effects of several mutations in other portions of TAR RNA were also tested for their ability to compete for TRP-185 binding. Addition of a TAR RNA bulge mutant at nucleotide +23, that prevents the binding of Tat to TAR RNA (Dingwall et al, 1989; Cordingley et al, 1990; Dingwall et al, 1990; Roy et al, 1990; Calnan et al, 1991), did not alter TRP- 185 binding. However, a TAR RNA containing a deletion of the entire bulge structure did not compete for TRP-185 binding. Addition of a TAR RNA with a three nucleotide disruption of the stem structure resulted in decreased competition for TRP- 185 as compared to a TAR RNA which restored TAR RNA stem base pairing by a compensatory mutation. In several other experiments the level of competition with this stem restoration mutant was nearly equivalent to that of wild-type TAR RNA. These results indicate that the binding of TRP- 185 is dependent on the primary sequence of the loop in addition to the maintenance of the overall TAR RNA tern and bulge structures.

EXAMPLE 13 PARTIALLY PURIFIED RNA POLYMERASE II BINDS TO TAR RNA

The present example demonstrates the binding of RNA polymerase II to TAR

RNA. The example also demonstrates the utility of the methods with partially purified RNA polymerase II, as these studies demonstrate binding of even partially purified preparations of RNA polymerase II to TAR RNA.

Wild-type and mutant HIV mRNAs were constructed by fusing a synthetic linker containing a T7 RNA polymerase promoter to DNA fragments of the indicated TAR constructs from +1 to +80 (Wu et al, 1991). Transcription of these constructs was performed after they were linearized with Hind III (+80) by using T7 RNA polymerase resulting in transcripts consisting of nucleotides +1 to +80 of the HIV LTR. RNA synthesis, labeling and purification were performed by using the reagents and procedures of the Riboprobe System II (Promega, Madison, WI)

Approximately 1.5 ng of TAR RNA probe was mixed with RNA polymerase II

(0.04 to 0.4 μg), poly (I)-poly(C) (0.2 μg), and a final concentration of 10 mM Tris-Cl (pH

7.4), 0.1 mM EDTA, 50 mM KCl, 1 mM DTT, 0.5 mM PMSF, 1.5 mM Pefabloc 5C

(AEBSF) and 10% glycerol in 30 μl total volume. For competition analysis, 50 ng of each unlabeled in vitro transcribed competitor TAR PNAS was mixed with probe and binding was performed. Binding was performed at room temperature for 20 minutes, and the samples were loaded onto a 5% polyacrylamide gel containing 1 x Tri-borate-EDTA (TBE) with 2% glycerol, and subject to gel electrophoresis at 180 V in 1 X TBE at room temperature. The binding assays with recombinant TRP- 185 were performed as described for endogenous TRP-185 (Wu et al, 1991). The amount of protein used in these assays were (50 ng) recombinant TRP-185 and cellular cofactor fraction (0.4 μg). Dephosphorylation of TRP- 185 and RNA polymerase II was performed by treating 50 ng of either TRP- 185 or with 20 units of alkaline phosphate (Boehringer-Mannheim) in binding reaction conditions for 20 min at room temperature and then cofactor fraction and TAR RNA probe were added and incubated for an additional 15 minutes at room temperature. ATP was included at a final concentration of 1 mM in binding reaction when necessary. For the antibody supershift studies, 1 μg of each of the antibodies was purified following chromatography on protein A sepharose and added to the binding reactions 10 minutes after the start of reaction and continued for an additional of 20 minutes at room temperature. The TRP-185 polyclonal and monoclonal antibodies used in this study were raised against a portion of TRP- 185 corresponding to amino acids 1409 to 1541 fused in frame to GST. The antibody directed against the C-terminal domain of RNA polymerase II 8WG16 was previously described (Thompson et al, 1989). Since both native and recombinant TRP- 185 bound specifically to TAR RNA, the present inventors investigated whether complexes comprised of TRP- 185 and other transcription factors may exist in HeLa nuclear extract and be capable of binding to TAR RNA. This analysis was facilitated by the presence of specific antibodies which were generated against TRP- 185 that could be used to analyze the components of the gel retarded complexes bound to TAR RNA. HeLa nuclear extract, chromatographed on heparin agarose and hydroxyapatite columns, was analyzed following chromatography on a

Superdex 200 FPLC column. This purification scheme (FIG. 1) allowed the detection of

TRP-185 and also removed a variety of nonspecific double-stranded RNA binding proteins which were present in HeLa nuclear extract.

Gel retardation was performed with wild-type TAR RNA on different fractions eluted from the Superdex 200 column. The binding of two closely migrating species in early fractions and a slower migrating species in later fractions was detected. Gel retardation with these fractions was performed without the addition of cellular cofactors since they copurified during the initial stages of the chromatography. The proteins which were responsible for these gel retarded species were found to be further purified and subject to amino acid microsequence analysis. The proteins which comprised the slower mobility complex that bound to TAR RNA corresponded to TRP- 185. The proteins that gave rise to the two faster mobility gel retarded species were found to be RNA polymerase II by a combination of U.V. crosslinking and gel retardation analysis and Western Blot studies using specific antibodies directed against the largest subunit of RNA polymerase II.

To further demonstrate the identity of these gel retarded species, antibody supershift experiments were performed with chromatographic fractions which gave rise to either the slower or faster mobility complexes bound to TAR RNA. The protein which generated the slower mobility complex was supershifted by a monoclonal antibody against the TRP- 185. However, there was no change in the mobility of this species upon the addition of a monoclonal antibody 8WG16 directed against the C-terminal domain of RNA polymerase II. In contrast to these results, the addition of monoclonal antibody directed against TRP- 185 did not alter the mobility of the two faster mobility species bound to TAR RNA. However, the addition of the 8WG16 monoclonal antibody directed against the

RNA polymerase II CTD prevented the binding of these two species to TAR RNA.

Alkaline phosphatase treatment indicated that these two species were the hypophosphorylated (II_a and hyperphosphorylated (ii₀) forms of RNA polymerase II (Cisek et al, 1989; Young et al, 1991). Due to the fact that native gels were used in the gel retardation analysis, the TAR RNA complex containing RNA polymerase II exhibited a faster mobility than TRP-185 even though the polymerase complex is of higher molecular weight than that of TRP- 185 (Young et al, 1991). UV crosslinking confirmed that the largest subunit of RNA polymerase II (210 kDa) was present in the complex bound to TAR

RNA. These data indicate that following initial chromatography to remove a variety of nonspecific double stranded RNA binding proteins that two proteins, RNA polymerase II and TRP-185, were the only two species that bound specifically to wild-type TAR RNA.

EXAMPLE 14 RNA POLYMERASE II AND TRP-185 COMPETE

FOR BINDING TO TAR RNA

The present example demonstrates that TRP- 185 and RNA polymerase II competed with each other for binding to TAR RNA. Gel retardation analysis with wild-type TAR RNA and purified preparations of RNA polymerase II, recombinant TRP- 185, and cofactors were performed. When the amount of RNA polymerase II was kept constant and increasing amounts of TRP-185 were added, a slower mobility species became predominant which was consistent with that of TRP- 185 alone. When the amount of TRP - 185 was kept constant and increasing amounts of RNA polymerase II were added, the faster mobility species which became predominant was consistent with that of RNA polymerase II. No slower migrating complexes were detected in gel retardation assays which would be consistent with a complex comprised of TRP-185 and RNA polymerase II.

These results support the observation that RNA polymerase II and TRP-185 mutually exclude the binding of each other to TAR RNA. The present inventors also sought to determine whether changes in the phosphorylation state of either TRP-185 or RNA polymerase II altered their binding to TAR RNA. Both TRP-185 and RNA polymerase II were treated with alkaline phosphatase and their ability to bind to TAR RNA before and after treatment was tested. Alkaline phosphatase treatment of TRP-185 markedly decreased its binding to TAR RNA. In contrast, treatment of RNA polymerase II with alkaline phosphatase slightly increased its ability to bind to TAR RNA. There was no effect of alkaline phosphatase treatment on the amount of the TRP- 185 protein as determined by Western blot analysis or that of RNA polymerase II. While not intending to be bound by any particular theory or mechanism of action, the effect of the alkaline phosphatase may have been due to dephosphorylation of RNA polymerase II and TRP-185, rather than on the cofactor fraction, since dephosphorylation had opposite effects on the binding of these proteins to TAR RNA. EXAMPLE 15

TRP-185 CAN BIND TO TAR RNA AS BOTH

A MONOMER AND A DIMER

The present inventors sought to determine whether TRP- 185 was capable of binding to TAR RNA as either a heterodimer or a homodimer using gluteraldehyde crosslinking. Gluteraldehyde crosslinking has been used in a number of studies to detect the dimerization of leucine zipper containing proteins and TRP- 185 contains a putative leucine zipper structure. The crosslinking reactions were performed under identical conditions as the gel retardation binding reactions described above, except glutaraldehyde was added at final concentrations ranging from 0.0004% to 0.01% (v/v) and then incubated at room temperature for 30 minutes. For crosslinking in the presence of HIV-1 TAR RNA, TRP- 185 was first bound to the labeled RNA probe for 20 minutes at room temperature prior to the addition of glutaraldehyde. The reactions were then incubated at room temperature for 25 minutes. For immunoprecipitation, the glutaraldehyde crosslabeled TRP-185 was immunoprecipitated with either 3 μg of NK 5.18 or 12CA5 antibody overnight at 4°C. All reactions above were stopped by the addition of SDS-β mercaptoethanol and subject to electrophoresis on a 7% polyacrylamide gel with 0.1% SDS followed by either Western blot analysis or and autoradiography as needed.

Recombinant TRP- 185 prepared as described in example 1 1, was treated with increasing amounts of gluteraldehyde in the presence of either labeled wild-type TAR RNA or a TAR RNA loop mutant and then subject to SDS-PAGE followed by Western blot analysis with 12CA5 antibody (Boehringer-Mannheim). Both monomer and dimer forms of TRP- 185 were detected in Western blot analysis in the presence of either the wild-type TAR RNA or the TAR RNA loop mutant. Thus, TRP- 185 could dimerize in the presence of either wild-type or a mutant TAR RNA. Autoradiography of these same gels was then performed to detect the position of the labeled wild-type and loop mutant TAR RNAs to determine whether the monomer or dimer forms of TRP- 185 bound directly to TAR RNA. A gel retarded species migrating at approximately 200kDa was detected with the wild-type TAR RNA. In addition another species of approximately 370 kDa was also noted with wild-type TAR RNA. No binding of either of these species was detected using the labeled TAR RNA loop mutant. These results indicate that both the monomer and dimer forms of TRP- 185 bound specifically to labeled wild-type but not a mutant TAR RNA. To even further demonstrate that both the monomer and dimer forms of TRP- 185 were capable of binding to wild-type TAR RNA, immunoprecipitation of gluteraldehyde crosslinked TRP-185 bound to labeled wild-type TAR RNA was performed.

Autoradiography again revealed that both the monomer and dimer forms of TRP-185 bound to labeled TAR RNA when these samples were directly subjected to SDS-PAGE.

This same pattern was detected when the gluteraldehyde crosslinked TRP- 185 bound to labeled TAR RNA was immunoprecipitated with 12CA5 antibody followed by SDS-PAGE and autoradiography. No labeled TAR RNA was detected following immunoprecipitation with β-galactosidase monoclonal antibody. These results demonstrate that both the monomer and dimer forms of TRP- 185 were capable of binding specifically to a labeled wild-type but not a TAR RNA loop mutant.

In another study, a monoclonal antibody directed against TRP-185 reacted with both the 185kDa TRP-185 protein and a 350kDa form. An unrelated monoclonal antibody did not react with either species. Further, UV crosslinking of each of these individual species to ³²P labeled TAR RNA was performed and the products subjected to electrophoresis on SDS-PAGE with subsequent autoradiography. Both forms of the

TRP 185 protein were labeled with ³²P by this crosslinking experiment, again indicating that both the monomer and dimer forms of TRP-185 bind specifically to wild-type TAR

RNA. EXAMPLE 16

TRP-185 GENE STRUCTURE AND EXPRESSION IN HUMAN AND RODENT CELLS

To determine the patterns of expression TRP- 185 RNA. the present inventors probed a Northern blot containing 2μg of poly A selected RNA prepared from a number of human tissues. Two RNA species of approximately 10 kb and 5 kb were detected in all tissues. A control blot using a GAPDH probe was used to standardize the amount of RNA from each tissue. In addition, TRP- 185 was detected in RNA prepared from HeLa cells and both resting and activated Jurkat T-lymphocytes. It was noted that there was an increased abundance of the lOkb as compared to the 5.0kb transcript in Jurkat cells as compared to HeLa cells.

It has been demonstrated that there is restricted HIV-1 gene expression in rodent cell lines as compared to human cell lines and this may be dependent on loop binding factors (Hart et al, 1993). The inventors thus performed a Southern blot analysis on genomic DNA isolated from human and murine lymphocytes. Using different portions of the TRP- 185 cDNA as probes, TRP- 185 was able to strongly hybridize to human, but not mouse, genomic DNA. This was true for each of three fragments which comprised the entire TRP-185 cDNA. However, using this same filter, both the human and mouse genomic DNA hybridized similarly to a probe consisting of a portion of the largest subunit of RNA polymerase II. The TRP-185 gene appeared to diverge significantly between human and mouse.

To further characterize the ability of TRP-185 to hybridize to DNA isolated from various species, the TRP- 185 cDNA was used to probe genomic DNA isolated from several different species. Again the TRP- 185 cDNA was able to strongly hybridize to human DNA and also hybridized to DNA isolated from the monkey, dog, and cow. but there was not detectable hybridization to DNA isolated from the rat, mouse, chicken and yeast. Furthermore, using RT-PCT, TRP- 185 transcripts were not detected in several rodent cell lines. These results indicate that the TRP- 185 has diverged significantly from human to rodent. Chromosomal mapping demonstrated that TRP-185 was present on human chromosome lp indicating that TRP- 185 alone was not the gene on human chromosome 12 that complemented HIV-1 gene expression in human mouse cell hybrids (Hart et al, 1993).

EXAMPLE 17

PURIFICATION OF CELLULAR COFACTORS

The present example provides a method for obtaining a cofactor fraction useful in the binding of RNA polymerase II to TAR RNA, and also for the binding of TRP- 185 to TAR RNA. Nuclear extract prepared from 60 liters of HeLa cells described (Dignam et a\., 1983) was applied to a heparin agarose column (2.5 x 9 cm) equilibrated with buffer A (20 mM Tris-Cl, pH 7.9, 20% glycerol (v/v), 0.2 mM EDTA) containing 0.1 M KCl, 0.5 mM PMSF and 0.5 mM DTT. However, any mammalian cell line susceptible to infection by HIV may be used to prepare the cofactor fraction as well as to isolate the various component peptides as described herein.

The column was washed in the same buffer and then eluted with buffer A with 0.4 M KCl, 0.5 mM PMSF, and 0.5 mM DDT. The 0.4 M KCl fractions were pooled and dialyzed against buffer A with 0.1 M KCl, 0.5 mM PMSF and 0.5 mM DDT and applied to a HTP Bio Gel (2.5 x 7 cm) which was equilibrated and washed with the same buffer. The Bio Gel may be substituted with any other gel that provides for the chromatographic separation of components in a dialysate. The column was then eluted with the same buffer containing 0.1 M potassium phosphate (pH 7.0). These fractions were pooled and precipitated with 70% ammonium sulfate followed by centrifugation at 12,000 rpm for 20 minutes. The precipitate was resuspended in 6 ml of buffer A with 0.1 M KCl and 1 mM DTT and loaded on a Superdex 200 FPLC (HiLoad 26/60 prep grade) column which was equilibrated in the same buffer. However, the Superex 200 FPLC column may be substituted with another separation column known to those of ordinary skill in the art in light of this disclosure. The flow through fractions containing cofactor activity were pooled and applied to a Q-Sepharose (1.5 x 4 cm) column equilibrated in the same buffer. The flow through fractions containing cofactor activity were pooled and fractionated further on a Bio-Rex 70 column (1 x 3 cm). Other separation columns may be used as well. Fractions eluted from this column that were able to reconstitute TRP- 185 binding activity to HIV-1 TAR RNA were pooled. This preparation gave approximately 20 μg of each of five cofactor species and was determined to be of approximately 80% purity on a silver-stained SDS protein gel.

Sucrose gradient ultracentrifugation analysis resulted in the separation of TRP- 185, which sedimented at approximately 200 kDa from a group of cellular proteins, designated cofactors, which sedimented at approximately 100 kDa (Wu et al. 1991). The sucrose gradient fractions, containing either TRP-185 alone or the cofactors alone, were each unable to bind to TAR RNA in gel retardation analysis. Addition of the sucrose gradient fractions containing the cofactors in conjunction with fractions containing TRP- 185 restored the ability of TRP- 185 to bind to TAR RNA (Wu et al. 1991). Furthermore, this cofactor fraction stimulates the binding of RNA polymerase II to TAR RNA (see Examples 1 and 13). To determine the mechanism by which these cofactors stimulated the binding of

TRP- 185 and RNA polymerase II to TAR RNA, the present inventors purified the proteins responsible for this cofactor activity. The ability of the cofactors to stimulate the binding of recombinant TRP- 185 to TAR RNA in gel retardation studies was used as an assay. To purify the cellular cofactors, HeLa nuclear extract prepared from 60 liters of cells was applied to a heparin agarose column. The cofactor activity was eluted with 0.4M KCl. applied to a hydroxyapatite column, and then eluted with 0.1M potassium phosphate. Following ammonium sulfate precipitation and chromatography on a Superdex 200 column, the active fractions were pooled and applied to a Q-sepharose column. The flow- through fractions were further fractionated on a Bio-Rex 70 column and the protein fractions which stimulated the binding of recombinant TRP-185 to TAR RNA were pooled. A flow-chart of this purification scheme is shown in FIG. 3.

The purified cofactor fraction was then assayed for its ability to stimulate TRP-185 binding to TAR RNA. Addition of increasing amounts of recombinant TRP-185 alone resulted in only minimal binding to TAR RNA. However, upon the addition of the purified cofactors to TRP-185, there was a marked increase in its binding to TAR RNA. There was no binding of the cofactor alone to TAR RNA. The enhancement of TRP-185 binding by the cofactors was not seen with equivalent amounts of other proteins such as GST or albumin. Finally, it was found that increasing the amount of cofactor fraction from 0.1 μg to 1.0 μg markedly increased the binding of TRP- 185 to TAR RNA. These results indicate that the purified cellular cofactors did not bind directly to TAR RNA by themselves, but acted to markedly stimulate the binding properties of TRP- 185 to TAR RNA.

EXAMPLE 18

CHARACTERIZATION OF CELLULAR COFACTORS AND CLONING OF THE

GENE ENCODING SRB The present example provides for characterization of the cellular factors of

Example 17 and cloning of the gene for SRB protein. The cofactor fraction was concentrated on a Centricon 10 membrane which was blocked with BSA. The concentrated sample (150 μl) was loaded into three lanes of a 10% polyacrylamide, 0.1% SDS protein gel and blotted overnight onto a nitrocellulose membrane (0.45 mm). The membrane was then treated and protein bands were excised as described with omission of the NaOH destaining step (Aebersold, 1987). A total of five protein species were excised from the nitrocellulose with approximate molecular weights of 36, 42, 53, 55, and 58 kDa respecting that were designated of CF36, CF42, CF53, CF55,and CF58. Following Lys C protease digestion of each of the proteins and HPLC separation, the amino acid sequence of each of these proteins was determined by N-terminal amino acid analysis using Edman degradation (Lane, 1991). The following peptides were obtained: a 24 mer with the amino acid sequence VETGVLKPGMVVTFAPVNVTTEVK (SEQ ID NO: 22) from CF36, an 11 mer with the amino acid sequence of IGGIGTVPVGR (SEQ ID NO: 23) from CF42, a 23 mer with amino acid sequence of KLPIDVTEGEVISLGLPFGK (SEQ ID NO: 24) and VSFSK (SEQ ID NO: 25) from CF55, and a 13 mer and 25 mer with amino acid sequence VVSQYSSLLSPMS (SEQ ID NO: 26) and

AFADAMEVIPSTLAENAGLNPISTV (SEQ ID NO: 27) from CF58.

Sequence comparison of these peptides using the Intelligenetics program demonstrated 100% amino acid homology of the peptides obtained from CF36. CF42, and CF53 with that of the previously described 462 amino acid protein elongation factor- 1 alpha (EF-lα) (Brands et al, 1986; Uetsuki et al, 1989). The CF36 and CF42 proteins were likely the proteolytic degradation products of CF53. A sequence comparison of the two peptides obtained from CF55 revealed 100% homology with the previously described 531 amino acid polypyrimidine tract-binding protein (PTB) (Gill et al, 1991). Primers were made according to the known sequence of EF-lα and PTB and PCR reactions were performed using HeLa cDNA. The full length cDNAs encoding EF-lα and PTB were obtained and verified by DNA sequence analysis followed by in vitro transcription of these cDNAs and translation with rabbit reticulocyte lysate.

Sequence comparison of the two peptides obtained from CF58 showed no significant homology with known proteins. To clone the gene encoding CF58. degenerate primers were made to the 5' and 3' amino acid sequences of the 25 mer peptide obtained from the CF58. PCR analysis was performed with HeLa cDNA and a 75 base pair fragment was generated which revealed the predicted amino acid sequence from the 25 mer peptide upon analysis of the DNA sequence. This fragment spanning nucleotides 1357- 1431 of SEQ ID NO:4 was used as a probe to screen a HeLa cDNA library (Clontech) and resulted in the identification of a cDNA of 2kb which encoded a 539 amino acid open reading frame (SEQ ID NO: 5) that was designated stimulator of TAR RNA binding protein (SRB).

EXAMPLE 19

EXPRESSION AND PURIFICATION OF RECOMBINANT COFACTORS

PCR primers were made to modify the 5' ATG and 3' end of the EIFl-α cDNA into Nco I sites, the PTB cDNA 5' ATG into Sph I and the 3' end into EcoRl sites, and the CF58 cDNA 5' ATG into Ncol and the 3' end into BamHi sites. These modified cDNAs were then cloned in a modified pTMl expression vector (Elroy-Stein et al, 1989) with sequences encoding the 12 amino acid influenza hemagglutinin epitope (Field et al, 1988) and 6 histidine residues at the carboxy-terminus of the protein coding sequence. Each of the constructs was then transfected onto 20 plates of HeLa plate cells (150 mm) followed by infection with a recombinant vaccinia virus which produced T7 polymerase. The cells were harvested 40 hours later and nuclear and SI 00 extracts were prepared as herein described. The SI 00 extract contained most of the over-expressed recombinant proteins as judged by Western blot analysis with the 12CA5 monoclonal antibody. The nuclear or SI 00 extracts were loaded onto a 2ml Q-sepharose column (1.5 x 2 cm) equilibrated with buffer A containing 0.1 M KCl, 0.5 mM PMSF, 1 mg/ml of leupeptin, aprotinin, and 10 mM B-mercaptoethanol. The columns were washed with the same buffer and the flow through fractions were pooled then loaded onto a 1 ml Ni-NTA agarose column (Qiagen) equilibrated with the same buffer. The flow through fractions were reloaded onto the columns a second time and they were washed with (1) 20ml of the buffer A, (2) 20 ml of the buffer A containing 1.0 M KCl and (3) 20 ml of the buffer A with 0.1 M KCl respectively. The columns were then eluted with 0.1 M KCl and 60 mM imidazole. The eluted fractions were then dialyzed vs. buffer A containing 0.1 M KCl and ImM DTT, assayed, and stored at -70 °C. A typical yield of each of the recombinant proteins from these preparations were approximately 100 μg with a purity of 85% as judged by silver staining of the 10% SDS polyacrylamide gel. The SRB amino acid sequence is shown as SEQ ID NO:5.

Cellular cofactors are ubiquitously expressed. RNA expression patterns of the different cofactors were examined. Northern analysis of the human multiple tissue (MTN) blot from Clontech was performed using each of the three cofactor cDNAs as probes according to the manufacturer's protocol (Sambrook et al, 1989). This membrane was probed with a nick translated protein of the CF58 cDNA encoding amino acids 331 to 539 of the PTB cDNA encoding amino acids 291 to 531 and EIF-l α full length cDNA. The probe was also removed following each hybridization and the filter stripped of the scanning probe prior to the next hybridization. The nick translated GAPDH cDNA was used as a control for the distribution of poly A RNA present in each lane of the tissue blot. Western blot analysis was performed using the 12CA5 monoclonal antibody (Field et al, 1988) and ECL reagents (Amersham). The amounts of protein used in Western blot was 300 ng of each of the recombinant proteins purified using nickel chromatography. Recombinant EF-lα, PTB, and CF58 had molecular weights of 54, a doublet of 58 and 62 Kd respectively.

Northern analysis was performed with a blot comprised of multiple tissue RNAs prepared from human tissues including heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. There were differences in the amount of RNA loaded in each sample as determined by hybridization to a GAPDH probe. The EF-lα probe hybridized to one transcript of approximately 2.0 kb (Brands et al, 1986; Uetsuki et al, 1989) while the PTB probe hybridized to two transcripts of approximately 5.0 kb and 3.5 kb (Gil et al, 1991). The SRB probe hybridized to a single transcript of 2.0 kb. Thus, each of the mRNAs encoding the cellular cofactors was ubiquitously expressed in a variety of human tissues.

Expression of recombinant cofactor proteins. To test the role of each of the cofactor proteins on the binding of TRP- 185 and RNA polymerase II to TAR RNA, the present inventors cloned cDNAs encoding each of the cofactors into the pTMl expression vector downstream of the T7 RNA polymerase promoter (Elroy-Stein et al, 1989). Following transfection of each of these cDNAs into HeLa cells, their expression was induced by infection with a recombinant vaccinia virus which expressed T7 polymerase. To facilitate the detection of these proteins, the twelve amino acid influenza hemagglutinin sequences was inserted in the carboxy terminus of these proteins to facilitate their purification using nickel chromatography. Following column chromatography, each of these recombinant proteins was eluted from nickel beads with imidazole and Western blot analysis was performed with the 12CA5 monoclonal antibody which recognizes the influenza hemagglutinin sequences on these proteins. The molecular weights of these three recombinant proteins of 54, 58, and 62 kDa were consistent with predicted molecular weights of EIF-lα, PTB, and SRB respectively.

5 EXAMPLE 20

RECOMBINANT COFACTORS RECONSTITUTE TRP-185 BINDING TO TAR RNA

The present example demonstrates that recombinant cofactors were able to l o reconstitute TRP-185 binding to the same extent as the native cofactors purified from HeLa cells.

The binding reactions used in the gel retardation assays were performed as described in Wu et al, (1991). The binding of TRP- 185 to HIV-1 TAR RNA was performed with 50 ng of each of the Ni column eluted recombinant cofactors. The 15 recombinant TRP- 185 protein (50ng) used in these experiments was produced using a pTMl expression vector, transfected into HeLa cells with a recombinant vaccinia virus producing T7 polymerase, and purified using Ni column chromatography. RNA polymerase II was purified as described (Reinberg and Roeder, 1987) and 50 ng of this protein was used in the binding reactions. The binding reactions with antibodies were 20 performed as described above except 1 μg of each of the protein A sepharose column purified antibodies was added to the gel retardation assays for 10 minutes prior to tel electrophoresis. The TRP- 185 monoclonal antibody (NK 5.18) used in this study was raised against a GST fusion of TRP- 185 corresponding to amino acids 1409 to 1541. The EIF-lα antibody was raised against a GST fusion of this protein containing amino acids 1 2₅ to 110 (Brands et al, 1986; Uetsuki et al, 1989). The PTB polyclonal antibody was raised against a GST fusion of the protein containing amino acids 291 to 531 (Gil et al, 1991). The SRB polyclonal antibody was raised against a GST fusion of the protein encoding amino acids 1 to 331.

A mixture of all three of the recombinant cofactors at the highest protein ₃₀ concentration used in these gel retardation studies did not generate a complex which bound to TAR RNA in the absence of added TRP- 185 or RNA polymerase II. Addition of each of the individual cofactors resulted in a low level of TRP- 185 binding to TAR RNA as compared to binding performed in the absence of the cofactors. The addition of SRB resulted in the greatest stimulation of TRP- 185 binding. Addition of the recombinant 35 cofactors in pairs resulted in some increase in the binding of TRP- 185 for most of the different combinations assayed.

The addition of all three cofactors was tested for their ability to increase the binding of TRP- 185 to TAR RNA. Addition of increasing amounts of all three cofactors resulted in a marked stimulation of TRP-185 binding to wild-type TAR RNA. This binding was equivalent to the maximal binding of TRP-185 seen in the presence of native cellular cofactors purified from HeLa cells. These results indicate that the addition of the recombinant cofactors will completely reconstitute TRP-185 binding. Since the native cellular cofactors in HeLa cells exist as a complex during purification, the ability of the individual recombinant cofactors to result in low-level stimulation of TRP- 185 binding to TAR RNA is likely due to cross contamination with other copurifying cofactors.

EXAMPLE 21

RECOMBINANT COFACTORS STIMULATE

RNA POLYMERASE II BINDING TO TAR RNA

Since the three recombinant cofactors functioned similar to native cellular cofactors to stimulate TRP- 185 binding to TAR RNA, the present inventors examined whether the recombinant cofactors were able to stimulate the binding of RNA polymerase II to wild- type TAR RNA. Unlike TRP- 185 which exhibited no detectable binding to TAR RNA in the absence of added cellular cofactors, RNA polymerase II alone exhibited low level binding to TAR RNA. Addition of each of the recombinant cofactors either alone or in any pair combination was able to stimulate the binding of RNA polymerase II to wild-type TAR RNA. The addition of increasing amounts of all three recombinant cofactors was able to markedly stimulate the binding of RNA polymerase II to TAR RNA reaching levels equivalent to that seen with the addition of native cofactors. There was no binding of either recombinant or purified cofactors to wild-type TAR RNA in the absence of RNA polymerase II. Addition of similar concentrations of other proteins such as albumin or glutathione S -transferase were unable to stimulate the binding of RNA polymerase II to wild-type TAR RNA. Thus, the recombinant cofactors were also able to markedly stimulate the binding of RNA polymerase II to TAR RNA. EXAMPLE 22

POLYPYRIMIDINE TRACT-BINDING PROTEIN

ASSOCIATES IN A COMPLEX WITH TAR RNA

5 The mechanism by which the cofactors were able to stimulate the binding of TRP-

185 and RNA polymerase II to TAR RNA was next investigated. To address whether the cofactors directly associated with proteins bound to TAR RNA during gel retardation analysis, rabbit polyclonal antibodies to glutathione S-transferase protein fusions comprised of portions of either EF-lα, polypyrimidine tract-binding protein (PTB), or SRB i o were raised. Western blot analysis was performed with each of these polyclonal antibodies and they each reacted specifically with vaccinia produced recombinant cofactors. Each of these antibodies was then affinity purified using protein A sepharose columns and tested for its ability to supershift a complex comprised of TRP- 185 or RNA polymerase II and cofactors in gel retardation analysis. Such a result would indicate that the individual

₁5 cofactors were present in the TAR RNA complex with either TRP- 185 or RNA polymerase

II.

Gel retardation analysis was performed with recombinant TRP- 185 and either the native cofactors or the recombinant cofactors. Gel retardation analysis indicated that a complex formed by the addition of TRP- 185 and purified cellular cofactors was ₂₀ supershifted by a monoclonal antibody directed against TRP- 185. Antibody directed against either EF-lα or SRB did not result in a similar supershifted complex. However, the addition of antibody directed against PTB resulted in a shift of the complex bound to TAR RNA similar to that seen with TRP- 185 monoclonal antibody. Addition of the PTB antibody alone with wild-type TAR RNA did not result in a detectable complex in gel 25 retardation analysis.

These gel retardation experiments were then repeated using recombinant cofactors and TRP-185. Antibodies directed against either TRP-185 or PTB were again able to supershift the complex bound to TAR RNA while antibodies directed against either EF-lα or SRB were unable to supershift this complex. These results indicated that PTB was able ₃0 to directly associate in a complex comprised of TAR RNA and TRP- 185. Either EF-lα or SRB may also directly associate with this complex, since subset of antibodies directed against the same protein are able to alter the mobility of the gel retarded complex resulting in either a loss or a shift in the complex. This was true with a panel of rabbit polyclonal antibodies directed against TRP-185 of which only two of five antibodies was able to 35 supershift this protein in gel retardation analysis with TAR RNA. Cofactors do not directly associate with RNA polymerase II. Gel retardation analysis was performed with purified RNA polymerase II and native cofactors purified from HeLa cells to determine whether any of the cofactor proteins were able to associate in a complex comprised of TAR RNA and RNA polymerase II. The addition of antibody directed against the C-terminal domain of RNA polymerase II disrupted the binding of

RNA polymerase II to TAR RNA. However, the addition of antibodies directed against either EF-lα, PTB, or SRB did not result in a super shifted species or disrupt the binding of the gel retarded complex. Similar results were seen in gel retardation assays by adding these antibodies with recombinant cellular cofactors and RNA polymerase II. Thus, differences in the nature of the TAR RNA complex comprised of cofactors and RNA polymerase II as compared to the cofactors and TRP-185 does not permit recognition following the addition of cofactor antibodies.

EXAMPLE 23 METHOD FOR IDENTIFYING BINDING PEPTIDES

OF POLYMERASE AND TRANSACTIVATING PROTEINS AS THERAPEUTIC AGENTS FOR THE TREATMENT OF AIDS

The present example teaches a method by which a peptide site on one protein having binding specificity and affinity for another protein may be identified and its amino acid sequence determined. In particular, the present inventors have determined that a basic region from amino acids 49-57 of Tat protein is the binding site for RNA polymerase II. Similarly, the peptide binding site on RNA polymerase II for Tat may be determined. The inventors envision the administration of either or both Tat peptides to inhibit RNA polymerase II activity and/or administration of the RNA polymerase II peptides to inhibit Tat activity. Similar methodologies may also be used to define interactions between Tat and the viral polymerase, reverse transcriptase. In a similar manner to the method described herein, other viral transactivator proteins having an affinity for RNA polymerase II may be inhibited, and therefore, the expression and replication of the virus may be inhibited.

To define the domains of Tat that interact with RNA polymerase II, bacterial Tat proteins, either wild-type or mutant in different domains, were used. These Tat proteins which were fused to glutathione S transferase were produced in bacteria and purified using glutathione agarose beads as described in Wu et al, (1991). Partially purified HeLa cell RNA polymerase II was incubated with glutathione agarose beads containing either wild- type Tat or mutants in either the cysteine or basic domains (Wu et al, 1991). Following extensive washing of the beads, they were subjected to Western blot analysis with a monoclonal antibody directed against the C-terminal domain of RNA polymerase II.

These results indicated that the polymerase bound tightly to the wild-type and cysteine region Tat mutant, it did not bind to the Tat basic region mutant. This indicates that the basic domain of Tat is the binding site for RNA polymerase II.

A similar technique will be used to define the region of the largest subunit of RNA polymerase II that contains this binding site. The cDNA encoding the largest subunit of

RNA polymerase II has been expressed using the same vaccinia virus expression system as used to express the TRP-185 cDNA. The wild-type and a variety of transaction mutants in the polymerase cDNA have been constructed and the twelve amino acid influenza hemagglutinin sequences which are recognized by the 12CA5 monoclonal antibody have been attached to the carboxy-terminus. Each of these constructs will be expressed following transfection of HeLa cells. Nuclear extracts will be prepared from each set of transfections and these extracts will be bound to wild-type and basic domain mutant glutathione S-transferase tat fusions coupled to glutathione beads. Following extensive washing, Western blot analysis will be performed with 12CA5 antibody. Thus, using these mutuant constructs, the site on the largest subunit of RNA polymerase that binds to the basic domain of Tat will be mapped. All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention.

More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

Acker, J., et al, (1992) J. Mol. Biol. 226, 1295-1299.

Aebersold, R.H. (1987) Proc. Natl. Acad Sci., 84, 6970-6974.

Bengal, E. & Aloni, Y. (1991) J Virol. 65, 4910-4918. Berkhout, B., et al, (1989) Cell 59, 273-282.

Berkhout. B. & Jeang, K. (1989) J. Virol. 63, 5501-5504.

Brands. H.G.M., et al, (1986) Eur. J. Biochem. 155:167-171

Broder, et al, AIDS Medicine, Williams - Wilkins, 1994

Cadena, D. & Dahmus, M. J. (1987) J. Biol Chem. 262, 12468- 12474.

Calnan, B. J., et al, (1991) Genes Dev. 5, 201-210.

Cisek, L. J. & Corden, J. L. (1989) Nature (London) 339, 679-684.

Corden, J.L., et al, (1985) Proc. Natl. Acad. Sci. 82,

7934-7938. Cordingley, M.G., et α/.,(1990) Proc. Natl. Acad. Sci., 87, 8985- 8989.

Dayton, A. I., et al, (1986) Cell 44, 941-947.

Dignam, J.D., et al, (1983) Nucl Acids Res., 11, 1475-1489

Digman, J.D., et al, (1983) Methods Enzymol. 101, 582-598.

Dingwall, C. et al, (1989) Proc. Natl. Acad. Sci., 86, 6925-6929. Dingwall, C, et al, (1990) EMBO J. 9, 4145-53.

Elangovan, B., et al, (1992) J. Virol. 66, 2031-2036.

Elroy-Stein, O., et al, (1989) Proc. Natl. Acad. Sci., 86, 6126-6130.

Emerman, J., et l, (1987) EMBO J. 6, 3755-3760.

Feinberg, M. B., et al, (1991) Proc. Natl Acad. Sci. USA 88, 4045-4049.

Feng, S. & Holland, E. C. (1988) Nature (London) 334, 165-167.

Feaver, W.J., et al, (1994) Cell 79, 1103-1109.

Field, J., el al, (1988) Mol Cell Biol. 8, 2159-2165. Fisher. A. G., et al, (1986) Nature (London) 320. 367-371

(lett.).

Garcia. J. A., et al, (1989) EMBOJ. 8, 765-778.

Gaynor, R. (1992) Λ/DS 6, 347-363. Gil, A., et al, ( 99 ) Genes & Dev., 5 1224-1236.

Ha, I., et al, (1991) Nature, 352, 689-695.

Hart, C.E., et al, (1993) J. Virol, 67:5020-5024.

Hodo, H.G. and Blatti, S.P. (1977) Biochemistry 16, 2334-2343.

Horikoshi. M., et al, (1991) Nucl. Acid Res 19, 5436. Janknecht, R., et al, (1991) Proc. Natl. Acad. Sc , 88, 8972- 8976.

Kao. S.-Y., et al, (1987) Nature (London) 330, 489-493.

Kato, H., et al, R. G. (1992) Genes Dev. 6, 655-666.

Kim. W.Y. and Dahmus, M.E. (1986) J. Biol Chem. 261,

14219-14225. Lane. W.S. (1991) J. Prot. Chem. 10, 151-160.

Laspia, M., et al, (1989) Cell 59, 283-292.

Lu, H., et al, (1992) Nature (London) 358, 641-645.

Lu, H., et al, (1991) Proc. Natl. Acad. Sci. USA 88,

10004-10008. Marciniak el al, Cell, 63:791-802, 1990

Marciniak, R. A. & Sharp, P. A. (1991) EMBOJ. 10, 4189-4196.

Modesti, et al, (1991) The New Biologist, 3:8, 759-768.

Ratnasabapathy, R., et al, (1990) Genes Dev. 4, 2061-2074.

Reinberg, D. & Roeder, R. G. (1987a) J. Biol Chem. 262, 3310- 3321.

Reinberg, D. and Roeder, R.G. (1987b) J Biol. Chem. 262,

3331-3337.

Rhim, H., et al, (1993) Protein Expression and Purification

4, 24-31. Rice, G. A., et al. , ( 1991 ) Proc. Natl. Acad. Sci. USA 88,

4245-4249.

Rosen, C. A., et al, (1985) Cell 41, 813-823.

Roy, S., et al, (1990) Genes Dev. 4, 1365-1373. Sambrook, J. et al, (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N Y

Sheline, C. T., et al, (1991) Genes Dev. 5, 2508-2520. Sopta. M., et al, (1985) J. Biol. Chem. 260, 10353-10360. Tanog, et al, (1995) Proc. Natl. Acad Sci., 92:4902-4906. Thompson, N. E., et al. , ( 1989) J. Biol. Chem. 264, 1 151 1-1 1520. Uetsuki, T. et l, (1989) J. Biol. Chem., 264, 5791-5798. Usheva, A., et al, (1992) Cell 69, 871-881. U.S. Patent No. 5,350,835, Gaynor, et al.

Weeks, K. M. & Crothers, D. M. (1991) Cell 66, 577-588. Wintzerith, M., et al, (1992) Nucleic Acids Res. 20, 910. Wu, F., et al, (1991) Genes Dev. 5, 2128-2140. Young. R. A. (1991 ) Ann. Rev. Biochem. 60, 689-715.

SEQUENCE LISTING

1) GENERAL INFORMATION:

(i) APPLICANT: BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM

(ii) TITLE OF INVENTION: COMPOSITIONS AND METHODS FOR

REGULATING HIV GENE EXPRESSION

(iii) NUMBER OF SEQUENCES: 35

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Board of Regents, The University of Texas System

(B) STREET: 201 West 7th Street

(C) CITY: Austin

(D) STATE: Texas

(E) COUNTRY: USA

(F) ZIP: 78701

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (512) 499-6200

(B) TELEFAX: (512) 499-6290

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Arg Lys Lys Arg Arg Gin Arg Arg Arg

1 5

;2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 219 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..216 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

ATG GAG CCA GTA GAT CCT AAT CTA GAG CCC TGG AAG CAT CCA GGA AGT 48

Met Glu Pro Val Asp Pro Asn Leu Glu Pro Trp Lys His Pro Gly Ser

1 5 10 15

CAG CCT AGG ACT GCT TGT AAC AAT TGC TAT TGT AAA AAG TGT TGC TTT 96

Gin Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe 20 25 30

CAT TGC TAC GCG TGT TTC ACA AGA AAA GGC TTA GGC ATC TCC TAT GGC 144 Thr Arg Lys Gly Leu Gly lie Ser Tyr Gly 40 45 CGA CGA AGA GCT CCT CAG GAC AGT CAG ACT 192 Arg Arg Arg Ala Pro Gin Asp Ser Gin Thr 55 60 AAG CAG TAA 219 Lys Gin

3 C (2) INFORMATION FOR SEQ ID NO: 3: m g (i) SEQUENCE CHARACTERISTICS: ^w (A) LENGTH: 72 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Met Glu Pro Val Asp Pro Asn Leu Glu Pro Trp Lys His Pro Gly Ser 1 5 10 15

Gin Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe

20 25 30

His Cys Tyr Ala Cys Phe Thr Arg Lys Gly Leu Gly lie Ser Tyr Gly

35 40 45

Arg Lys Lys Arg Arg Gin Arg Arg Arg Ala Pro Gin Asp Ser Gin Thr

50 55 60

His Gin Ala Ser Leu Ser Lys Gin 65 70

0 (2) INFORMATION FOR SEQ ID NO: 4: > 0 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 72 amino acids

(B) TYPE: amino acid > ri (C) STRANDEDNESS: S ) (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: 0 Met Glu Pro Val Asp Pro Asn Leu Glu Pro Trp Lys His Pro Gly Ser 1 5 10 15 ) Gin Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe 20 25 30

His Cys Tyr Ala Cys Phe Thr Arg Lys Gly Leu Gly He Ser Tyr Gly 35 40 45

Arg Lys Lys Arg Arg Gin Arg Arg Arg Ala Pro Gin Asp Ser Gin Thr 50 55 60

His Gin Ala Ser Leu Ser Lys Gin 65 70

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1617 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..1617 (0 m (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: O

H

H ATG CCC GAG AAT GTG GCA CCC CGG AGC GGG GCG ACT GCC GGG GCT GCC 48

S Met Pro Glu Asn Val Ala Pro Arg Ser Gly Ala Thr Ala Gly Ala Ala 75 80 85

<0 m GGC GGC CGC GGG AAA GGC GCC TAT CAG GAC CGC GAC AAG CCA GCC CAG 96 HI Gly Gly Arg Gly Lys Gly Ala Tyr Gin Asp Arg Asp Lys Pro Ala Gin -jg 90 95 100 c fπ ATC CGC TTC AGC AAC ATT TCC GCC GCC AAA GCG GTT GCT GAT GCT ATT 144 r He Arg Phe Ser Asn He Ser Ala Ala Lys Ala Val Ala Asp Ala He cn 105 110 115 120

AGA ACA AGC CTT GGA CCA AAA GGA ATG GAT AAA ATG ATT CAA GAT GGA 192 Arg Thr Ser Leu Gly Pro Lys Gly Met Asp Lys Met He Gin Asp Gly

125 130 135

AAA GGT GAT GTA ACC ATT ACA AAT GAT GGT GCT ACC ATT CTG AAA CAA 240

Lys Gly Asp Val Thr He Thr Asn Asp Gly Ala Thr He Leu Lys Gin

140 145 150

GAG CTG TCT AAG 288

GTA GTC ATC ATT 336 Val Val He He

CAG AAA GGG ATT 384

Gin Lys Gly He

200

CTT AGA GAT ATT 624

Leu Arg Asp He

280

TGT GAG TTG GTG 672 Cys Glu Leu Val

295

GAA GGG CTG GTT CTC ACC CAA AAA GTG TCA AAT TCT GGC ATA ACC AGA 720 Glu Gly Leu Val Leu Thr Gin Lys Val Ser Asn Ser Gly He Thr Arg

300 305 310

GTT GAA AAG GCC AAG ATT GGG CTT ATT CAG TTT TGC TTA TCT GCT CCC 768

Val Glu Lys Ala Lys He Gly Leu He Gin Phe Cys Leu Ser Ala Pro

315 320 325

AAA ACA GAC ATG GAT AAT CAA ATA GTG GTT TCT GAC TAT GCC CAG ATG 816

Lys Thr Asp Met Asp Asn Gin He Val Val Ser Asp Tyr Ala Gin Met 330 335 340

GAC CGA GTG CTG CGA GAA GAG AGA GCC TAT ATT TTA AAT TTA GTG AAG 864 cO Asp Arg Val Leu Arg Glu Glu Arg Ala Tyr He Leu Asn Leu Val Lys CD 345 350 355 360

H CAA ATT AAA AAA ACA GGA TGT AAT GTC CTT CTC ATA CAG AAA TCT ATT 912

H Gin He Lys Lys Thr Gly Cys Asn Val Leu Leu He Gin Lys Ser He π> 365 370 375

CO

X m CTA AGA GAT GCT CTT AGT GAT CTT GCA TTA CAC TTT CTG AAT AAA ATG 960 ϋ} Leu Arg Asp Ala Leu Ser Asp Leu Ala Leu His Phe Leu Asn Lys Met 380 385 390 c fn AAG ATC ATG GTG ATT AAG GAT ATT GAA AGA GAA GAC ATT GAA TTC ATT 1008 r Lys He Met Val He Lys Asp He Glu Arg Glu Asp He Glu Phe He * 395 400 405

TGT AAG ACA ATT GGA ACC AAG CCA GTT GCT CAT ATT GAC CAA TTT ACT 1056

Cys Lys Thr He Gly Thr Lys Pro Val Ala His He Asp Gin Phe Thr 410 415 420

GCT GAC ATG CTG GGT TCT GCT GAG TTA GCT GAG GAG GTC AAT TTA AAT 1104

Ala Asp Met Leu Gly Ser Ala Glu Leu Ala Glu Glu Val Asn Leu Asn 425 430 435 440

GGT TCT GGC AAA CTG CTC AAG ATT ACA GGC TGT GCC AGC CCT GGA AAA 1152 Gly Ser Gly Lys Leu Leu Lys He Thr Gly Cys Ala Ser Pro Gly Lys

445 450 455

ACA GTT ACA ATT GTT GTT CGT GGT TCT AAC AAA CTG GTG ATT GAA GAA 1200

Thr Val Thr He Val Val Arg Gly Ser Asn Lys Leu Val He Glu Glu

460 465 470

GCT GAG CGC TCC ATT CAT GAT GCC CTA TGT GTT ATT CGT TGT TTA GTG 1248 Ala Glu Arg Ser He His Asp Ala Leu Cys Val He Arg Cys Leu Val 475 480 485

AAG AAG AGG GCT CTT ATT GCA GGA GGT GGT GCT CCA GAA ATA GAG TTG 1296

CcO Lys Lys Arg Ala Leu He Ala Gly Gly Gly Ala Pro Glu He Glu Leu m 490 495 500

CO

H

H GCC CTA GCA TTA ACT GAA TAT TCA CGA ACA CTG AGT GGT ATG GAA TCC 1344

S Ala Leu Ala Leu Thr Glu Tyr Ser Arg Thr Leu Ser Gly Met Glu Ser . m 505 510 515 520

CO m TAC TGC GTT CGT GCT TTT GCA GAT GCT ATG GAG GTC ATT CCA TCT ACA 1392 m H Tyr Cys Val Arg Ala Phe Ala Asp Ala Met Glu Val He Pro Ser Thr

525 530 535 c

£ CTA GCT GAA AAT GCC GGC CTG AAT CCC ATT TCT ACA GTA ACA GAA CTA 1440 ro Leu Ala Glu Asn Ala Gly Leu Asn Pro He Ser Thr Val Thr Glu Leu ot 540 545 550

AGA AAC CGG CAT GCC CAG GGA GAA AAA ACT GCA GGC ATT AAT GTC CGA 1488 Arg Asn Arg His Ala Gin Gly Glu Lys Thr Ala Gly He Asn Val Arg 555 560 565 AAG GGT GGT ATT TCC AAC ATT TTG GAG GAA CTG GTT GTC CAG CCT CTG 1536

Lys Gly Gly He Ser Asn He Leu Glu Glu Leu Val Val Gin Pro Leu 570 575 580

TTG GTA TCA GTC AGT GCT CTG ACT CTT GCA ACT GAA ACT GTT CGG AGC 1584 Leu Val Ser Val Ser Ala Leu Thr Leu Ala Thr Glu Thr Val Arg Ser

585 590 595 600

ATT CTG AAA ATA GAT GAT GTG GTA AAC ACT CGA 1617

He Leu Lys He Asp Asp Val Val Asn Thr Arg

605 610

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 539 amino acids

<2 (B) TYPE: amino acid

01 (D) TOPOLOGY: linear

CO

H (ii) MOLECULE TYPE: protein c (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

CO m Met Pro Glu Asn Val Ala Pro Arg Ser Gly Ala Thr Ala Gly Ala Ala ϋj 1 5 10 15

C Gly Gly Arg Gly Lys Gly Ala Tyr Gin Asp Arg Asp Lys Pro Ala Gin £ 20 25 30

•2 He Arg Phe Ser Asn He Ser Ala Ala Lys Ala Val Ala Asp Ala He

35 40 45

Arg Thr Ser Leu Gly Pro Lys Gly Met Asp Lys Met He Gin Asp Gly 50 55 60

Lys Gly Asp Val Thr He Thr Asn Asp Gly Ala Thr He Leu Lys Gin

65 70 75 80

Met Gin Val Leu His Pro Ala Ala Arg Met Leu Val Glu Leu Ser Lys

85 90 95

Ala Gin Asp He Glu Ala Gly Asp Gly Thr Thr Ser Val Val He He 100 105 110

Ala Gly Ser Leu Leu Asp Ser Cys Thr Lys Leu Leu Gin Lys Gly He 115 120 125

His Pro Thr He He Ser Glu Ser Phe Gin Lys Ala Leu Glu Lys Gly 130 135 140

He Glu He Leu Thr Asp Met Ser Arg Pro Val Glu Leu Ser Asp Arg 145 150 155 160

CO c Glu Thr Leu Leu Asn Ser Ala Thr Thr Ser Leu Asn Ser Lys Val Val σ co 165 170 175

H

H C Ser Gin Tyr Ser Ser Leu Leu Ser Pro Met Ser Val Asn Ala Val Met i 180 185 190 m - Leu Arg Asp He 205 Cys Glu Leu Val

to Glu Gly Leu Val Leu Thr Gin Lys Val Ser Asn Ser Gly He Thr Arg 225 230 235 240

Val Glu Lys Ala Lys He Gly Leu He Gin Phe Cys Leu Ser Ala Pro

245 250 255

Lys Thr Asp

Asp Arg Val Leu Arg Glu Glu Arg Ala Tyr He Leu Asn Leu Val Lys 275 280 285

Lys Lys Thr Gly Cys Asn Val

295

Asp Ala Leu Ser Asp Leu Ala 310

Met Val He Lys Asp He Glu 325

Thr He Gly Thr Lys Pro Val 340 345 Leu

Thr

Ser

Leu

Gly 425

Ala Leu Thr Glu Tyr Ser Arg 435 440

Val Arg Ala Phe Ala Asp Ala

455

Glu Asn Ala Gly Leu Asn Pro

Arg Asn Arg His Ala Gin Gly Glu Lys Thr Ala Gly He Asn Val Arg

485 490 495

Lys Gly Gly He Ser Asn He Leu Glu Glu Leu Val Val Gin Pro Leu 500 505 510

Leu Val Ser Val Ser Ala Leu Thr Leu Ala Thr Glu Thr Val Arg Ser 515 520 525

He Leu Lys He Asp Asp Val Val Asn Thr Arg 530 535

<2 (2) INFORMATION FOR SEQ ID NO: 7: co (i) SEQUENCE CHARACTERISTICS: q (A) LENGTH: 539 amino acids

Sj (B) TYPE: amino acid m (C) STRANDEDNESS:

W (D) TOPOLOGY: linear m 21 ;xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7

3J

C Met Pro Glu Asn Val Ala Pro Arg Ser Gly Ala Thr Ala Gly Ala Ala m 1 5 10 15 3 Gly Gly Arg Gly Lys Gly Ala Tyr Gin Asp Arg Asp Lys Pro Ala Gin 20 25 30

He Arg Phe Ser Asn He Ser Ala Ala Lys Ala Val Ala Asp Ala He 35 40 45

Arg Thr Ser Leu Gly Pro Lys Gly Met Asp Lys Met He Gin Asp Gly

50 55 60

Lys Gly Asp Val Thr He Thr Asn Asp Gly Ala Thr He Leu Lys Gin

65 70 75 80

Met Gin Val Leu His Pro Ala Val Glu Leu Ser Lys

85 95

Ala Gin Asp He Glu Ala Gly Ser Val Val He He 100 110

Ala Gly Ser Leu Leu Asp Ser Leu Gin Lys Gly He 115 125

His Pro Thr He He Ser Glu Ala Leu Glu Lys Gly 130 135 140

CcO He Glu He Leu Thr Asp Met Glu Leu Ser Asp Arg

00 145 150 160 CO

Glu Thr Leu Leu Asn Ser Ala Asn Ser Lys Val Val c

H 165 175 m Ala Val Met 190 Arg Asp He

Glu Leu Val

Glu Gly Leu Val Leu Thr Gin Ser Gly He Thr Arg 225 230 240

Val Glu Lys Ala Lys He Gly Cys Leu Ser Ala Pro

245 255

Lys Thr Asp Met Asp Asn Gin Asp Tyr Ala Gi n Met 260

270

Asp Arg Val Leu Arg Glu Glu Arg Ala Tyr He Leu Asn Leu Val Lys 275 280 285

Gin He Lys Lys Thr Gly Cys Asn Val Leu Leu He Gin Lys Ser He 290 295 300

Leu Arg Asp Ala Leu Ser Asp Leu Ala Leu His Phe Leu Asn Lys Met 305 310 315 320

Lys He Met Val He Lys Asp He Glu Arg Glu Asp He Glu Phe He

325 330 335

Cys Lys Thr He Gly Thr Lys Pro Val Ala His He Asp Gin Phe Thr O 340 345 350 D O Ala Asp Met Leu Gly Ser Ala Glu Leu Ala Glu Glu Val Asn Leu Asn 355 360 365

Gly Ser Gly Lys Leu Leu Lys He Thr Gly Cys Ala Ser Pro Gly Lys c o 370 375 380 I

Thr Val Thr He Val Val Arg Gly Ser Asn Lys Leu Val He Glu Glu 385 390 395 400 - π Ala Glu Arg Ser He His Asp Ala Leu Cys Val He Arg Cys Leu Val

405 410 415

Lys Lys Arg Ala Leu He Ala Gly Gly Gly Ala Pro Glu He Glu Leu 420 425 430

Ala Leu Ala Leu Thr Glu Tyr Ser Arg Thr Leu Ser Gly Met Glu Ser

435 440 445

Tyr Cys Val Arg Ala Phe Ala Asp Ala Met Glu Val He Pro Ser Thr 450 455 460

Leu Ala Glu Asn Ala Gly Leu Asn Pro He Ser Thr Val Thr Glu Leu

465 470 475 480

Arg Asn Arg His Ala Gin Gly Glu Lys Thr Ala Gly He Asn Val Arg

485 490 495

Lys Gly Gly He Ser Asn He Leu Glu Glu Leu Val Val Gin Pro Leu 500 505 510

Leu Val Ser Val Ser Ala Leu Thr Leu Ala Thr Glu Thr Val Arg Ser 515 520 525

ι GGGGUUCCUC UGGUUAGACC AGAUCUGAGC CUGGGAGCUC UCUGGCUAAC UAGGGAACCC 60

ACU 63

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1621 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Met Glu Trp Val Leu Ala Glu Ala Leu Leu Ser Gin Ser Arg Asp Pro

1 5 10 15

Arg Ala Leu Leu Gly Ala Leu Cys Gin Gly Glu Ala Ser Ala Glu Arg

20 25 30

Val Glu Thr Leu Arg Phe Leu Leu Gin Arg Leu Glu Asp Glu Glu Ala

35 40 45

Leu Ala Gly Trp Arg Ala Pro Gly Ala Glu Ala Ala Val Glu Val Leu 130 135 140

Ala Ala Val Gly Pro Cys Leu Arg Pro Arg Glu Asp Gly Pro Leu Leu

145 150 155 160

Glu Arg Val Ala Gly Thr Ala Val Ala Leu Ala Leu Gly Gly Gly Gly

165 170 175

Asp Gly Asp Glu Ala Gly Pro Ala Glu Asp Ala Ala Ala Leu Val Ala 180 185 190

Gly Arg Leu Leu Pro Val Leu Val Gin Cys Gly Gly Ala Ala Leu Arg 195 200 205

Ala Val Trp Gly Gly Leu Ala Ala Pro Gly Ala Ser Leu Gly Ser Gly 210 215 220

Arg Val Glu Glu Lys Leu Leu Val Leu Ser Ala Leu Ala Glu Lys Leu 225 230 235 240

Lys Lys Asp Glu Leu Leu Lys Phe Trp Glu Asn Tyr He Leu He Met

325 330 335

Glu Thr Leu Glu Gly Asn Gin He His Val He Lys Pro Val Leu Pro 340 345 350

Lys Leu Asn Asn Leu Phe Glu Tyr Ala Val Ser Glu Glu Asn Gly Cys 355 360 365

Trp Leu Phe His Pro Ser Trp His Met Cys He Tyr Lys Arg Met Phe 370 375 380

Glu Ser Glu Asn Lys He Leu Ser Lys Glu Gly Val He His Phe Leu 385 390 395 400

Glu Leu Tyr Glu Thr Lys He Leu Pro Phe Ser Pro Glu Phe Ser Glu

405 410 415

Phe He He Glv Pro Leu Met Asp Ala Leu Ser Glu Ser Ser Leu Tyr 420 425 430

Asp Val He His Cys Thr Met He Thr His Gin He Leu Leu Arg Gly 515 520 525

Ala Ala Gin Cys Tyr Leu Leu Gin Thr Ala Met Asn Leu Leu Asp Val 530 535 540

Glu Lys Val Ser Leu Ser Asp Val Ser Thr Phe Leu Met Ser Leu Arg 545 550 555 560

c CO

CD CO

rn co x m m x c ι- m

Leu Tyr Leu Met Val Leu Thr Glu Leu He Asn Leu His Leu Lys Val 755 760 765

Gly Trp Lys Arg Gly Asn Pro He Trp Arg Val He Ser Leu Leu Lys 770 775 780

Asn Ala Ser He Gin His Leu Gin Glu Met Asp Ser Gly Gin Glu Pro 785 790 795 800

Thr Val Gly Ser Gin He Gin Arg Val Val Ser Met Ala Ala Leu Ala

805 810 815

Ser Phe Leu Leu Lys Lys Tyr His Thr Leu He Pro Thr Thr Gly Ser 900 905 910

Glu He Leu Glu Pro Phe Leu Pro Ala Val Gin Met Pro He Arg Thr

915 920 925

Leu Gin Ser Ala Leu Glu Ala Leu Thr Val Leu Ser Ser Asp Gin Val 930 935 940

Leu Pro Val Phe His Cys Leu Lys Val Leu Val Pro Lys Leu Leu Thr 945 950 955 960

Ser Ser Glu Ser

He Ser Ser Leu Ser Asn Thr Gin Leu He Phe Trp Ala Asn Leu Lys 980 985 990

Ala Phe Val Gin Phe Val Phe Asp Asn Lys Val Leu Thr He Ala Ala 995 1000 1005

Val Gin Thr Phe He Glu Asn Leu Gly His Asp Cys Ala Ala Asn He 1090 1095 1100

Val Met Glu Asn Thr Lys Arg Glu Asp His Tyr Val Arg He Cys Ala 1105 1110 1115 1120

Val Lys Phe Leu Cys Leu Leu Asp Gly Ser Asn Met Ser His Lys Leu

1125 1130 1135

Phe He Glu Asp Leu Ala He Lys Leu Leu Asp Lys Asp Glu Leu Val 1140 1145 1150

Ser Lys Ser Lys Lys Arg Tyr Tyr Val Asn Ser Leu Gin His Arg Val 1155 1160 1165

Lys Asn Arg Val Trp Gin Thr Leu Leu Val Leu Phe Pro Arg Leu Asp 1170 1175 1180

Gin Asn Phe Leu Asn Gly He He Asp Arg He Phe Gin Ala Gly Phe 1185 1190 1195 1200

Asn His Asn Phe Ser Val Arg Leu Tyr Ala Leu Val Ala Leu Lys Lys

1285 1290 1295

Leu Trp Thr Val Cys Lys Val Leu Ser Val Glu Glu Phe Asp Ala Leu 1300 1305 1310

Thr Pro Val He Glu Ser Ser Leu His Gin Val Glu Ser Met His Gly 1315 1320 1325

Ala Gly Asn Ala Lys Lys Asn Trp Gin Arg He Gin Glu His Phe Phe 1330 1335 1340

Phe Ala Thr Phe His Pro Leu Lys Asp Tyr Cys Leu Glu Thr He Phe 1345 1350 1355 1360

Tyr He Leu Pro Arg Leu Ser Gly Leu He Glu Asp Glu Trp He Thr 1365 1370 1375

He Asp Lys Phe Thr Arg Phe Thr Asp Val Pro Leu Ala Ala Gly Phe 1380 1385 1390

Gin Trp Tyr Leu Ser Gin Thr Gin Leu Ser Lys Leu Lys Pro Gly Asp O

C 1395 1400 1405 CD CO

H Trp Ser Gin Gin Asp He Gly Thr Asn Leu Val Glu Ala Asp Asn Gin

H 1410 1415 1420

C H m Ala Glu Trp Thr Asp Val Gin Lys Lys He He Pro Trp Asn Ser Arg co x 1425 1430 1435 1440 m m H Val Ser Asp Leu Asp Leu Glu Leu Leu Phe Gin Asp Arg Ala Ala Arg

3 1445 1450 1455 c rπ Leu Gly Lys Ser He Ser Arg Leu He Val Val Ala Ser Leu He Asp t 1460 1465 1470

Lys Pro Thr Asn Leu Gly Gly Leu Cys Arg Thr Cys Glu Val Phe Gly 1475 1480 1485

Ala Ser Val Leu Val Val Gly Ser Leu Gin Cys He Ser Asp Lys Gin 1490 1495 1500

Phe Gin His Leu Ser Val Ser Ala Glu Gin Trp Leu Pro Leu Val Glu 1505 1510 1515 1520

Val Lys Pro Pro Gin Leu He Asp Tyr Leu Gin Gin Lys Lys Thr Glu

1525 1530 1535

Gly Tyr Thr He He Gly Val Glu Gin Thr Ala Lys Ser Leu Asp Leu 1540 1545 1550

Thr Gin Tyr Cys Phe Pro Glu Lys Ser Leu Leu Leu Leu Gly Asn Glu 1555 1560 1565

Arg Glu Gly He Pro Ala Asn Leu He Gin Gin Leu Asp Val Cys Val 1570 1575 1580

3 (2) INFORMATION FOR SEQ ID NO: 10:

C m (i) SEQUENCE CHARACTERISTICS: g (A) LENGTH: 63 base pairs

-' (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: GGGGUUCCUC UGGUUAGACC AGAUCUGAGC CCAAAAGCUC UCUGGCUAAC UAGGGAACCC 60

ACU 63

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 63 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GGGGUUCCGA ACUGUAGAGC ACAUCUGAGG CCAAAACCUC UGUGCCUACA GUUGGAACCC 60 ACU 63

CO c σ co

H (2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS: rri (A) LENGTH: 63 base pairs

CO (B) TYPE: nucleic acid m (C) STRANDEDNESS: single jj (D) TOPOLOGY: linear

_C (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: r~

_N GGGGUUCCGA ACUGUAGAGC ACAUCUGAGG CUGGGACCUC UGUGCCUACA GUUGGAACCC 60 3

ACU 63

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 63 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: GGGGUUCCUC UGGUUAGACC AGAUCUGAGC AUGGGAGCUC UCUGGCUAAC UAGGGAACCC 60

ACU 63

(2) INFORMATION FOR SEQ ID NO: 14:

3J (2) INFORMATION FOR SEQ ID NO: 15: m g (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 63 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: GGGGUUCCUC UGGUUAGACC AGAUCUGAGC AUGGUAGCUC UCUGGCUAAC UAGGGAACCC 60 ACU 63

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 63 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: GGGGUUCCUC UGGUUAGACC AGAACUGAGC CUGGGAGCUC UCUGGCUAAC UAGGGAACCC 60

£ ACU 63

CD CO

H

H (2) INFORMATION FOR SEQ ID NO: 17:

^m (i) SEQUENCE CHARACTERISTICS:

CO

X (A) LENGTH: 60 base pairs mm (B) TYPE: nucleic acid

H (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: t GGGGUUCCUC UGGUUAGACC AGAGAGCCUG GGAGCUCUCU GGCUAACUAG GGAACCCACU 60

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 63 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

GGGGUUCCUC UGGUUAGACU CGCUCUGAGC CUGGGAGCUC GCGAGCUAAC UAGGGAACCC 60

ACU 63

(2) INFORMATION FOR SEQ ID NO: 19:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

Leu Lys Pro Gly Asp Trp Ser Gin Gin Asp He Gly Thr Asn Leu Val

1 5 10 15

Glu Ala Asp

(2) INFORMATION FOR SEQ ID NO: 21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

Thr Glu Gly Tyr Thr He He Gly Val Glu Gin Thr Ala Lys S 1 5 10

09 CO

"H (2) INFORMATION FOR SEQ ID NO: 22:

H ^m (i) SEQUENCE CHARACTERISTICS:

X (A) LENGTH: 5173 base pairs

[Jj (B) TYPE: nucleic acid

™ (C) STRANDEDNESS: single

9 D) TOPOLOGY: linear

C m (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:

ATGGAGTGGG TGCTCGCGGA AGCGCTGCTC TCGCAGAGCC GGGACCCCCG GGCCCTGCTT 60

GGGGCGCTGT GCCAAGGGGA GGCATCCGCG GAGCGCGTGG AGACGCTGCG CTTCCTTCTG 120

CAGCGGCTCG AGGACGAGGA GGCGCGCGGC AGCGGGGGCG CAGGCGCGCT CCCGGAGGCG 180

GCGCGCGAGG TGGCTGCAGG GTACCTCGTG CCACTGCTGC GGAGCCTGCG CGGACGCCCC 240

GCGGGCGGCC CGGACCCCAG TCTGCAGCCT CGCCACCGCC GGCGCGTGCT GAGGGCGGCG 300

GGCGCGGCCC TGCGCTCGTG CGTCCGCCTG GCCGGGCGTC CGCAGCTGGC GGCCGCGCTG 360

GCTGAGGAGG CGCTGCGCGA TCTGCTCGCC GGGTGGCGCG CGCCTGGCGC CGAGGCTGCC 420

GTGGAAGTGC TAGCAGCCGT CGGGCCATGT TTGCGGCCCC GCGAGGACGG GCCGCTACTG 480

GAGCGGGTGG CGGGGACCGC CGTCGCCCTG GCGCTGGGCG GGGGCGGGGA CGGGGATGAG 540

GCCGGGCCTG CCGAGGACGC GGCGGCGCTG GTGGCCGGGC GACTGCTGCC AGTGCTGGTC 600

CAATGTGGCG GGGCGGCGCT GCGGGCCGTG TGGGGCGGGC TGGCCGCGCC TGGGGCGTCC 660

CTGGGGTCCG GCCGCGTAGA GGAGAAGCTG CTGGTCCTGA GCGCCCTGGC CGAGAAGCTG 720

<2 TTGCCCGAGC CCGGCGGCGA CCGCGCCCGC GGCGCGCGCG AGGCGGGCCC GGACGCCCGG 780 σ co

H CGCTGCTGGC GCTTCTGGAG GACGGTGCAG GCGGGGCTGG GCCAGGCGGA CGCCCTGACG 840

H H C CGCAAGCGAG CGCGCTACCT GCTGCAGAGG GCGGTGGAGG TGTCGGCGGA GCTGGGGGCC 900 m v

M GACTGCACCT GCGGGCCCCA GGAAGGAAAC GGCCCAAGTC TGTTTTGGTG GTCTGAGAGG 960 rn

AAAAAAGATG AGCTTCTAAA GTTTTGGGAA AATTATATTT TAATTATGGA GACTTTAGAA 1020 GGAAATCAGA TACATGTTAT AAAGCCAGTT TTACCAAAGC TAAACAATCT GTTTGAATAT 1080

_N) GCGGTGTCAG AGGAAAATGG ATGTTGGCTC TTTCACCCAT CCTGGCATAT GTGTATTTAT 1140 at

AAAAGAATGT TTGAAAGTGA AAACAAAATC CTGTCCAAAG AAGGTGTTAT CCATTTTTTG 1200

GAGCTGTATG AAACAAAGAT TCTTCCATTT TCACCAGAAT TTTCTGAGTT TATTATTGGA 1260

CCATTAATGG ATGCGCTTTC AGAGAGCTCT CTGTATAGCA GGTCCCCAGG CCAGCCAATA 1320

GGAAGCTGTT CTCCATTGGG ACTGAAATTA CAGAAGTTTT TAGTCACTTA TATTTCTCTT 1380

CTTCCAGAAG AAATAAAGAG TAGCTTCCTA TTGAAGTTTA TTCGGAAGAT GACAAGTAGG 1440

CATTGGTGTG CTGTTCCCAT TTTGTTTCTA TCTAAGGCTT TGGCAAATGT CCCAAGACAT 1500

AAGGCCCTGG GTATAGATGG GCTTCTTGCT CTCAGGGATG TTATTCATTG CACTATGATC 1560

ACACATCAGA TTCTCCTGAG AGGGGCAGCC CAATGCTACC TTCTTCAAAC AGCTATGAAT 1620

TTGCTAGATG TGGAGAAAGT GTCACTTTCT GATGTCTCAA CTTTTCTCAT GTCTCTGAGA 1680

CAAGAGGAAT CCTTAGGACG AGGAACTTCA TTGTGGACAG AGCTGTGTGA CTGGCTACGT 1740

GTTAATGAAA GCTATTTTAA GCCATCCCCT ACGTGTAGCT CCATTGGACT TCACAAGACA 1800

CO TCTTTAAATG CTTATGTAAA GAGCATTGTT CAAGAGTATG TTAAGTCATC TGCTTGGGAA 1860 c /l ACAGGAGAAA ACTGCTTTAT GCCTGATTGG TTTGAAGCCA AGCTTGTTTC TCTGATGGTC 1920

H jjj TTGCTGGCTG TGGATGTGGA AGGAATGAAG ACTCAGTATA GCGGAAAGCA GAGAACAGAG 1980

H

JJ! AATGTATTGC GGATATTCTT AGACCCTCTT CTGGATGTGC TTATGAAGTT TAGTACCAAT 2040

X

JJJ GCCTACATGC CCTTGCTGAA GACTGACAGA TGCCTCCAGC TGCTGTTGAA GCTGTTGAAC 2100 H

3 ACATGCAGGT TGAAAGGTTC CAGTGCCCAA GATGATGAGG TGTCTACTGT TCTTCAGAAC 2160

C m TTTTTCATGT CTACTACAGA GAGCATTTCT GAATTTATTC TCAGAAGACT TACTATGAAT 2220 ro

OI

^*"' GAGCTAAATA GTGTTTCAGA TCTGGATCGT TGCCATTTAT ACCTGATGGT GTTAACTGAG 2280

CTTATAAATC TGCATTTGAA GGTTGGGTGG AAAAGGGGTA ACCCTATCTG GAGAGTTATT 2340

TCTCTTTTGA AAAATGCATC CATTCAGCAT CTTCAAGAGA TGGACAGTGG ACAGGAGCCA 2400

ΛCAGTTGGAA GTCAGATTCA GAGAGTAGTG AGCATGGCTG CCTTGGCCAT GGTGTGTGAG 2460

GCCATAGACC AGAAGCCTGA GCTGCAGCTG GACTCTCTCC ATGCTGGGCC CCTGGAAAGC 2520

TTCCTTTCCT CTCTTCAGCT CAATCAGACG CTGCAGAAGC CCCACGCAGA GGAGCAGAGC 2580

AGTTATGCTC ACCCCTTGGA GTGCAGCAGT GTTTTGGAAG AATCGTCATC TTCCCAAGGA 2640

TGGGGAAAAA TAGTTGCACA ATATATTCAT GATCAATGGG TGTGCCTCTC TTTCCTGTTG 2700

AAAAAATATC ACACCCTTAT ACCAACCACA GGGAGTGAAA TTCTGGAACC GTTTCTACCT 2760

GCCGTTCAGA TGCCAATAAG GACTTTGCAG TCTGCACTAG AAGCCCTCAC AGTTCTTTCT 2820

TCTGATCAAG TTTTACCAGT GTTCCATTGC TTGAAAGTGT TGGTTCCCAA GCTTCTGACT 2880

TCCTCTGAAT CACTCTGCAT AGAGTCTTTT GACATGGCGT GGAAAATTAT ATCTTCTTTA 2940

<2 AGCAACACTC AGCTGATATT CTGGGCTAAT TTAAAAGCTT TTGTTCAGTT TGTTTTTGAT 3000

OB f2 AACAAAGTTC TTACCATTGC TGCCAAAATC AAGGGCCAGG CATATTTCAA AATAAAAGAG 3060

ATTATGTACA AGATAATTGA AATGTCTGCT ATAAAGACTG GAGTCTTCAA TACACTGATA 3120 m v

W AGTTACTGCT GTCAGTCTTG GATAGTGTCT GCTTCAAATG TGTCCCAAGG ATCTTTATCA 3180 m ϊ!| AGTGCTAAAA ATTATAGCGA ACTTATCCTT GAGGCTTGTA TATTTGGAAC TGTGTTTAGG 3240 CGTGATCAAA GACTTGTTCA GGATGTACAG ACCTTCATAG AAAACCTTGG ACATGACTGT 3300 rrπ _t GCGGCAAATA TTGTTATGGA AAATACTAAG AGAGAAGACC ATTATGTGAG AATTTGTGCT 3360 σ»

GTCAAATTCC TGTGTTTATT AGATGGCTCC AATATGTCCC ACAAGTTGTT TATTGAGGAT 3420

CTTGCAATCA AGCTATTAGA TAAAGATGAA TTAGTGTCCA AGTCCAAAAA ACGCTACTAT 3480

GTGAATTCTC TACAGCACAG AGTGAAAAAC CGAGTCTGGC AGACTCTGCT GGTACTTTTC 3540

CCTAGACTTG ACCAGAATTT CTTGAATGGA ATTATTGACA GGATTTTCCA GGCTGGTTTC 3600

ACCAACAATC AAGCATCCAT AAAATATTTT ATAGAATGGA TTATTATATT GATTCTTCAT 3660

AAATTCCCTC AATTTCTTCC AAAGTTCTGG GATTGTTTTT CTTATGGTGA AGAAAATCTT 3720

AAAACAAGCA TTTGTACATT TTTAGCAGTT TTATCACATT TAGACATTAT TACTCAAAAT 3780

ATTCCAGAAA AGAAACTAAT TCTGAAGCAA GCCCTTATAG TTGTGCTGCA GTGGTGTTTC 3840

AATCACAATT TTAGTGTTCG ACTGTATGCT TTAGTTGCTC TTAAGAAACT CTGGACTGTG 3900

TGTAAAGTGT TAAGTGTTGA AGAATTTGAT GCCCTGACTC CTGTGATTGA ATCCAGCCTC 3960

CATCAAGTGG AAAGCATGCA CGGAGCAGGG AATGCCAAGA AGAATTGGCA ACGCATTCAG 4020

to σt TGCAGGACCT GTGAGGTATT TGGGGCTTCA GTGCTCGTTG TTGGCAGCCT TCAGTGTATC 4500

AGCGACAAAC AGTTTCAGCA CCTCAGTGTC TCTGCAGAAC AGTGGCTTCC TCTAGTGGAG 4560

GTAAAACCAC CTCAGCTAAT TGATTATCTG CAGCAGAAGA AAACAGAAGG TTATACCATC 4620

ATTGGAGTGG AACAAACTGC CAAAAGTTTA GACCTAACCC AATATTGCTT TCCTGAGAAA 4680

TCTCTGCTCT TGTTGGGAAA TGAACGTGAG GGAATTCCAG CAAATCTGAT CCAACAGTTG 4740

GACGTTTGTG TGGAAATTCC TCAACAGGGC ATTATCCGCT CCCTGAATGT CCATGTGAGT 4800

GGAGCCCTGC TGATCTGGGA GTACACCAGG CAGCAGCTGC TCTCGCACGG AGATACCAAG 4860

CCATGATGTG CCTTCCTTAG TGAACTGCTG CTGCTGTTCA GACTTTTTTA AAAAAAACTA 4920

TTTGGACTAA AGAAACAGAT TCTGAAATTT ATTGTGATAA TTTGTATTTC TTTTTTCTTG 4980

CAATTTAATG CCAAAAGTTT GCCATGTGCC TTAAACATAT TACTATATAT TTTCCCCTTT 5040

AATAAACACT TTTTGTTAAA TTGTATTCTT CCTTTAATAA AATATTTTAA GCAATTGTCC 5100

AATAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 5160 AAAAAAAAAA AAA 5173

!2) INFORMATION FOR SEQ ID NO: 23:

• (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 435 base pairs '

{J| (B) TYPE: nucleic acid

-t (C) STRANDEDNESS: single (D) TOPOLOGY: linear c nϊ (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: t CTAAAACCAG GTGACTGGTC TCAGCAAGAC ATAGGTACTA ATTTGGTTGA AGCAGAT AC 60

CAAGCAGAGT GGACCGACGT TCAGAAGAAG ATTATCCCGT GGAACAGTCG TGTTTCCGAC 120

TTAGACCTGG AGCTCCTGTT TCAGGATCGT GCTGCCAGAC TTGGAAAGTC AATTAGTAGA 180

CTCATCGTTG TGGCCTCGCT CATCGACAAA CCGACCAATT TAGGAGGACT GTGCAGGACC 240

TGTGAGGTAT TTGGGGCTTC AGTGCTCGTT GTTGGCAGCC TTCAGTGTAT CAGCGACAAA 300

CAGTTTCAGC ACCTCAGTGT CTCTGCAGAA CAGTGGCTTC CTCTAGTGGA GGTAAAACCA 360

CCTCAGCTAA TTGATTATCT GCAGCAGAAG AAAACAGAAG GTTATACCAT CATTGGAGTG 420

GAACAAACTG CCAAA 435

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 amino acids

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

He Gly Gly He Gly Thr Val Pro Val Gly Arg

1 5 10

(2) INFORMATION FOR SEQ ID NO: 26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

( D) TOPOLOGY : linear

( xi ) SEQUENCE DESCRI PTION : SEQ I D NO : 26 :

Lys Leu Pro He Asp Val Thr Glu Gly Glu Val He Ser Leu Gly Leu 1 5 10 15

J2 Pro Phe Gly Lys

OB 20

CO

H

Ξj (2) INFORMATION FOR SEQ ID NO: 27: m

Jg (i) SEQUENCE CHARACTERISTICS: m (A) LENGTH: 5 amino acids ϋj (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear r (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:

■3

Val Ser Phe Ser Lys 1 5

(2) INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:

Val Val Ser Gin Tyr Ser Ser Leu Leu Ser Pro Met Ser 1 5 10

(2) INFORMATION FOR SEQ ID NO:29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

CO (D) TOPOLOGY: linear 00

H (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:

H

H Ala Phe Ala Asp Ala Met Glu Val He Pro Ser Thr Leu Ala Glu Asn 1 5 10 15

CO

X m Ala Gly Leu Asn Pro He Ser Thr Val

H 20 25

(2) INFORMATION FOR SEQ ID NO: 30: t

" (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: Tyr Ser Pro Thr Ser Pro Ser

^{1 5}

(2) INFORMATION FOR SEQ ID NO: 31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:

Leu Val Pro Arg Gly Ser 1 5

(2) INFORMATION FOR SEQ ID NO: 32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:

Arg Arg Ala Ser Val 1 5

(2) INFORMATION FOR SEQ ID NO: 33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:

Arg Arg Gin Arg Arg Arg 1 5

(2) INFORMATION FOR SEQ ID NO: 34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:

Gly Gly Ala Gly Gly Gly 1 5

;2) INFORMATION FOR SEQ ID NO: 35: (1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: Modifled-site

(B) LOCATION: 2..3

(D) OTHER INFORMATION: /product= "OTHER" /note= "Xaa = Any amino acid"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:

Cys Xaa Xaa Cys

1

Claims

WHAT IS CLAIMED IS:

1. A method for inhibiting expression from the TAR region of HIV- 1 , comprising administering a composition comprising a pharmacologically active amount of a Tat peptide having binding affinity for RNA polymerase II, an RNA polymerase II peptide having binding affinity for a Tat peptide, or an RNA polymerase II peptide having binding affinity for TAR RNA in the presence of a cofactor fraction, or a mixture thereof.

2. The method of claim 1 where the Tat peptide has a sequence of amino acids corresponding to a basic region of Tat.

3. The method of claim 2 where the basic region of Tat includes an amino acid sequence, Arg Lys Lys Arg Arg Gin Arg Arg Arg.

4. The method of claim 1 wherein the composition comprises a Tat peptide having binding affinity for RNA polymerase II.

5. The method of claim 1 wherein the composition comprises an RNA polymerase II peptide having binding affinity for Tat protein.

6. The method of claim 1 where the cofactor fraction is further defined as comprising elongation factor- 1 alpha, polypyrimidine tract-binding protein, and a peptide stimulator of TAR RNA binding proteins.

7. The method of claim 1 where the RNA polymerase II peptide having binding affinity for TAR RNA is from the about 210 kDa subunit of RNA polymerase II.

8. A method for inhibiting expression from a TAR region of HIV- 1 , comprising administering a pharmacologically active amount of an oligonucleotide having a nucleotide sequence from a TAR region of HIV- 1 LTR and having binding affinity for RNA polymerase II or Tat.

9. The method of claim 1 where expression is inhibited in vivo.

10. The method of claim 8 wherein the oligonucleotide comprises about 25 nucleotides from an about +18 to an about +44 region of TAR RNA.

11. A method of screening for a candidate substance that is capable of inhibiting the binding of RNA polymerase II to TAR RNA in the presence of a cofactor fraction, the method comprising: obtaining an RNA polymerase II protein and a cofactor fraction; admixing a candidate substance with the RNA polymerase II protein and the cofactor fraction in the presence of a nucleic acid sequence encoding a TAR RNA; selecting a candidate substance that inhibits the binding of RNA polymerase II to TAR RNA in the presence of a cofactor fraction.

12. A substantially purified SRB peptide capable of enhancing TRP- 185 binding to TAR RNA in the presence of elongation factor- 1 alpha or polypyrimidine tract-binding protein.

13. The peptide of claim 12 having an amino acid sequence substantially as shown in SEQ ID NO: 5.

14. A substantially purified SRB peptide capable of enhancing TRP-185 binding to TAR RNA in the presence of elongation factor- 1 alpha or polypyrimidine tract-binding protein, said SRB peptide prepared by a process of: obtaining a purified nucleic acid encoding an SRB peptide. and; expressing the nucleic acid to obtain an SRB peptide.

15. The substantially purified SRB peptide of claim 14 where the nucleic acid has a nucleotide sequence as defined by SEQ ID NO:4.

16. An oligonucleotide having binding affinity for RNA polymerase II and capable of inhibiting expression from an HIV-1 TAR region.

17. The oligonucleotide of claim 16 defined as having a nucleotide sequence corresponding to a position +18 to a position +44 of an HIV-1 TAR RNA region.

18. A method of inhibiting viral replication in a subject comprising: administering a pharmacologically active amount of a transactivator peptide having binding affinity for RNA polymerase II, or an RNA polymerase II peptide having binding affinity for a transactivator protein, or a mixture thereof, to the subject.

19. A method of inhibiting retroviral replication in a subject comprising: administering a pharmacologically active amount of a Tat peptide having binding affinity for reverse transcriptase, or a reverse transcriptase peptide having binding affinity for a Tat protein, or a mixture thereof, to the subject.

20. The method of claim 19 where the Tat peptide includes a sequence of amino acids corresponding to Arg Lys Lys Arg Arg Gin Arg Arg Arg.