WO2000040606A2

WO2000040606A2 - Modulation of hiv replication using sam68

Info

Publication number: WO2000040606A2
Application number: PCT/US2000/000324
Authority: WO
Inventors: Flossie Wong-Staal; Thipparthi Reddy; Weidong Xu
Original assignee: The Regents Of The University Of California
Priority date: 1999-01-06
Filing date: 2000-01-06
Publication date: 2000-07-13
Also published as: AU2493300A; WO2000040606A3

Abstract

The invention features methods and products for modifying retrovirus replication or expression in a host cell. Specifically, the invention relates to the use of cellular nuclear export proteins such as Sam68 and homologs, analogs, and derivatives thereof that can effect retroviral replication or expression in a host cell. Depending on whether desired enhanced or impeded, different specific applications and embodiments apply. For the latter, various negative transdominant mutants of Sam68 are disclosed that are likely to have great utiliy as research implements in, e.g., cell culture, transgenic animals, and in medicine as pharmaceutical compositions and techniques for ameliorating retroviral disease symptoms and etiology in afflicted organisms, i.e., humans.

Description

MODULATION OF HIV REPLICATION USING SAM68

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to Wong-Staal et al., United States provisional application serial number 60/1 14,848, filed January 6, 1 999, entitled "Inhibition of HIV Replication by Dominant Negative Mutants of Sam68", and also claims priority to Wong-Staal et al., United States provisional application 60/140,524, filed June 22, 1 999, entitled "Method for Increased Production of Proteins and Vectors Derived From Complex Retroviruses". Each of the foregoing applications is herein incorporated by reference in its entirety including all drawings.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

This work was supported in part by NIH grants GM056089, P30AI 36214-06, and RR04050. The United States Government may have rights in the invention.

BACKGROUND OF THE INVENTION

The invention relates to the fields of virology and medicai research, specifically as applied to controlling retroviruses and disease etiologies and symptoms associated with the same, e.g., Acquired Immunodeficiency Syndrome (AIDS).

The defining feature of a retrovirus is its ability, upon infection, to convert its RNA genome to a DNA proviral intermediate through the use of the virally encoded enzyme reverse tanscriptase. Following conversion to DNA, the viral enzyme integrase catalyzes the DNAs incorporation into the host chromosome, where it is established as a provirus. Once positioned in the host chromosome, transcription of the provirus initiates from a 5' repeat end and proceeds through to the polyadenylation (polyA) signal located in the 3' repeat end of the proviral genome.

An important difference between retroviral and cellular mRNA pertains to their splicing and nuclear export ability. For cellular mRNA, these two processes are tightly coupled and only completely spliced mRNA species are exported to the cytoplasm. However, retroviruses need to bypass this regulation in order to utilize unspliced viral RNA, both as messenger RNA for protein synthesis and as genomic RNA for progeny virions.

Retroviruses assume at least two different types: complex and simple. Complex retroviruses include the lentiviruses, typified by HIV-1 , the operative infectious agent contributing to human acquired immunity deficiency syndrome (AIDS). HIV encodes an essential regulatory protein Rev, which mediates the nuclear export of unspliced or incompletely spliced viral RNA. Rev is a 1 1 6-amino acid phosphoprotein composed of a basic, nuclear localization sequence (NLS) and a leucine-rich nuclear export sequence (NES). The basic domain also constitutes an RNA-binding domain that specifically interacts with the cognate target sequence, Rev response-element (RRE), a highly structured complex retrovirus RNA element that is present in singly spliced env mRNA and full-length genomic RNA. These incompletely spliced products necessarily require export to the cytoplasm for successful viral reproduction, and Rev provides that function.

Rev is conserved in all HIV and simian immunodeficiency virus isolates, and it has been shown that RREs from different primate immunodeficiency viruses are largely and functionally interchangeable. Furthermore, Rev specifically interacts with RRE in multimerized form, directing it and that to which is attached to a nuclear export pathway specified by its leucine-rich NES (activation domain) . The high affinity binding of Rev for RRE owes primarily to recognition in the latter of an extensive stem loop secondary structure. Several cellular cofactors including CRM-1 , elF-5A, RIP/Rab, and RHA have been implicated in Rev-mediated export by virtue of cross-linking and yeast 2-hybrid screening studies. Thus far, however, no cellular counterpart or functional replacement for Rev has been identified.

Another subfamily of complex retroviruses, typified by HTLV, has evolved a mechanism similar to HIV. Instead of Rev, however, these viruses encode a protein called Rex which binds the cis viral sequence, RXRE (aka RexRE), to promote the export of unspliced transcripts.

In contrast to complex retroviruses, simple retroviruses, typified, e.g., by Mason-Pfizer Monkey Virus (MPMV) and Simian Retroviruses Type-1 and 2 (SRV-1 , SRV-2), do not encode a Rev or Rex like protein, nor do they possess RRE or RXRE cis sequences. Instead, the genomic RNAs of simple retroviruses possess structurally and functionally equivalent cis structures.

For MPMV and SRV-1 , e.g., this element is called CTE (constitutive transport element). This acronym reflects the belief that simple retroviruses tap into a constitutive cellular pathway by interacting with a cellular factor or factors that may normally be used for the transport of cellular mRNA from the nucleus to the cytoplasm.

Interestingly, a CTE element engineered into HIV Rev-/RRE- mutants has been demonstrated to rescue the mutant virus. This activity, again, is presumed mediated by a cellular export protein or proteins, and, indeed, at least two cellular proteins have been demonstrated that bind CTE, namely

RHA and TAP. Thus far, however, the exact roles for these factors remain undefined.

Curiously, CTE and RRE would appear to characterize two distinct

RNA export pathways that are cooperative in host systems. For instance, while Rev/RRE uses a leptomycin B-sensitive export pathway, CTE in the same system utilizes a distinct mRNA export pathway that is independent of

CRM-1 and leptomycin B inhibition.

In controlling retroviruses and, more specifically, in developing therapies that combat retrovirus-mediated diseases, it would be highly desirable to use a broad-spectrum agent that could bridge the mechanistic differences between simple and complex retroviruses. Thus far, attention has primarily centered on HIV and AIDS therapy, with specific focus on Rev. For example, substitutions of leucine and glutamic acid residues within the nuclear export sequence of Rev yields a mutant (RevM 1 0) having a dominant negative phenotype. RevM 10 confers human CD4 cells with antiviral resistance in cell culture and preferential survival in HIV-1 -infected patients.

However, theoretical and demonstrable shortcomings exist in using mutant

Rev proteins as therapeutic agents. Namely, such therapies 1 ) fail to address the multiplicity of alternative export pathways, 2) fail to address the mechanistic differences in complex and simple retroviruses, and 3) fail to address immunogenicity problems associated with the use of non-self proteins.

The Applicants here identify and demonstrate that the use of a particular cellular protein, Sam68, overcomes the above limitations, and with extraordinary effect. Specifically, Sam68 is demonstrated to bind RRE both in vitro and in vivo, and to functionally mimic and replace Rev in RRE- mediated gene expression and virus replication. Surprisingly, Sam68 is also demonstrated to synergize with Rev when expressed or supplied above basal levels, and C-terminal deletion and point mutants of Sam68 are demonstrated to be potent inhibitors of HIV replication. Moreover, preliminary data described herein suggests that the effect and applicability of Sam68 is not limited to Rev-dependent complex retroviruses, but also embraces HTLV (Rex/RxRe) and simple retroviruses such as SIV. These findings suggest a new, general use for Sam68 and homologous genes and gene products in research and therapy directed to retroviruses.

Even more broadly, and in borrowing from the knowledge that differential splicing and regulation of mRNA nuclear export occur in cellular genes as well, it is anticipated that Sam68 may have an even more general and broad application to any system, viral or cellular, that makes use of these regulatory mechanisms.

SUMMARY OF THE INVENTION

It is an object of the invention to supply a nuclear export protein or viral protein mimetic capable of modulating differential splicing activity and gene expression in vivo or in vitro, preferably in retroviruses, and most preferably in retrovirus associated with diseases such as AIDS.

It is another object of the invention to supply transdominant negative mutants of the above that are capable of overriding or outcompeting wild- type cellular and/or viral analogs to effect inhibition of splice-dependent mechanisms, and preferably those that affect retrovirus replication and expression of proteins encoded or controlled thereby.

It is a further object of the invention to enhance the production and supply of lentiviral or retroviral vectors as implements in, e.g., gene therapy, and research applications, to modulate the expression of transgenes contained therein upon transfection.

In a first aspect, the invention features a method of altering the replication of a retrovirus in an eukaryotic host cell. A first step of the method includes establishing, providing or supplying an eukaryotic host cell infected with a retrovirus. The retrovirus is capable of producing an RNA transcript in the cell that has a sequence characteristic of unspliced, incompletely spliced, or minimally spliced retroviral RNA. This sequence is capable of a more or less specific association with human Sam68 (Seq I.D. No. 1 ) or an analog or homologous sequence thereto that promotes translocation of the transcript to the cytoplasm in the unspliced or minimally spliced state to express retroviral genes. The second step takes advantage of the innate properties of Sam68 and exploits them to alter natural retroviral replication, and the expression of proteins encoded or controlled thereby.

In certain embodiments, increased virus production is desired, e.g., for harvesting vector or expressing proteins contained or encoded within for further research or commercial characterization and/or exploitation. Lentiviral vectors, e.g., HIV and FIV, offer certain advantages in transgenic applications in which one or more foreign genes are to be transferred to an appropriate host cell or organism. Namely, they are capable of infecting non-dividing cells. By exceeding the basal or normal level or amount of cellular nuclear protein expression within an infected cell, or within a cell in which future infection is contemplated, enhanced viral replication occurs thereby resulting in a higher than normal yield or titer of vector, and proteins encoded or controlled thereby. Such proteins may be natural or recombinant, and in the case of gene therapy applications, therapeutic to an afflicted individual. In preferred embodiments, the retrovirus is a delivery vehicle and devoid of certain genes necessary for wild-type viral replication within an infected cell. In this way, wild-type retrovirus production is negated while enjoying the benefits of its infectious ability to deliver transgenes. Initial production of the recombinant virus is accomplished using helper virus or equivalent means to package the recombinant nucleic acid sequences.

The Sam68 protein, homolog, or analog can be directly added to the cell, with or without modification to enhance expression and/or function. Alternatively, a nucleic acid capable of expressing the same may be introduced to the cell. In the latter case, increasing the level of nuclear export protein within the cell may comprise transfecting the cell with a construct having a gene encoding an autologous gene or gene analog encoding the nuclear export protein or derivative thereof.

In certain embodiments, the gene is capable of expression to a greater degree within said host cell than is a corresponding native gene owing, e.g., to more potent regulatory elements such as stronger or constitutive promoters or other regulatory elements. This can also include engineering increased stability into genes, gene transcripts, and products thereof through modification or elimination of various destabilizing sequences commonly known in the art. In other, not necessarily exclusive embodiments, codon- usage is optimized for a given host, taking advantage of redundancy within the genetic code.

In additional embodiments, domain swapping is envisioned where, e.g., the NLS domain of native Sam68 or equivalent structure is appropriately replaced or supplemented with a functionally equivalent domain (or dysfunctional, as discussed for embodiments below in which reduced expression is desired). The NLS domain of Sam68 has been localized to about residues 366 to 443 of the native protein (Seq. ID. No. 1 ). Functionally equivalent NLS domains are exemplified by, but not limited to, those enumerated in Ishidate et al. (1 997), FEBS Letters 409: 237-241 . Another domain, the NES functional domain, has typical and atypical configurations as exemplified in Otero et al., J. Virol. 72(9)7593-7 that can similarly be used Each of these references is herein incorporated by reference.

In preferred embodiments, the enhancement of retrovirus production and/or gene product encoded within is at least one order of magnitude higher, and preferably 2 orders of magnitude higher than conventional production methods.

In contrast to embodiments that enhance retrovirus production, e.g., in the creation of retroviral vectors for gene therapy purposes, or as general transfection agents, other embodiments contemplate that replication be quelled, attenuated, impeded, retarded, eradicated or prophylactically guarded against. In preferred instances, this is accomplished through modification of the Sam68 protein such that the protein substantially possesses at least some feature or features characteristic of its native ability to foster retrovirus replication, but lacks another feature or features without which the retrovirus cannot reproduce as well. In most preferred embodiments, modifications are introduced within the carboxy terminal 1 50 amino acids (roughly 1 /3 of the protein) of Sam68 such as to specifically or generally alter the ability of the protein to shuttle between the nucleus and cytoplasm at normal kinetic rates. In some preferred embodiments the changes are a truncation, e.g., the C-terminal sequences following residue 330, 420, or anything in between or following. Truncations at other locations, and deletions within such locations, are also contemplated.

In other preferred embodiments, point mutations are introduced at the genetic level that change or eliminate a critical amino acid implicated in successful nuclear localization or, more generically, shuttling between the nucleus and cytoplasm. A specific example of this is residue 429 of the wild-type human Sam68 translated gene product. This residue normally corresponds to an arginine, but conversion to alanine results in a marked decrease in shuttling ability, and hence decreased retrovirus expression. A more preferred example is changing residue 439, a proline, to, e.g., arginine. The above-noted changes result in a dominant negative effect on retrovirus replication within the host cell.

Homologous (homolog) or analog species to human Sam68 are anticipated to exist for other eukarytotic organisms, e.g., animal, and plant, and to have a similar effect on the propagation of species specific retroviruses. Evidence for this is found in preliminary data provided herewith by the Applicants in Figure 1 1 , panel A, that suggests that human Sam68 can exert a similar synergistic effect on the expression of Equine Infectious Anemic Virus (EIAV), which is the equivalent of human HIV. The success in transfecting human cells with eRev and eRRE reporter constructs suggests cross-species utility and importance of the Sam68 protein, and also demonstrates the feasibility of domain-swapping, as described above. These findings further compliment those of Fridell et al., ( 1 993) J. Virol. 67:731 7- 7323, and Mancuso et al., ( 1 994) J. Virol. 68: 1 998-2001 that describe the structural and functional similarity and interchangeability of Rev and RRE isotypes in the human HIV, feline FIV, and equine infectious anemia virus (EIAV) retroviral systems, but which references fail to identify or even remotely correlate Sam68 in the process, much less modify it and/or modulate its expression to achieve a desired effect. The Applicants herein further demonstrate by data presented in Figure

1 1 , Panel B, that the invention has utility and application with HTLV and its genetic elements. This confirms, less the implication of Sam68 in the process, the functional interchangeability of REX and REV elements as between HIV and HTLV as described by Hope et al., ( 1 991 ) J. Virol. 65:6001 -6007. Some of the same (or very similar) reporter vector constructs used here are used there, and their original constructions described (pRev, pRex, pCAT RRE and pCAT XRE). Hope et al. ( 1 990) Proc. Natl. Acad. Sci. USA 87:7787-7791 is also instructive for the balance of vector constructions used in the present study.

The generic concept of manipulating nuclear/cytoplasmic shuttling ability may be extended to other proteins besides Sam68, and may be exploited to modulate replication or expression of a variety of retroviruses, and in a variety of contexts, as the person of ordinary skill in the art understands. In especially preferred embodiments, the retrovirus of interest is human HIV-1 , but the invention is anticipated applicable to all retroviruses, i.e., lentiviruses, oncoviruses, simple, and complex retroviruses alike, that make use of retrovirus-characteristic cis sequences to regulate differential splicing, and hence gene expression. Retroviruses within the scope of the invention include but are not limited to those identified in or from Coffin et al. eds. ( 1 997) Retroviruses, Cold Spring Harbor, NY: Cold Spring Harbor Lab. Press.; Li et al., Proc. Natl. Acad. Sci. USA 96:709-14 ( 1 997), and Malim et. al., Cell 58:205-1 4 (1 989). Simple retroviruses, e.g., SRV and MPMV, are also within the scope of the invention. (See Bray et al., Proc. Natl. Acad. Sci. USA 91 : 1 256-60 ( 1 994); Gruter et al., Mol. Cell 1 :649-59 (1 998); Tang et al., Science 276: 1 41 2-1 5 ( 1 997); and Zolotukhin et al., J. Virol. 68:7944-52 ( 1 994).

At a fundamental level, e.g., cell culture or transgenic mouse models, methods of the invention may be employed to study the effect of other natural or synthetic modulators of retrovirus replication. These can be proteins or other ligands that exist or occur naturally in infected host cells and which react, interact, or associate with Sam68. These can further be synthetic molecules/ligands specifically designed or generally identified from a population, e.g., by selective means, and according to techniques well known in the art.

In preferred embodiments of the invention, Sam68 or its homolog or analog recognizes and binds a cis sequence, e.g., RRE or CTE, within the unspliced or minimally spliced retrovirus transcript. These sequences and, it is known and/or envisioned, other cis analogs can adopt similar secondary structures recognized at the transcript level by Sam68, which in turn mediates translocation to the cytoplasm to foster retrovirus expression and replication. See, e.g., Otero et al., (1 998) J. Virol. 72(9) 7593-7 (citing Ernst et al., ( 1 997) RNA 3:21 0-222; Ernst et al., (1 997) Mol. Cell. Biol. 1 7: 1 35-1 44; Tabernero et al., ( 1 996) J. Virol. 70:5998-601 1 ), the disclosures of which are herein incorporated by reference.

Depending on the exact retrovirus , and as understood by one of skill in the art, other specific shuttle proteins, e.g., Rev (and mutants thereof, e.g., RevM 10), can also be used in conjunction. RNA and nuclear export protein complexes can be trapped in the cytoplasm and unable to return to the nucleus when, e.g., a trans dominant negative mutant such as described above is employed.

In preferred embodiments, it is desirable that where the cellular nuclear export protein possesses a KH domain (implicated in RNA binding) or equivalent thereof, that the function of this domain be substantially preserved, or at least cognizable. This does not rule out changes that either increase or decrease the functionality of the domain, as long as the overall desired effect is had, e.g., increased or decreased retrovirus production or expression, and of genetic components encoded therein and/or thereby.

In further embodiments it is desirable that the host cells be other than human, e.g., murine, to pave the way for compliance with Food and Drug Administration criteria and requirements, or else simply to facilitate in vivo studies in mammalian systems and hosts. To this end, transgenic organisms are also contemplated, e.g., mice that are transgenic for human Sam68 or a functional or dysfunctional analog or homolog thereof. Such transgenic organisms can be useful as molecular models in the study and development of new drug therapies for humans and other mammals.

Because of biochemical idiosyncrasies characteristic and peculiar to the host subject of interest, e.g., a human, it may be necessary, for example, to humanize or otherwise render conformity or functionality in the model system of choice, e.g., a mouse or rat. Examples of human genes or analogs thereof whose gene-products are required for human retrovirus replication include CD4, CCR5, and Cyclin T. Any or all of these may be functionally engineered into a mouse in addition to human Sam68 and/or synthetic or mutant analogs thereof for modeling and research purposes. The subcombinations have utility in identifying new substitute analogs or co- factors that might synergize in combined effect or otherwise contribute to understanding and controlling retroviruses, especially those associated with pernicious disease etiologies and symptoms such as AIDS. ln a further aspect that is not necessarily exclusive of the first and its embodiments, the invention includes a recombinant nucleic acid or host cell bearing a recombinant nucleic acid that encodes Sam68 or an analog or homolog thereof used for control of retrovirus replication. In preferred embodiments, the protein is engineered to influence retrovirus replication above or below levels that occur in natural infection.

In most preferred embodiments of this aspect, the cellular nuclear export protein or analog thereof is human Sam68 possessed of any combination of features discussed above which makes the product desirable as a research or therapy implement. For this and for the preceding method aspect and embodiments, the alteration of retroviral replication may occur as a result of administration of a pharmaceutical composition embodying the cellular nuclear localization protein or analog thereof, or means for production of such, e.g., recombinant DNA or RNA, or (dys)functional analogs, homologs, or hybrids thereof.

A third aspect of the invention features any of the above reagents supplied in kit format, preferably diagnostic. The kit optionally contains at least one additional reagent selected from the group consisting of controls, Rev or nucleic acid vector encoding the same, and reporter vectors harboring, e.g., an RRE or CTE response element, or an equivalent thereof.

In yet a fourth aspect, and because it is anticipated that most, if not all retroviruses possess sequences capable of interaction with Sam68, which may prove useful in both cellular and viral splice-dependent processes, the general use of Sam68 and analogs, homologs, and derivatives thereof to modify or affect splice-dependent processes generally, and especially in retroviruses, is claimed. Any of the above features and combinations of features may be employed to effect this. Combinational applications, e.g., using RRE and REV type cis and trans element mutants per the discussion in WO 97391 28-A and WO 9202228-A are also envisioned. In more prophetic aspects of the invention, gene therapy, pharmaceutical compositions, and general medicinal and diagnostic uses are contemplated that make use of the above combinations, as well as procedures and products identified therefrom, however indirect. In the latter respect, it is postulated that yeast 2-hybrid studies using Sam68 and domains thereof as bait are exemplary and will lead to fruitful discoveries of novel cellular and viral factors that can as well be harnessed in the mode of the other inventive aspects and embodiments.

In addition to the above aspects and embodiments, the latter of which may be used singularly or combined in any useful form, uses and combinations are further embodied or implicit from the claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 A shows the domain array within wild-type Sam68 (top) and specific mutants thereof (bottom) used in this study;

Figure 1 B shows the effects of wild-type and mutant Sam68 on RRE- mediated CAT expression in transfected 293T cells;

Figure 1 C shows RRE-independent expression in CAT assays of cells transfected with CTE-CAT or HIV LTR-CAT Sam68 Δ96 and/or Tat ( + , present; -, absent);

Figure 2A shows that microinjection of a Sam68 Δ96 expression plasmid activates RRE-mediated reporter gene (pCM228 (RRE-lacZ)) expression in human HS68 cells;

Figure 2B shows a reporter construct having minimal basal activity (3XUASp36LacZ) injected with and without Sam68;

Figure 3A shows the interaction of Sam68 with RRE in vitro using an RNA gel mobility shift assay;

Figure 3B shows the interaction of Sam68 with RRE in vivo using various reporter constructs; Figure 4A shows how Sam68 synergizes with Rev in CAT assays of

293T cells co-transfected with RRE-CAT, Sam68 Δ96 and/or Rev;

Figure 4B shows how Sam68 synergizes with Rev in RRE directed rescue of rev(-) HIV-1 expression in transfected 293T cells;

Figure 5A shows Sam68-Rev-RRE mediated CAT activity in transfected 293T cells;

Figure 5B shows dose-dependent inhibition of Rev and Sam68 function in transfected 293T cells using mutant forms of Sam68;

Figure 5C shows inhibition of wild-type HIV replication in 293T cells using Sam68 mutants; Figure 5D shows comparative inhibition of HIV replication in 293 T cells co-transfected with wild-type HXB-2 and Sam68 or RevM 1 0 mutant plasmids;

Figure 5E is analogous to experimentation and results achieved in Figure 5A, but using a single point mutant, 439, changed from a proline to an arginine;

Figure 6A shows the interaction of Sam68C'Δ330-443 with RRE in vitro using gel mobility shift assays;

Figure 6B shows a dose-dependent gel mobility shift assay using increasing amounts of mutant Sam68 and derivatives thereof;

Figure 6C shows purified GST-Sam68 and/or GST-Sam68C'Δ330-443 fusion proteins ( 1 g) bound to GST beads in affinity matrix to assess the in vitro binding of Rev;

Figure 7 shows the co-localization of Rev and Sam68 with transdominant mutant CΔ330-443;

Figure 8 shows how, using RRE-CAT reporter assays, administration of Sam68 to NIH3T3 mouse cells can overcome species-specific barriers and permit Rev to function;

Figure 9 is cumulative to results demonstrated in Figure 8 and demonstrates the same using cells co-transfected with cyclin T and HXB-2 and subjected to p24 antigen capture;

Figure 1 0 shows the effect of Sam68 on CTE-Gag expression in 293T cells using a p24 antigen capture assay;

Figure 1 1 A shows CAT assay results using equine RRE (eRRE) and Rev (eRev) with human Sam68;

Figure 1 1 B shows CAT assay results using RxRE and Rex from HTLV- 1 in the presence of human Sam68;

Figure 1 2A shows a schematic for the essential features of the plasmid vectors pRex and pRev; and Figure 1 2B shows a schematic for the features of the plasmid vectors

RRE-CAT (pCMV1 28), CTE-CAT (pCMV1 38), and RXRE-CAT (pCMV 1 28- XR) that are used to practice the invention.

The present invention will be better understood from the following detailed description of preferred embodiments of the invention, taken in conjunction with the accompanying drawings. DESCRIPTION OF THE PREFERRED EMBODIMENT

Definitions

As used in the claims, the following terms have the following meanings:

By "altering" means changing or modulating, either with increased or decreased affect relative to a natural or basal state.

By "replication" means expression or duplication, such as would normally occur in an infected host cell, and is not limited to production of wild-type virion complexes and virus, but also to individual genes or gene products encoded within. Thus, the term also embraces the expression of recombinant virus and genes and gene products encoded within, including the situation not only where the viral particles are self-sufficient and sustaining of their own replication, but also the situation in which such is only possible upon the co-supply or presence of a helper virus or entity, e.g., in situations where pathological genes essential for replication are eliminated and replaced with useful therapeutic genes, or for biological containment purposes. By "expression" can refer to either wild-type or recombinant viral expression or subcoding entity expression from a retrovirus or noninfectious vector of retroviral origin, or to the activity, presence or expression of a protein encoded thereby or therefrom, e.g., in binary or trans systems. Hence the meaning of the claim term "of or from a retrovirus" . Embraced by the use of the term "retrovirus" can also be constructs that have minimal retrovirus features but that otherwise borrow from the features necessary to the invention, i.e., the associational cis elements described in the claims, exemplified by RRE and CTE, and possessing suitable splice sites.

By "gene product encoded therein" is meant a polypeptide product that is encoded in the genome of the subject retrovirus, whether the product or retrovirus be natural or recombinant, infectious or not, or one whose genome has been engineered to possess and/or be capable of expressing one or more transgenes.

By "providing" means supplying, either directly or indirectly. The indirect sense may include, e.g., certain prophylactic applications of the transdominant negative cellular nuclear export protein to host cells and organisms.

By "capable of producing" means directly or indirectly. The latter case may include, but is not limited to, the situation, e.g., where a helper virus is necessary to the application, i.e., one that includes transgenic or recombinant infection of a mammalian subject or cell culture.

By "sequence characteristic of unspliced retroviral RNA" takes cognizance that retroviruses rely, in large part, on the functional association of certain cis-active sequences, e.g., RRE or CTE, with viral and/or cellular nuclear export proteins such as Rev and Sam68, and that such cis sequences normally are found in intron (noncoding) sequences and, upon complete splicing, effectively or substantially eliminated from the spliced RNA product.

The terms "incompletely" or "minimally" spliced describe the situations where differential splicing short of complete elimination of intron sequences has occurred.

By "capable of association" may refer to the ability, directly or indirectly, to engage or attract an opposing entity or complex, such as the propensity of Rev and Sam68 to bind RRE sequences in certain unspliced or minimally spliced retroviral transcripts. The association may include close, i.e., tight binding, or else less stringent relationships. Analogies may be drawn to ligand:receptor interactions as known in the art, and to the ability to cross-link certain molecular species when they communicate with one another or are otherwise in juxtaposition with one another.

By "wild-type" is meant functionally and/or structurally resembling the natural state, either perfectly or imperfectly.

By "cellular nuclear export protein" is meant a protein that originates from a cell and is nonviral in origin. Sam68 is an example, as contrasted with the viral protein, Rev. The protein may be a recombinant chimeric derivative or a wild-type molecule in its natural state. Furthermore, the gene can be transgenic with respect to a given cell line or organism, meaning that it may or may not be native to that species, and either stable or transient in presence or function when engineered, introduced or modified in such organism or cell.

By "Sam68" is meant the human amino acid sequence corresponding to Seq. ID. No. 1 or a homolog thereof, or encoded by the human cDNA coding sequence defined in Seq. ID. No. 2 (see GenBank accession NM006559 for full-length cRNA, i.e., noncoding regions), or a homolog thereof. The terms "homolog", "analog", and "derivative" as used in the claims to qualify Sam68 are not necessarily redundant of one another, although they can be as will be clear to one of skill in the art in reading this disclosure. As concerns nucleic acid sequences, the term 'homolog" is primarily meant to embrace mRNAs capable of conversion to cDNAs, or genomic DNAs, that bear great homology to Sam68, whether from the same or different species, and whether or not from a species that is already transgenic for the same gene, homolog thereof, or analog thereof. In the case of nucleic acids, homologs as defined herein have a hybridizable, and hence isolatable, affinity for a human Sam68 anti-sense molecule or portion thereof using standard non-stringent and stringent hybridization techniques as described, e.g., in 1 989, "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory, and Ausubel et al., 1 994, "Current Protocols in Molecular Biology" , John Wiley & Sons. A representative example is murine Sam68 (GenBank accession U 1 7046), and it is anticipated that equivalents in other organisms are isolable using the same techniques without undue experimentation. Other examples include allocations and allowances for any minor sequencing errors or differential splicing mechanisms that may be present or operative, and also for allelic variations that may be naturally present in the species population or else introduced thereto.

The term "homolog" as embraces amino acid sequences can mean the translated species of the nucleic acid homologs described above, and/or functional or structural equivalents that occur in and are isolatable using routine experimentation, e.g., by immunoprecipitation or antigenic recognition using human or murine Sam68 specific antibodies, or else by traditional biochemical purification techniques that employ a functional assay. By "analog" can mean the same but is more akin to synthetic man- made variations of Seq ID Nos 1 and 2 and homologs thereto and thereof, e.g., those introduced by mutation (point, deletion, truncation, or addition), substitution of stabilizing or destabilizing peptides or other functional entities, general codon usage adjustments (in nucleic acid embodiments), or general derivatization as discussed more fully in the detailed description section to follow. Domain swapping or supplementation is also embraced by the term analog, as are hybrid fusion proteins. A "derivative" is consonant with the term "analog" although it may also embrace the situation where changes are subsequently made to or based on an analog as described above.

By "promote" or "promoting" means assisting, but not necessarily essential or sufficient to the cause or goal.

By "translocation" means the changing of location, whether by diffusion or by active transport, e.g., as between the nucleus and the cytoplasm, and vice-versa.

By "manipulating the activity of" signifies modification of the level of, or activity of, the protein or homolog within the cell. In the case of the latter, this can arise by supply of a competitive or allosteric inhibitor or activator of the native entity. While this may embrace nonpeptide molecules, it preferably embraces peptides of like or similar constitution to the native entity, i.e., having one or more functions associated therewith in common but otherwise lacking or else possessing an enhanced or altogether new function, such as by manipulation of a domain, e.g., the nuclear localization domain, of Sam68. The embodiments may also, and preferably does, embrace the situation where the natural promoter/enhancer sequence is replaced with a stronger one for overexpression of the protein. By "analog" means resembling short of complete identity. This can either be at the nucleic acid or protein level, such as by variations as occur naturally within corresponding genes and gene products (mRNA and proteins) of the same or different organisms, or else by artificial changes induced or otherwise implemented by man. By "functional" and "dysfunctional" analog is meant the respective ability of a molecule having a natural or wild- type counterpart to function in a given respect. The molecule may possess one or more functions, and not all of these functions may be changed in the analog. The term "equivalent" can have a similar, although not necessarily identical, meaning. By "construct" is meant a nucleic acid entity capable of directly or indirectly expressing an encoded genetic element into a given protein or peptide upon or following transfection or infection of a cell. The construct may be DNA, RNA, circular, linear, viral, plasmid or genomic in nature, or have any combination of features compatible thereof, and will usually have regulatory components associated with it. The term "transfection" takes many forms and one of skill in the art knows that many alternatives exist to accomplish this, e.g., viral infection mediated, chemical, i.e. calcium-phosphate mediated, electroporation, biolistic gun, lipid-vesicle, i.e., liposome- mediated, etc. By "capable of expression" means that the construct can either directly or indirectly express on its own. The latter case may be exemplified by the situation in which an inducing molecule needs be supplied to de- repress expression, or else where all the components necessary for replication and expression are not provided on the same genetic entity, but are included in distinct entities, e.g., in a helper-virus context.

By "corresponding native gene" means that a functional and/or structural variant or equivalent already exists in the host cell genome, whether naturally or by virtue an earlier transgenic manipulation. The native gene may also encode the exact same product as administered by the methods described herein, only at a lesser level. Hence, the difference in concentration, and not necessarily function, may be important in some embodiments of the invention, e.g., in increasing retrovirus titer.

By "RRE or a functional equivalent thereof" is meant any one of a distinct number of cis sequences that are known or capable of adopting secondary structures characteristic of unspliced or minimally spliced transcripts of retroviruses, e.g., complex and simple, usually noncoding in nature, and capable of interaction with Sam68 or a homolog, analog, or derivative hereof, whether cellular or viral in origin. Primary sequence variants of these structures are also contemplated within the scope of the invention as long as overall RNA binding and/or multimerization abilities are not sacrificed. The simple retrovirus CTE element is especially embraced.

By "dominant negative effect" means that supply in a biological system overcomes, overwhelms or dominates the effect of a natural analog in the system, e.g., where mutant Sam68 outcompetes wild-type Sam68 in the same system to exert a negative or attenuating effect on retrovirus replication. This contrasts with a further aspect of the invention, which is the oversupply or expression of wild-type Sam68 product to synergistically enhance retrovirus replication or the expression of gene products associate therewith. By "modifying" means changing, either structurally or functionally, and directly or indirectly. The term can be synonymous with altering. By "carboxy terminal portion" is meant, in its broadest sense, the relative orientation within a polypeptide of the individual amino acids that comprise it. A polypeptide is usually linear and has both an amino and a carboxy end. As many amino acid residues constitute a polypeptide, the term can be a relative one used to describe the relative position or direction of one residue with respect to another in a larger chain of residues. In terms of Sam68, the phrase preferably denotes the approximate terminal one-third end of the molecule, which comprises about 1 10 residues, but does not rule out longer stretches. By "impedes" can mean blocks, disrupts or attenuates to varying degrees, including but not limited to absolute or total impairment.

By "elimination" can mean either the physical deletion or conversion or substitution of one residue for another. Thus, point mutations, truncations, and internal deletions are contemplated, either at the nucleic acid level, or else at the synthetic chemistry level for either peptides or nucleic acids. The term can also include the situation where an enzymatic cleavage effects the change, e.g., exo- or endo-nuclease or protease activity, and either in or outside a cell (in vivo versus in vitro applications) .

By "about" denotes moderate flexibility within the permissible bounds of the patent laws.

By "substantially preserved" suggests flexibility, but more importantly, operativity. Thus, when used, e.g., in describing the function of the KH domain that is to bind RNA, at least some level of RNA binding over, e.g., a negative control must be maintained. By "transgenic" means possessing a gene sequence, e.g., one encoding the cellular nuclear export proteins of the invention, that are not normally found in the prototypical host cell specimen or species, or else are redundant over genes already to be found there, e.g., increasing the gene number. The latter is typified, e.g., where the natural Sam68 coding sequence is placed under control of a stronger promoter, thereby enhancing retrovirus replication, or else where more optimal codon usage is employed in a synthetic or mutant gene that encodes the same gene-product as does the natural gene. An organism or cell that is transgenic does not lose its transgenic character after it replicates or reproduces. By "recombinant DNA or gene product encoded by the same" is meant a DNA or gene product that originates outside the cell or organism of interest, and has usually although not necessarily been cloned, i.e., propagated at the genetic level in a different cell culture first by virtue of certain genetic elements which have been incorporated or added to it. In this sense, the term can reflect a natural or native nucleic acid species or product thereof under control of a new promoter, enhancer, and/or 3 regulatory entitie(s), or else it can denote a mutant or synthetic sequence that encodes the same or a variant structure or product. In any event, when introduced to a host cell or organism the effect is different from that of the cell or organism s unadulterated state, however small the difference. By "implicated in" means previously, or by novel virtue of the invention, identified, directly or indirectly, with the feature that follows.

By "engineered to" means modified by man-made means, either directly or indirectly, e.g., by genetic or protein engineering as known in the art. By "influence" means changes away from the basal or natural state.

This includes the situation where the basal or natural state is an etiologically diseased one, in which case the term can connote a change to a more normal level.

By "delivery" is meant a broad means of introducing into, e.g., by different chemical and/or physical means such as transfection, electroporation, microscopic injection, liposomes, biolistic gun use, or chemical transduction, e.g., by the use of calcium-phosphate.

By "upon or following" connotes the permissibility of a temporal variation in the sequence of events or effects, which may occur at a substantially instantaneous or contemporaneous time with one another, or else at some more remote point in time, e.g., after other entities have also been delivered to or eliminated from the system.

As noted, the majority of poly-adenylated cellular pre-mRNAs must be completely spliced before being exported to the cytoplasm. However, unspliced and partially spliced RNAs expressed by human immunodeficiency virus (HIV) bypass the requirement for splicing and move into the cytoplasm to be translated or packaged into new virions. Nuclear export of these RNAs is dependent on Rev, a 1 9-kDa HIV regulatory protein. Rev comprises a basic RNA binding domain which specifically interacts with the cognate target sequence, RRE, and a leucine-rich nuclear export sequence, RRE, and a leucine-rich nuclear export sequence (NES) which binds to the export receptor CRM-1 .

Cytoplasmic accumulation of partially spliced HIV-1 RNAs in the absence of Rev in lymphoid cell lines has previously been reported. Other viral or cellular cofactor proteins might then compensate for the absence of Rev. In fact, the patentees have proven that Sam68, a cellular protein, can substitute for Rev, and with surprising results and implications for improved therapeutic strategies.

Sam68 is a src binding protein that has been implicated in mitosis. The Applicants unexpectedly and surprisingly hit upon Sam68 as also important to retrovirus replication when using a yeast-2-hybrid screening system that employed the carboxy terminal portion of RNA Helicase A (RHA) as bait (data not shown). RHA had previously been observed to bind to the CTE element of type D retroviruses, the carboxy terminal portion of which was identified as possessing a bidirectional nuclear transport domain. Tang et al., (1 997) Science, 276: 141 2-1 5. Furthermore, the Applicants subsequently noted the same RHA protein's ability to bind Rev. Li et al., ( 1 999) Proc. Natl. Acad. Sci. USA 96:709-14. Positives were culled from a library screen and sequenced, wherein identity with human Sam68 Seq ID. No. 2 was determined using standard bioinformatics computer analysis and comparison with existing nucleic acid sequence databases. The Applicants show here that not only does Sam68 specifically interact with RRE in vivo and redistribute to the cytoplasm in response to overexpression of RRE-containing RNA, but also that overexpressed Sam68 can functionally substitute for, and even synergize with, Rev in RRE mediated gene expression. Sam68 also can rescue Rev-deficient HIV-1 replication.

Sam68 binds directly to Rev in vitro. Two Sam68 truncated mutants ((330-443) and (41 0-443)) were constructed and found to be capable of binding RNA and translocating it to the cytoplasm, but incapable of cycling back to the nucleus. In effect, these Sam68 truncated mutants are trapped in non-functional form in the cytoplasm. Both of these mutants inhibit Rev and wild-type Sam68 in RRE mediated gene expression, and are capable of inhibiting wild-type HIV-1 (HBX2) replication. Hence the negative transdominant character of these mutants. The Applicants also found that strategically positioned point mutations can do the same. For example, a conversion of Sam68 proline residue 439 to an arginine has a similar transdominant negative effect to the truncations above.

Use of Sam68 has distinct advantages over the use of viral factors like Rev to increase or moderate complex retrovirus production. Transdominant mutants of viral proteins act by competitively interfering with wild-type protein activity. Dominant negative proteins were first studied by yeast geneticists. For HIV, a transdominant mutant of Rev, M 1 0, has received considerable interest. Preclinical studies as well as a Phase 1 gene therapy trial has been completed in HIV-1 infected patients. The Applicants have discovered that Sam68 has an analogous function in HIV replication, and can replace Rev or synergize with Rev in activating HIV replication. Therefore, it may not be sufficient or best to target Rev alone to inhibit HIV. The Applicants have specifically found that mutations in the C-terminus of Sam68 inhibit both wild-type Sam68 and Rev function, and result in a more effective inhibition of HIV replication. Another advantage is that since Sam68 is a cellular protein, the mutants are less likely to elicit an immune response (e.g., CTL) which destabilize gene-modified cells bearing or expressing non-self proteins. Therefore, Sam68 mutant genes, because they comprise or resemble self, have great potential in HIV gene therapy for AIDS patients.

Prior to the invention, other cellular modulators of Rev activity were known, e.g., CRM-1 and Rab/hRIP, but not cellular components that could actually substitute for Rev, and certainly not that could work effect across both the complex and simple retrovirus species. The Applicants are the first to discover the ability of Sam68 to do so, and the first to harness it to accomplish useful ends.

Preliminary data suggests that therapeutic strategies targeting inhibition of Rev alone may not be enough. The Applicants discovery can significantly enhance this ability, either alone or in combination with the mutant Rev approach. Although the endogenous level of Sam68 is too low to support a high level of virus replication, it is conceivable that virus mutants that utilize this pathway can more efficiently emerge under selective pressure. The attractiveness of being able to block two pathways instead of one bodes large for effective suppression and inhibition of retroviruses. The examples to follow illustrate how effective Sam68 transdominant negative mutants can be in inhibiting Rev, wild-type Sam68, and retrovirus replication.

Importantly, overexpression of the Sam68 mutants was not found to induce cytotoxicity (data not shown). While a transdominant mutant of Rev is currently under development by Novartis as a gene therapy implement for HIV, the use of a cellular protein such as Sam68 has a distinct advantage in that is does not provoke host immune responses. Such responses would remove cells expressing the foregoing proteins and thus hinder the effectiveness of anti-HIV therapy. Therefore, cellular transdominant mutants are better-suited vehicles for therapy than are viral-based proteins such as Rev and nucleic acids encoding the same. By administering such transdominant mutants to autologous T-cells or hematopoietic progenitor cells that give rise to CD4, e.g., by AAV or retroviral vectors, i.e., lentiviral vectors, HIV-resistance can be introduced and the cells re-infused into an autologous host, e.g., by an ex vivo procedure as commonly known in the art.

According to the invention, transdominant Sam68 mutant genes can be delivered in HIV target cells or stem cells by available gene therapy vectors (AAV, MLV, or lentiviral vectors) to confer resistance to HIV infection. The in vitro selected resistant cells can be re-infused into autologous recipients. Initial preclinical studies are to be carried out in primary cells from uninfected or HIV-infected donors. Studies may also be performed in Rhesus macaques to test for safety and efficacy.

The advantages of the Applicants' invention as concerns retrovirus inhibition applications, in sum, are that 1 ) two pathways retroviral reproduction pathways are effectively blocked or impeded instead of one, and that 2) unwanted host immune responses are avoided that would otherwise compromise the effectiveness of therapy by killing targeted cells instead of merely conforming them. As concerns the invention aspect in which retrovirus replication is actually enhanced, wild-type Sam68 can unexpectedly synergize with, and actually substitute for, Rev and Rex, which has great merit in enhancing transgene delivery and expression using complex retroviruses as vectors. It is anticipated that exceeding basal Sam68 expression in a given transfected cell or cell line will also enhance the expression of and from simple retroviruses and vector constructions based on the same. Detailed description of the drawings

Figure 1 A shows the domain array within wild-type Sam68 (top) and specific mutants thereof (bottom) used in this study. P, Proline-rich motifs; KH, K homology domain; RGG, Arg-Gly-Gly; 1 and 443, amino acid numbers. Figure 1 B shows the effects of wild-type and mutant Sam68 on RRE- mediated CAT expression in 293T cells transfected with RRE-CAT alone (I); RRE-CAT plus Sam68 (II), Sam68 Δ96 (III), Sam68 Δ96 ΔKH (IV), Sam68C'Δ330-443 (V), Sam68C'Δ41 0-443 (VI), Sam68 Δ42-329 (VII) and/or Rev (VIII). Data represent fold increase relative to basal levels (RRE- CAT alone) .

Figure 1 C shows RRE-independent expression in CAT assays of cells transfected with CTE-CAT or HIV LTR-CAT Sam68 Δ96 and/or Tat ( + , present; -, absent). DNA was equalized to 2.5 μg for each transfection. Figure 2A shows that microinjection of a Sam68 Δ96 expression plasmid activates RRE-mediated reporter gene (pCM228 (RRE-lacZ)) expression in human HS68 cells. Phase contrast images (LacZ expression) and corresponding immunofluorescence photographs (injected cells) demonstrate typical experimental results. Figure 2B shows a reporter construct having minimal basal activity

(3XUASp36LacZ) injected with and without Sam68.

Figure 3A shows the interaction of Sam68 with RRE in vitro. Left, GST-Sam68, GST-Sam68 Δ1 -329, GST, gp1 20 and/or Rev proteins were assessed by RNA gel mobility shift assay. Arrows, muitimeric (upper) and monomeric (lower) forms of RRE RNA. For RNA competition (right), increasing amounts (wedges above blot) of unlabeled (cold) RRE, poly-U and tRNA were included in the pre-incubation mixture.

Figure 3B shows the interaction of Sam68 with RRE in vivo. 293T cells were transfected with RRE-CAT plus Rev (lanes 1 and 2), RRE-CAT alone (lanes 3-6), (lane 7), CTE-CAT (lane 8) antisense-CTE-CAT (lane 9)/or HIV 1 LTR-CAT alone. Lane 10, TAR (TAT activation-responsive); lane 1 1 , RRE RNA PCR; lane 1 2, CTE RNA PCR (PC, positive control) . Antibodies against Rev (lanes 1 and 2) or Sam68 (lanes 3 and 4) were used to immunoprecipitate interacting components from cell lysates. Non-immune IgG was used as a negative control (lanes 5 and 6) . RNA extracted from these complexes was subjected to RT-PCR with ( + ; lanes 1 ,3,5 and 8-1 2) or without (-) reverse transcriptase (RT) using RRE-CTE-and/or TAR specific primers. M, DNA markers.

Figure 4A shows how Sam68 synergizes with Rev in CAT assays of 293T cells co-transfected with RRE-CAT, Sam68 Δ96 and/or Rev; DNA was equalized to 2.5 μg for each transfection. Data represent fold increase relative to basal levels (RRE-CAT alone).

Figure 4B shows how Sam68 synergizes with Rev in RRE directed rescue of rev(-) HIV-1 expression. 293T cells were co-transfected with rev(-) proviral DNA, Sam68 Δ96 and/or Rev expression plasmids; DNA was equalized to 2 μg for each transfection. Cell-free supernatants collected 48 h later were analyzed by p24 antigen capture assay.

Figure 5A shows Sam68-Rev-RRE mediated CAT activity in 293T cells co-transfected with the indicated (below graph) combinations of RRE CAT, Rev, Sam68 (0.1 ug) Sam68 Δ42-329 (1 ug) and increasing amounts (wedges; 0.1 25, 0.25, 0.5 and 1 ug) of Sam68C'Δ or Sam68C'Δ41 0-443; DNA was equalized to 1 .5 μg for each transfection. Relative CAT activities are expressed as percent of activity of RRE-CAT in the presence of Rev and wild-type Sam68.

Figure 5B shows inhibition of Rev and Sam68 function in 293T cells co-transfected with the indicated combinations (below graph) of rev(-) proviral DNA (0.25 ug), Rev (0.025 ug), Sam68 (0.1 ug) and increasing amounts of (wedges; 0.1 25, 0.25, 0.5 and 1 ug) of Sam68C'Δ330-443 or

Sam68C'Δ 41 0-443 analyzed by p24 antigen capture assay.

Figure 5C shows inhibition of wild-type HIV replication in 293T cells co-transfected with wild-type HXB-2 and Sam68C'Δ30-443 or Sam68C'Δ410-443 analyzed by p24 antigen capture assay. *, complete inhibition.

Figure 5D shows comparative inhibition of HIV replication by CΔ41 0- 443 and RevM 1 0 in 293 T cells con-transfected with wild-type HXB-2 and Sam68C'Δ41 0-443 or RevM 1 0 mutant plasmids (wedges; 0.1 25 and 1 ug) and analyzed by p24 antigen capture assay; DNA was equalized to 1 .5 μg for each transfection. Cell-free supernatants collected 72 h later and compared with expression levels seen in the presence of endogenous levels of Sam68 (100%). Figure 5E is analogous to experimentation and results achieved in Figure 5A, but uses a point mutant of wild-type Sam68, corresponding to change of residue 439 from a proline to an arginine.

Figure 6A shows the interaction of Sam68C'Δ330-443 with RRE in vitro. GST-Sam68, GST-Sam68 Δ1 70-208, and GST-Sam68C'Δ330-443 were assessed in gel mobility shift assays.

Figure 6B shows a gel mobility shift assay using increasing amounts (wedges 0.1 , 0.4, 0.8, and 1 .6 ug) of Sam68C'Δ330-443, Sam68C'Δ1 -329 or GST added to the incubation mixtures containing RRE and GST-Sam68. Figure 6C shows purified GST-Sam68 and/or GST-Sam68C'Δ330-443 fusion proteins ( 1 μg) bound to GST beads in affinity matrix to assess the in vitro binding of Rev. Controls, GST alone or GST-Sam68C'Δ proteins (50 ug) bound to G-Sepharose beads. The binding of Rev to Sam68 was assessed by western blot analysis with rabbit polyclonal antibodies against Rev. Arrow, positioning of Rev protein.

Figure 7 shows co-localization of Rev and Sam68 with transdominant mutant CΔ330-443. GFP- CΔ330-443 fusion expression vector was transfected separately or in conjunction with wild-type Sam68 or Rev expression plasmids into HeLa cells. Cells were fixed and stained with antibodies against Sam68 or Rev 48 h later and were visualized by confocal microscopy, a, Wild-type Sam68 stained with antibodies against Sam68. b, GFP-CΔ330-443. c, Rev stained with antibodies against Rev. d-f, Cells expressing GFP-CΔ330-443 and Rev. f, solid line, cell boundary; broken line, rim of the nucleus, g-l, Cells expressing GFP-CΔ330-443 and wild-type Sam68. d and g, green fluorescence from GFP-Sam68C'Δ330-443; e and h, rhodamine staining with antibodies against Rev and Sam68 respectively; f and i, composite of the two colors to demonstrate co-localization of the

CΔ330-443 mutant protein (green) with Rev (red) or wild-type Sam68 (red) .

Figure 8 shows how, using RRE-CAT reporter assays, administration of Sam68 to NIH3T3 mouse cells can overcome species-specific barriers and permit Rev to function.

Figure 9 is cumulative to results demonstrated in Figure 8 and demonstrates the same using cells co-transfected with cyclin T and HXB-2 and subjected to p24 antigen capture. Figure 1 0 shows the effect of Sam68 on CTE-Gag expression in 293T cells using a p24 antigen capture assay. This contradicts the earlier CTE-CAT assay data of Figure 1 C and suggests that Sam68 may indeed be important in the expression and replication of simple retroviruses. The constitution of the CTE-Gag reporter is analogous to CTE-CAT but for the difference in reporter coding sequences. Figure 1 1 A shows CAT assay results using equine RRE (eRRE) and

Rev (eRev) with human Sam68. The EIAV genome in which these elements exist are known and published, e.g., GenBank accession AF033820. The individual elements can readily be isolated or created therefrom, e.g., using PCR or synthetic oligo linkage, to recreate functionally equivalent vectors to those used here.

Figure 1 1 B shows CAT assay results using RxRE and Rex from HTLV- 1 in the presence of human Sam68. The RxRE and Rex sequences are published, e.g., GenBank accession L03561 and J02029, and in Hanly et al. ( 1 989) Genes & Development. Figure 1 2A shows a schematic for the essential features of the plasmid vectors pRex and pRev. (construction detailed in U.S. Patent No. 5,871 ,958 citing Rimsky et al., Nature 335:738-740 ( 1 988) and Malim et al. Nature 335: 1 81 -1 83 (1 988)) which are otherwise available or reproducible in equivalent or enhanced effect by those of ordinary skill in the art using routine techniques and commercially available starting materials. A similar construct was used in which equine Rev as described by Harris et al., ( 1 998) Mol. Cell. Biol. 1 8:3889-3899 was substituted for HIV Rev.

Figure 1 2B shows a schematic for the essential features of the plasmid vectors RRE-CAT (pCMV1 28), CTE-CAT (pCMV1 38), and RXRE- CAT (pCMV 1 28-XR). RRE-CAT is widely known (see, e.g., U.S. Patents Nos. 5,935,776, 5,922,856, and 5,989,81 4 (citing Chen and Frankel, Biochemistry 33:2708-1 5 ( 1 994) for details of construction) and otherwise available or reproducible in equivalent or enhanced effect by those of ordinary skill in the art using routine techniques and commercially available starting materials. See also U.S. Patent No. 5,733,543 (describing an RSV- driven equivalent and citing Hope et al., Proc. Natl. Acad. Sci. USA 87: 7787-7791 ( 1 990)) . The precise RRE sequence used in the present examples corresponds to bases 7305-7546 of HIV-1 (GenBank accession K03455). The precise CTE sequence used corresponds to bases 8007-8240 of Simian Mason Pfizer D-type retrovirus (GenBank accession M 1 2349) . The terms SD and SA denote, respectively, the splice donor and splice acceptor cites which positions are flank the reporter gene CAT.

I. Experimental Examples

The following examples reflect the use of chimeric constructs having vectors whose exact identity is not important and merely illustrative to practice of the invention. One of ordinary skill in the art recognizes that many possible vectors exist that are commercially available or that can be fashioned without undue experimentation, and which can substitute for those described below to reproduce the invention. What is important is the identity, characterization, and function of Sam68 as a modulator of retrovirus activity.

The experiments below are reproduced in Reddy et al., Nature Medicine, Vol. 5, No. 6, pp. 635-642 ( 1 999), which is herein incorporated by reference in its entirety including drawings. What follows is intended to further describe and/or to supplement what is shown in the preceding drawings and figure legends.

Example 1 : Sam68 enhances RRE-directed reporter gene expression

To explore a potential role for Sam68 in RRE-mediated transactivation, we assessed the effect of exogenously expressed Sam68 under the control of the CMV promoter on an RRE-regulated reporter gene (RRE-CAT; chloramphenicol acetyltransferase) in transient co-transfection assays. To more easily distinguish Sam68 transgene expression for endogenous Sam68, we also constructed a mutant with a 96-amino acid deletion for the N terminus (Sam68Δ96). Deletion of 103 amino acids for the N terminus does not affect the RNA binding and multimerization of Sam68 . We studied both wild-type and a variety of mutant Sam68 constructs (Fig. 1 a) . Sam68 is expressed at low levels but ubiquitously in human cells (data not shown) . Both wild-type Sam68 and Sam68Δ96 were over-expressed in transfected cells (Fig. 1 a), and both induced a 1 2-fold to 1 5-fold increase in RRE- mediated CAT reporter gene expression over basal levels, whereas Rev expression yielded a 20-fold to 30-fold increase (Fig. 1 b). The increase in CAT expression mediated by Sam68Δ96 was dose dependent, and at times was as efficient as that with Rev (data not shown). Therefore, we used Sam68Δ96 instead of full-length gene in most of our subsequent studies. We also assessed the effect of a mutant in which the KH domain was deleted (Sam68Δ96ΔKH). The KH domain of Sam68 is important for self- association as well as RNA binding. Sam68Δ96ΔKH failed to enhance the RRE-mediated transactivation (Fig.1 b). Similarly, mutants deleted at the N terminus Δ42-329) and C terminus (Δ330-443 and Δ41 0-443) did not transactivate RRE-directed gene expression (Fig. 1 b). As negative controls, we examined the effect of hnRNP A1 , another RNA binding protein, on RRE- directed transactivation (data not shown), as well as the effect of Sam68Δ96 on other expression constructs including CTE-CAT (constitutive transport element of type-D retroviruses, a functional analogue of RRE) and HIV LTR-CAT (CAT reporter gene linked to the HIV long terminal repeat; Fig. 1 c). [ The results suggest that transactivation of RRE by Sam68 is specific. However, results achieved using a CTE-Gag construct indicate that that specificity likely also embraces CTE (see Figure 1 0 and discussion in Example 2, below)].

We obtained independent confirmation of the specific effects of Sam68Δ96 on RRE-dependent activation by microinjection assays. Co- injection of pSam68Δ96 expression vector into the nuclei of HS68 cells transactivated the expression of RRE-directed lacZ (pCM228) as efficiently as pRev expression vector at the same concentration (Fig. 2a). Of the pRev- and pSam68Δ96-injected cells, 39% and 44%, respectively, stained blue. In contrast, pSam68Δ96 did not stimulate the expression of a control reporter plasmid (3XUASp36LacZ), which has low basal activity (Fig. 2b), again indicating the Sam68Δ96 does not activate reporter gene expression through a general transcriptional activation mechanism

Example 2: Interaction of Sam68 with RRE in vitro and in vivo, and with CTE

The specificity of Sam68 or RRE-mediated gene expression prompted us to examine the potential interaction of Sam68 with RRE in vitro and in vivo. We used a gel mobility assay to assess the direct interaction between Sam68 and in ^'tro-transcribed, ³²P-labeled RRE RNA. Labeled RRE RNA bound to GST-Sam68 and Rev but not to GST or gp1 20 protein (Fig. 3a). Unlabeled RRE RNA, but not to tRNA, competitively inhibited the formation of the Sam68 and RRE RNA complex, indicating the Sam68 is not a general RNA binding protein (Fib. 3a). Sam68 binds to poly-U-rich sequences. Unlabeled poly-U RNA was not as efficient as unlabeled RRE RNA in the competition (Fig 3a), indicating a greater affinity of Sam68 for RRE RNA than for polyU RNA.

For the in vivo binding studies, we used antibodies against Sam68 to immunoprecipitate interacting component form 293T cells transfected with RRE-CAT (pCMV1 28), CTE-CAT (pCMV1 38), α-CTE-CAT (antisense) or HIV LTR-CAT plasmids. We extracted RNA from these complexes and subjected it to RT-PCR with RRE-, CTE-, and TAR-specific primers. As positive and negative controls, we used Rev antibodies (and co-transfection with pRev) and control IgG, respectively. RRE RNA was precipitated by antibodies to Sam68 and by antibodies to Rev when Rev was co-expressed, but not by control IgG (Fig. 3b), indicating that RRE does interact with endogenous Sam68 in vivo.

Although CTE, α-CTE and TAR RNAs were not detected using their respective primers (Fig. 3b), indicating that Sam68 interacts specifically with RRE in vivo, these results are contradicted by experiments in which the retroviral coding element Gag was substituted for CAT (see Figure 1 0), and also experiments in which CTE is otherwise observed to bind Sam68 in vitro (data not shown). Similarly, and as depicted in Figures 1 1 A and B, human Sam68 can functionally recognize both equine RRE/Rev, and also RxRE/Rex from HTLV-1 . This suggests a broad-spectrum utility for Sam68 in the general modulation of retrovirus expression.

Example 3: Synerqistic effects of Sam68 and Rev

In addition to Rev-independent transactivation or RRE-mediated gene expression, Sam68Δ96 also exerted a significant synergistic effect when co- expressed with Rev (1 20-fold more than the basal level; Fig. 4a) . This synergy was also evident in a virus rescue assay. We co-transfected 293T cells with a rev(-) proviral DNA plasmid and the Sam68Δ96 expression vector, with or without a Rev expression vector, and measured the expression of p24 antigen in the cell-free supernatants. Sam68Δ96 alone increased the level of rev(-) virus expression fivefold to sixfold more than the basal rev(-) replication (Fig. 4b). Expression of Rev yielded a 25-fold to 30- fold increase, whereas co-expression of Sam68Δ96 and Rev resulted in an increase in p24 expression more than 1 20-fold over basal levels (Fig. 4b). These results indicate that Sam68 can partially substitute for and synergize with Rev in mediating RRE-directed gene expression in the context of a provirus.

Example 4: Differential inhibition of Samββ and Rev activities

To determine whether Sam68 and Rev might use a common nuclear export pathway, we assessed the effects of leptomycin B on Sam68/RRE- mediated CAT gene expression and rev(-) virus rescue. LMB inhibits Rev by disrupting the complex formation of CRM1 , RanGTP and Rev. LMB inhibited as much as 77% of Rev/RRE-mediated CAT expression and 64% of p24 expression in the virus rescue assay, consistent with previous observations (Table). In contrast, LMB did not substantially inhibit Sam68/RRE-mediated reporter gene expression or rev(-) virus rescue (Table). However, LMB partially inhibited the synergistic enhancement exerted by both Sam68Δ96 and Rev (40% and 30%, respectively, for CAT and p24). This partial inhibition may be attributed to the repression of the Rev function alone. These results also indicate that the pathways of Sam68 and Rev are distinct but not mutually exclusive in mediating RRE nuclear export. The transdominat Rev mutant M10 inhibited both reporter gene expression and virus replication mediated by Sam68Δ96/RRE, Rev/RRE or a combination of both, to the same extent.

In addition to being tyrosine phosphorylated by c-Src, Sam68 is also phosphorylated on threonine residues by Cdc2 during mitosis. The drug olomoucine selectively inhibits Cdc2 kinase activity. Treatment with olomoucine (75 μM) inhibited 70% of the Sam68Δ96 effect, and only 25% of the Rev effect, on RRE-dependent activation (Table). Iso-olomoucine, an isoform of olomoucine that does not inhibit Cdc2 kinase, did not have any inhibitory effect. These results indicate that phosphorylation by Cdc2 kinase may be important for the observed function of Sam68 . Basal RRE-CAT expression was inhibited by both olomoucine and RevM 10, indicating that the endogenous Sam68 may be responsible for this Rev-independent activity. Example 5: Inhibition of virus replication by Sam68ΔC mutants

We next assessed the Sam68 mutants that failed to transactivate RRE-mediated gene expression for their ability to act as dominant negative mutants. In these studies, we measured inhibition of the synergistic transactivation by both Rev and Sam68. There was a steady decrease (up to 95%) in RRE-mediated CAT reporter gene expression in the presence of increasing amounts of the Sam68C'Δ330-443 or Δ41 0-443 mutant DNA plasmids (Fig 5a). In contrast, no inhibition was seen with the Δ42-329 mutant (Fig 5a). We also assessed the effects of olomoucine and isolomoucine on the transdominant inhibitory activity of Sam68C'Δ41 0-443. This mutant inhibited 80% of the Rev activity on RRE-dependent activation. Olomoucine slightly increased the inhibition to 93%, whereas iso-olomoucine had no effect. These results indicate that the transdominant inhibitory activity of Sam68C'Δ41 0-443 is independent of phosphorylation. The greater inhibition seen in the presence of olomoucine could be due to inhibition of endogenous Sam68 by this compound.

We also confirmed the inhibitory effects of these mutants using a virus rescue assay (Fig 5b). Co-transfection of increasing concentrations of CΔ330-443 and Δ420-443 mutants in conjunction with Rev and Sam68 resulted in a complete inhibition of p24 expression from the rev(-) provirus (Fib. 5b) . To determine whether the inhibition would extend to Rev function in native HIV, we co-transfected Sam68C'Δ330-443 or Sam68C'Δ41 0-443 expression vectors with HXB-2 proviral DNA (GenBank accession K03455). We used RevM 1 0 as a parallel control. Both Sam68C'Δ330-443 and Sam68C'Δ420-443 exerted a profound, dose-dependent inhibition on viral replication (Fig 5c), similar to that seen with RevM 1 0 (Fig. 5d). These results indicate that the C-terminal deletion mutants of Sam68 show a dominant negative phenotype for HIV replication.

Example 6: in vitro binding studies with Sam68C'Δ330-443

To explore the underlying mechanism of inhibition by the C-terminal deletion mutants of Sam68, we assessed their binding propertied to RRE, Rev and wild-type Sam68. Despite the presence of the KH domain in the

Sam68C'Δ330-443 mutant, it binds very poorly to RRE (Fig 6a). However, addition of increasing amounts of Sam68C'Δ330-443 resulted in a reduction of mobility of Sam68-RRE complex, indicating the Sam68C'Δ330-443 may displace wild-type Sam68 in a muitimeric complex on RRE. In contrast, GST or GST fused to a non-transdominant mutant (Δ1 -329) did not affect the gel mobility of the Sam68-RRE complex (Fig. 6b). For direct protein-protein binding, we mixed affinity matrices of Sam68, C'330-443 and Δ1 -329 GST fusion proteins with recombinant Rev protein. After being washed extensively, proteins were eluted by boiling and assessed by western blot analysis using antibodies against Rev. Rev was retained by the full-length as well as by the C'Δ330-443-Sam68 fusion proteins but not by GST or GST fused with Δ1 -329 (Fig 6c). These results indicate that the formation of a non-functional complex with Rev and Sam68 by the CΔ330-443 mutant protein may contribute to its transdominant phenotype.

Example 7: Cellular localization of transdominant mutants

We transfected HeLa cells separately with Rev, Sam68 and GFP- CΔ330-443 expression plasmids. Then 48 hours after transfection, we stained the cells with appropriate antibodies and examined them with a confocal microscope. Sam68 and Rev were both localized in the nucleus, but Sam68 was excluded from the nucleoli (Fig 7a and c). In contrast, Sam68C'Δ330-443 was localized in the cytoplasm (Fig 7b). GFP alone was distributed evenly in the cytoplasm as well as nucleus (data not shown). When the cells were co-transfected with Rev and GFP-Sam68C'Δ330-443 expression vectors, the two proteins completely co-localized, mainly in the nucleoli and in the cytoplasmic area near the nucleus (Fig. 7d-f) . In cells co- transfected with Sam68 and GFP-Sam68C'Δ330-443 expression vectors, both proteins were localized in the nucleus (Fig. 7 g-i). These results indicate that the in vivo binding of Sam68C'Δ330-443 to Rev impedes the normal nuclear import of Rev, thus rendering it nonfunctional. In contrast, the nuclear localization sequence of wild-type Sam68 was sufficient to direct the complexed mutant protein to the nucleus, even though the complex was still nonfunctional.

Example 8: Methods and Reagents Used in Above Examples Construction of plasmids. The Sam68 expression plasmids (pcSam68 and pcSam68 Δ96) were constructed by cloning the BamHI-EcoR1 fragments embracing the full-length Sam68 coding sequence from a BlueScript SK + library clone (Stratagene, La Jolla, CA) into the cognate sites of the pcDNA3 vector (Invitrogen, Carlsbad, CA) . We constructed Sam68 Δ96 ΔKH mutant according to the procedures of Xu and McFadden (W.X., McFadden, B.A., Zang, Y., T.R.R. and F.W.-S., manuscript submitted). For Sam68 CΔ330- 443 and Δ410-443, full-length pSam68 expression plasmid was digested with Xhol and/or Hindlll, and the resultant Sam68 'backbone' vector was re- ligated. For Sam68 Δ42-329, we created an Agel restriction site at amino acid 329 in the open reading frame of pSam68; the resultant plasmid was digested with Agel, followed by re-ligation. GST-CΔ330-443, Δ1 70-208 and Δ1 -329 were constructed by cloning the EcoRI fragments from the respective Sam68 expression plasmids into the cognate sites of the pGEX-4T (Amersham Pharmacia Biotech, Piscataway, NJ). For GFP-Sam68 C'Δ 330- 443, the EcoRI fragment from the Sam68 CΔ330-443 expression vector was cloned in-frame into the pcDNA-GFP plasmid.

Random mutations were generated pursuant to the method described in US provisional application 60/1 3761 9, filed June 4, 1 999, and herein incorporated by reference. Briefly, a 1 .8 kb Sam68 cDNA fragment was subcloned into a zero-background mutagenesis vector, pZerO-2.1 5, which carries a bacterial toxic ccdB gene regulated by lac promoter (Xu and McFadden, 1 998). The resultant plasmid pZerO-Sam68 was subjected to site-directed mutagenesis to generate 1 7 random mutations covering through the entire Sam68 gene. Briefly, in a 20 ul reaction volume, 100 ng of pZerO- Sam68, 1 00 ng of selection primer and 300 ng of mutagenic primers were denatured and annealed. On Unit of T4 DNA polymerase and 1 Unit T4 DNA ligase were added to the DNA mixture and then incubated at 37 C for 2 hours. Ten ul of the reaction mixture was transferred to mutS-defective strain of BMH71 -1 8 and the transformants were plated on 50 ug/ml Hanamycin plates containing 2 mM IPTG to select against the parental pZerO-Sam68 plasmids. Only cells carrying the mutant plasmids could survive after IPTG selection. Since a specific restriction sequence was included in each mutagenic primer, all mutant plasmids were verified by restriction digestion. For each random mutation, a restriction Agel site was designed so that internal truncation mutants could be expressed by removing the Agel fragment between amino acid 60 and each random mutation position. An EcoRI restriction was also introduced right upstream of the start codon or at the position of amino acid 310 in order to construct GST or GFP fusion proteins with full-length Sam68 or it C-terminal domain. For construction of pCDNA-Sam68 mutants, the 1 .7 kB Kpnl-EcoRI fragments containing different mutations were removed from mutated pZerO- Sam68 and subcloned back into pCDNA3 between Kpnl and EcoRI sites. Expression vectors of GST or GFP fusion proteins were constructed by subcloning the EcoRI fragment containing full-length Sam68 or its C-terminal gene into the unique EcoRI sites of pGEX-4T or pcGFP per Reddy et al., Oncogene 1 4: 2785-2792 (1 997), hereby incorporated by reference.

Specific details of the construction of all plasmids are available from the authors, and are otherwise detailed in sufficient manner in the figures and figure descriptions to enable one of ordinary skill in the art using routine knowledge and techniques to duplicate the essence of the invention without exercising undue experimentation. Such techniques are well illustrated in such common laboratory manuals as Sambrook et al., 1 989, "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory, and Ausubel et al., 1 994, "Current Protocols in Molecular Biology" , John Wiley & Sons, and the genetic elements which comprise the various vectors described herein are publicly known and available. For example, the HXB2 HIV genome is well known, published, and the individual genetic elements, i.e., Rev, gag, RRE, and the pertinent splice junctions delineated, e.g., in the discussion and pictorials accompanying GenBank accession K03455 and Ratner et al., Nature 31 3: 277-284 ( 1 985). Other isolates are also fully known and delineated, e.g., GenBank accession U4601 6, and as published by Saliminen et al. (1 996), AIDS Res. Hum. Retroviruses 1 2( 1 4) 1 329-1 339.

Using the above, and given the abundance of AIDS patients and infected cells that exist, one of skill can easily generate PCR primers flanking the pertinent genetic elements, amplify the material between, purify it, and/or combine the products as appropriate to provide suitable vectors that allow duplication of the invention. Alternatively, or combined, one can have the individual genetic elements synthesized as oligos, and then join them appropriately. Western blot analysis of plasmid expression. Wild-type and mutant

DNA plasmids (2 ug) were transfected into 293T (1 x 10⁵) cells. Then, 48 h after transfection, cell extracts were prepared and 40-μg samples were separated by SDS-PAGE and assessed by western blot analysis using antibodies raised against Sam68 protein (Santa Cruz Biotechnology, Santa Cruz, California). Cells, transfections and CAT assays. Human 293T (commonly known and available in the art; prepared by transfecting an SV40 large T antigen cDNA into a 293 cell (transformed primary embryonal kidney, human; American Type Culture Collection, Manassas, Va; accession number CRL- 1 573) and HeLa cells (American Type Culture Collection, Manassas, Va) were maintained in DMEM supplemented with 1 0% fetal bovine serum. 293T cells ( 1 x 1 0⁵) were transfected with the plasmid constructs by the calcium phosphate method. Unless otherwise indicated, amounts transfected for each plasmid were: 0.1 25 μg RRE-CAT, 0.5 μg Sam68, 0.5 μg Sam68 Δ96, 0.5 μg Sam68 Δ96 ΔKH, 0.5 μg CΔ330-443, 0.5 μg CΔ41 0-443, 0.5 μg Δ42- 329, 0.025 μg Rev, 0.1 25 μg CTE-CAT, 0.1 25 μg HIV LTR-CAT, 0.1 μg Tat and 0.5 μg RevM 1 0 (from T. Hope). For treatment with soluble inhibitors, 1 8 h after transfection, cells were incubated in medium containing 4 nM LMB (From M. Yoshida), 75 uM olomoucine or 75 uM iso-olomoucine (Calbiochem). Then, 48 h after transfection, cells were collected, washed with phosphate buffered saline and then resuspended in 75-1 50 ul of 0.25 M Tris, pH 7.8. The cell extracts were prepared by three 'freeze-thaw' cycles (-70 C, and 37 C) followed by a brief centrifugation to remove cell debris. CAT assays and the separation of reaction products was done as described, with the minor modification of using acetyl-CoA in place of butyryl-CoA. The increase in transactivation was quantified by scintillation counting of products separated from the reaction.

Microinjection. Microinjection analysis was done essentially as by Torchia, J. et al. Nature 387: 677-684 (1 997). Before being injected, primary HS68 human fibroblasts were rendered quiescent by incubation in serum-free medium for 24-36 h. Microinjection experiments were then done, and overnight expression was allowed before fixation and staining. Plasmids were injected into the nuclei of cells at a concentration of 1 00 ug/ml. Preimmune IgG was also injected in all samples to allow the detection of injected cells by indirect immunofluorescence. B-galactosidase activity was detected by incubation with 5-bromo-4-chloro-3indolyl-beta-D-galactosidase (X-gal). Injected cells were identified by staining with secondary antibodies conjugated with tetramethylrhodamine. In cells expressing high levels of B- galactosidase, the blue staining tended to 'quench' rhodamine fluorescence. For this reason, injected cells were counted as those with either nuclear rhodamine fluorescence or blue X-gal staining or both. All cells showing any trace of blue staining were scored as positive for expression, to avoid any possible subjectivity in the analysis. Experiments were analyzed and photographs were taken using a Zeiss Axiophot epifluorescence microscope. in vitro transcription and RNA gel mobility shift assay. The plasmid pcRRE was constructed by inserting PCR-amplified HIV-1 (H x B-2) RRE sequence into the Hindlll and BamHI cloning site of pcDNA3 (Invitrogen, Carlsbad, California). Unlabeled ('cold') and ³²P UTP-labeled RRE were synthesized by in vitro transcription with T7 RNA polymerase according to the manufacturer's protocols (Promega, Madison, Wisconsin) using BamHI- linearized pcRRE plasmid as template. RNA-protein binding reactions were pre-incubated for 1 0 min at room temperature in a binding buffer containing 60mM NaCl, 1 2 mM HEPES pH 7.9, 1 2 mM DTT, and 50 U RNasin. Typically, 1 x 1 0⁴ cpm of ³ P-labeled RNA and 1 00 ng of protein were used. The binding reaction (Final volume, 30 ul) was allowed to proceed for 1 5 min at room temperature and then the mixture was separated by 4.5% non-denaturing PAGE in 1 x Tris-borate- EDTA buffer. The gel was dried and exposed directly to X-ray film. For the competition assay, ³²P-labeled RRE was first pre-incubated with increasing amounts of cold RRE, poly-Urich RNA (0.01 5, 0.1 5 and 1 .5 ug) and/or yeast tRNA (0.1 5 and 1 .5 ug) in the binding buffer for 1 0 min before the protein was added.

Immunoprecipitation and RNA PCR. 293T cells (in 1 00-mm dishes) were transfected with pCMV1 28 (40 ug) plus either pRev (20 ug) or pcDNAS (20 ug). Then, 48 h later, cells were lysed in 1 ml of 0.65% Nonidet P-40 lysis buffer (containing 1 50 mM NaCl, 1 0 mM Tris-HCI, pH7.8 and 1 .5 mM MgCI₂) . Cell lysates were 'pre-cleared' with normal rabbit serum by overnight incubation at 4 C with 20 ul of normal rabbit serum conjugated agarose beads. The resultant lysate from the cells co-transfected with pCMV1 28 and pRev was mixed with 40ul protein A and protein G agarose beads plus 2 ul rabbit antiserum against Rev. The lysate from the cells co-transfected with pCMV1 28 and pcDNA3 was divided into two aliquots. Each was mixed with 40 ul protein A and protein G agarose beads plus 1 0 ul mouse IgG antibody against Sam68 and/or normal mouse IgG. After overnight incubation at 4 C, the beads were washed three times with lysis buffer. RNA was extracted from the beads with Ultraspec (Biotecx Laboratories, Houston, Texas) reagent according to the manufacturer's protocols. RT-PCR used a pair or RRE-specific primers. DNA products generated by RT-PCR were analyzed by 2% agarose gel electrophoresis. For specificity studies, 293T cells cultured in 1 00-mm dishes were transfected with pCMV1 38 (CTE), antibody against pCMV1 38 (anti-CTE) and/or pHIV1 LTR-CAT. Antibodies against Sam68 were used to immunoprecipitate interacting components from cell lysates. RNA was extracted from these complexes and subjected to RT-PCR analyses using CTE and TAR-specific primers.

Although from the results described in Example 2 above, Sam68 would appear to be specific for Rev and RRE , and not for CTE, such results are contradicted by experiments in which the retroviral coding element Gag was substituted for CAT (see Figure 1 0), and also experiments in which CTE is otherwise observed to bind Sam68 in vitro (data not shown). This suggests a broad-spectrum utility for Sam68 in modulating the expression of all retroviruses. p24 antigen capture assay. 293T cells were co-transfected using the

CaPO⁴ transfection method. Unless otherwise indicated, the following amounts were transfected: 0.25 or 0.4 μg HIV-1 rev(-) proviral DNA, 0.5 μg Sam68 Δ96, 0.025 μg Rev expression vector (Sadaie et a!., Science 239: 91 0-91 4 ( 1 988), 0.1 μg Sam68, 0.025 μg wild-type HXB-2. Where indicated, pcDNA3 was used to equalize the amount of DNA input for each transfection. Also where indicated, cells were incubated with 4 nM LMB 1 8 h after transfection. Then, 48 or 72 h after transfection, cell-free supernatants were collected and subjected to p24 antigen assay (Coulter, Hialeah, Florida), in vitro binding of Rev to Sam68. The same buffer ( 1 0 mM Tris, pH

8.0, 1 50 mM Na Cl, 1 mM EDTA containing 1 mM DTT, 1 mM PMSF and 5% glycerol) was used in all binding studies unless otherwise stated. Recombinant Rev protein (0.25 ug) was added to 25 ul of a fusion protein slurry of glutahione-bound GST-Sam68 ( 1 ug), GST-Sam68C'Δ330-443 ( 1 ug) or GST-Sam68 Δ1 -329 (50 ug); this was incubated at 4 C for 3 h in 25 ul column buffer. The beads were washed four times with 0.25 ml column buffer at each time. Washed beads were suspended in SDS buffer, boiled for 5 min and assessed by western blot analysis using rabbit polyclonal antibodies raised against Rev.

Indirect immunoflourescence. To determine the subcellular distribution of Sam68, Sam68C Δ330-443 and Rev, HeLa cells were transfected with 1 0 μg each of expression plasmids and cells were cultivated on glass slides. To assess the co-localization of the proteins, HeLa cells were transfected with Sam68C'Δ330-443 and either Sam68 or Rev at a ratio of 1 0 μg Sam68C'Δ330-443 to 1 μg expression plasmid. Cells were prepared for immunofluorescence as described in Reddy et al., Oncogene 1 4, 2785-2792 ( 1 997) . Confocal microscopy was done in a Bio Rad MRC 600 laser scanning confocal microscope attached to a Zeiss Aniovert 35M microscope and viewed using a 40xc 1 .3-na oil objective.

Discussion

We have demonstrated here that a cellular protein, Sam68, binds to Rev and RRE independently in vitro and in vivo and partially substitutes for Rev in several assays. This included reporter gene expression by co- transfection and microinjection as well as rescue of a Rev-defective proviral DNA clone (Figs. 1 , 2 and 4). The relative efficiency of Sam68 compared with that of Rev was greater in the RRE CAT assays than in the virus rescue assays, possibly because of the presence of multiple inhibitory nucleotide sequences in the proviral DNA. It is possible that Sam68 is not as effective as Rev in overcoming these inhibitory sequences and this difference is amplified in the virus assay. The Us1 1 protein of HSV-1 binds to and activates RRE and the HTLV-1 RxRE from an envelope expressing construct. However, unlike Sam68, it is unable to rescue the expression of a Rev- deficient HIV-1 . Sam68 was identified as a Src binding protein in mitosis, and belongs to a family of proteins that contain KH domains. The KH domain is highly conserved in several RNA binding proteins such as hnRNP-K, GRP33, fragile X mental retardation gene product FMR-1 and the C. elegans germline- specific tumor suppressor GLD-1 . The N terminus of Sam68 also contains an RGG box (a domain containing several Arg-Gly-Gly motifs), another characteristic of RNA binding proteins. However, the RGG box is dispensable for RNA binding and the multimerization of Sam68, as well as for the activation of RRE-mediated reporter gene expression and viral replication shown here. In contrast, a deletion in the KH domain completely abolished its RRE-transactivation activity indicating that RNA binding as well as multimerization through the KH domain may be important for the observed transactivation (and corresponding transinhibition when the C-terminal mutants of the invention are used in conjunction therewith).

Sam68/RRE-mediated transactivation is insensitive to leptomycin B. The differential sensitivity of Rev and Sam68 to LMB indicates that they use two distinct nuclear export pathways, which is consistent with the synergistic effects seen when both are overexpressed. RevM 10 inhibits both Rev and Sam68 activities. Perhaps RevM 10 either competes with Sam68 for binding to RRE or multimerizes with Sam68 to form an inactive complex. Inhibition of Sam68 function by olomoucine further indicates that phosphorylation of threonine residue(s) is important for its transactivating function. Phosphorylation of Sam68 at tyrosine or serine/threonine residues it tightly coupled with cell cycle progression, thus raising the possibility that post transcriptional regulation of HIV may also be cell cycle dependent. The relative activities of Sam68 and Rev overexpression varied, but exogenous Sam68 was found to be as active as Rev in the microinjection experiments, in which cells were routinely synchronized by serum starvation before being injected. Although the endogenous level of Sam68 is too low to support a high level of virus replication in the absence of Rev, it is conceivable that virus mutants that use this pathway more efficiently can emerge under selective pressure. Therefore, interventions directed at Rev alone may not be effective in the long term.

The C-terminal deletent and point mutants of Sam68 exerted a transdominant phenotype, and inhibited not only Sam68 transactivation of RRE, but also Rev function and wild-type HIV replication (Fig. 5). Unlike the transdominant mutant RevM 1 0, which localizes to the nucleus and competes with wild-type Rev for binding to RRE the CΔ330-443 Sam68 mutant is mainly cytoplasmic, and binds RRE very poorly (Figs. 6 and 7). However, it retains the ability to bind Rev (Fig. 6) . Therefore, its mechanism of inhibition seems to be trapping Rev in the cytoplasm by direct protein-protein interaction, as shown by confocal microscopy (Fig. 7). The multimerization domain of Rev residues in the basic domain, a region that also contains the nuclear localization sequence. It is possible that the complex formation of Sam68 and Rev results in masking the Rev nuclear localization sequence, such that the nuclear localization sequence in Sam68 at the C terminus is required for nuclear import of the complex. In contrast, the wild-type Sam68 and CΔ330-443 complex was localized in the nucleus, indicating that the nuclear localization sequence and multimerization domains are non- overlapping. However, this complex is not functional, as CΔ330-443 also inhibits wild-type Sam68 transactivation. The mechanism of this inhibition remains to be determined. Transdominant viral proteins have been exploited for antiviral gene therapy. In particular, transfer of the RevM 10 gene into primary lymphocytes effectively inhibit HIV-1 replication in vitro and prolongs cell survival in patients. In parallel, gene transfer of RevM 1 0 into hematopoietic stem cells is also being pursued as a gene therapy strategy. One potential problem with Rev M 1 0 and any others transdominant viral proteins is its immunogenicity. Cells chronically expressing such proteins are likely to be targeted by immunosurveillance of the host. Thus, a transdominant cellular protein, if it is not toxic to the cell, would be preferable. Preliminary indications are favorable, at least in cell culture.

II. Prophetic Examples

1 . Pharmaceutical Compositions

Those of skill in the art are familiar with the principles and procedures discussed in such widely known and available sources as Remington's Pharmaceutical Science, 1 7th Ed., Mack Publishing Co., Easton Pa. ( 1 985) and Goodman and Gilman's The Pharmacological Basis of Therapeutics, 8th Ed., Pergamon Press, Elmsford, New York (1 990), each of which is herein incorporated by reference.

Generally, compositions of the invention will comprise a therapeutically effective amount of a nucleic acid or gene product thereof encoding a cellular nuclear export protein of the invention in a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Various delivery systems are known and can be used to administer a therapeutic of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules and the like.

The composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings or other mammals. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ameliorate any pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. The therapeutics of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

The amount of the therapeutic of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, suitable dosage ranges for intravenous administration are generally about 20-4000 micrograms of active compound per kilogram body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

Those of skill in the art, applying routine pharmacological techniques, can readily determine a suitable formulation without exercising undue experimentation.

a. Gene Therapy

The pharmaceutical compositions of the invention need not be supplied in peptide or protein form, but instead may be administered as a nucleic acid species which can then be conveniently expressed in the afflicted host. This is especially so for embodiments that do not contemplate extraneous chemical modification that is not programmable at the nucleotide level. For example, those of skill know that certain destabilizing amino acid sequences can be introduced into a peptide, e.g., targeted protease cleavage points, such that the overall peptide is more readily degraded and does not persist to generate unwanted side-effects. The opposite is also true in that stabilizing amino acid may also be incorporated. Those of skill in the art are familiar with the possibilities. It is also envisioned that a genetic construct within the bounds of the invention may be capable of transient expression only, and that to the degree such expression is inadequate to completely fulfill the desired therapeutic function, additional transiently expressing constructs be administered to supplement or conclude the action. Thus, boluses of genetic construct may be delivered, just as may boluses of recombinant and/or purified gene product, e.g., native, modified, or synthetic Sam68.

The genetic constructs contemplated will embody any combination of DNA, RNA, hybrids thereof (referred hereinafter as nucleic acids) or chemically modified derivatives thereof that are operably linked to regulatory elements, e.g., promoters, enhancers, polyadenylation sequences, Kozak sequences, including initiation and stop codons, etc., needed for gene expression. Incorporation of such a DNA or RNA molecule into a living cell, by a variety of well-understood means, can result in the expression of nuclear cellular export protein and its desired effect within the cell.

Examples of promoters that may be useful in practice of such genetic applications include, but are not limited to Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV) such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV, Cytomegalovirus (CMV) such as the CMV immediate early promoter, Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters from other human genes such as human actin, human myosin, human hemoglobin, human muscle creatine and human metalothionein.

Examples of polyadenylation signals useful to practice the present invention, especially in the production of a genetic vaccine for humans, include but are not limited to SV40 polyadenylation signals and LTR polyadenylation signals. In particular, the SV40 polyadenylation signal that is in pCEP4 plasmid (Invitrogen, San Diego Calif.), referred to as the SV40 polyadenylation signal, can be used.

Examples of alternative enhancers may be selected from the group including but not limited to: human actin, human myosin, human hemoglobin, human muscle creatine and viral enhancers such as those from CMV, RSV and EBV.

In order to maximize protein production, regulatory sequences may be selected which are well suited for gene expression in the cells the construct is administered into. Moreover, codons may be selected which are most efficiently transcribed in the cell or tissue type, or mammalian host of interest, generally.

The genetic therapeutic may be administered directly into the individual or ex vivo into removed cells of the individual which are reimplanted after administration of the therapeutic product. By either route, the genetic material is introduced into cells that are present in the body of the individual. Routes of administration include, but are not limited to, intramuscular, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, intraoccular, and oral, as well as transdermally or by inhalation or suppository. Preferred routes of administration include intramuscular, intraperitoneal, intradermal and subcutaneous injection. Delivery of gene constructs that encode target proteins can confer mucosal immunity in individuals immunized by a mode of administration in which the material is presented in tissues associated with mucosal immunity. Thus, in some examples, the gene construct is delivered by administration in the buccal cavity within the mouth of an individual. Genetic constructs may be administered by means including, but not limited to, traditional syringes, needleless injection devices, or "microprojectiie bombardment gene guns" . Alternatively, the genetic vaccine may be introduced by various means into cells that are removed from the individual. Such means include, for example, ex vivo transfection, electroporation, microinjection and microprojectiie bombardment. After the genetic construct is taken up by the cells, they are reimplanted into the individual. It is contemplated that otherwise non- immunogenic cells that have genetic constructs incorporated therein can be implanted into the individual even if the vaccinated cells were originally taken from another individual. The genetic vaccines according to the present invention comprise about 1 nanogram to about 1000 micrograms of DNA. In some preferred embodiments, the vaccines contain about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the vaccines contain about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the vaccines contain about 1 to about 350 micrograms of DNA. In some preferred embodiments, the vaccines contain about 25 to about 250 micrograms of DNA. In some preferred embodiments, the vaccines contain about 100 micrograms DNA. The genetic vaccines according to the present invention are formulated according to the mode of administration to be used. One having ordinary skill in the art can formulate a genetic vaccine or therapeutic that comprises a genetic construct.

In cases where intramuscular injection is the chosen mode of administration, an isotonic formulation is preferably used. Generally, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstriction agent is added to the formulation. The pharmaceutical preparations according to the present invention are preferably provided sterile and pyrogen free.

b. Therapy By Administration of Purified Protein Protein purification has been made routine by the use of various short peptide tags that can be fused to the protein of interest at the nucleic acid level to facilitate protein purification via affinity chromatography directed against the tag. Such tags can be conveniently removed or, alternatively, left intact without normally affecting protein function. Many different tags exist and are embraced within the scope of this invention.

For example, the Flag octapeptide (Hopp et al., Bio/Technology 6: 1 204, 1 988; offered through Kodak, New Haven, Ct) can be positioned at the N-terminus and does not alter the biological activity of fusion proteins, is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody, enabling rapid detection and purification of the expressed fusion protein. The sequence is also specifically cleaved away by bovine mucosal enterokinase. A murine monoclonal antibody that binds the Flag sequence has been deposited with the ATCC under accession number HB 9259. Methods of using the antibody in purification of fusion proteins comprising the Flag sequence are described in U.S. Pat. No. 5,01 1 ,91 2, which is incorporated by reference herein.

Other types of linkers that can be used include, but are not limited to maltose binding protein (MBP), glutathione-S-transferase (GST), thioredoxin (TRX) and calmodulin binding protein (CBP). Kits for expression and purification of such fusion proteins are commercially available from, e.g., New England BioLabs (Beverly, Mass.), Pharmacia (Piscataway, N.J.), InVitrogen (Carlsbad, CA) and Stratagene (San Diego, CA), respectively.

In addition, it may be necessary to add between the individual portions of the hybrid protein a "linker" or "spacer" as is known in the art to ensure that the proteins form proper secondary and tertiary structures so as to endow the full-length molecule to be functional as a CD 1 4 receptor. Suitable linker sequences will adopt a flexible extended conformation, will not exhibit a propensity for developing an ordered secondary structure which could interact with the functional domains of fusion proteins, and will have minimal hydrophobic or charged character which could promote interaction with the functional protein domains. Typical surface amino acids in flexible protein regions include Gly, Asn and Ser. Virtually any permutation of amino acid sequences containing Gly, Asn and Ser would be expected to satisfy the above criteria for a linker sequence. Other near neutral amino acids, such as Thr and Ala, may also be used in the linker sequence. The length of the linker sequence may vary without significantly affecting the biological activity of the fusion protein. Exemplary linker sequences are described in U.S. Pat. Nos. 5,073,627 and 5, 1 08,91 0, herein incorporated by reference.

Described above are affinity chromatography methods of purification. Not to be overlooked as alternative or combined methodologies are those employing conventional growth and biochemical purification.

For example, supernatants from systems which secrete recombinant protein into culture media may be first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate may be applied to a suitable purification matrix. For example, a suitable affinity matrix may comprise a counter structure protein (i.e., a protein to which a polypeptide binds in a specific interaction based on structure) or antibody molecule bound to a suitable support. Alternatively, or conjunctively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred. Gel filtration chromatography also provides a means of purifying polypeptides.

Alternative Production and Modifications to the Proteins

It is envisioned that changes to the proteins and polypeptides of the invention other than as already described will also work, e.g., substitutions, deletions, and insertions. These changes can be incorporated at the nucleic acid level or else may be administered in therapy and pharmaceutical composition applications as purified proteins.

Preliminarily, it is to be expected that conservative additions may be made that preserve function. A "conservative substitution" in the context of the subject invention is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged for these regions. Other such conservative substitutions, e.g., include substitutions of entire regions having similar hydrophobicity characteristics, are well known. Mutagenic techniques for such replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Pat. No. 4,51 8,584).

a. Chemical Synthesis

In certain embodiments, polypeptides of the invention may be prepared synthetically. Synthetic formation of the polypeptide or protein requires chemically synthesizing the desired chain of amino acids by methods well known in the art. Chemical synthesis of a peptide is conventional in the art and can be accomplished, for example, by the Merrifield solid phase synthesis technique [Merrifield, J., Am. Chem. Soc, 85: 21 49-21 54 ( 1 963); Kent et al., Synthetic Peptides in Biology and Medicine, 29 f.f. eds. Alitalo et al., (Elsevier Science Publishers 1 985); and Haug, J. D., "Peptide Synthesis and Protecting Group Strategy", American Biotechnology Laboratory, 5 ( 1 ): 40-47 (January/February. 1 987)]. Techniques of chemical peptide synthesis include using automatic peptide synthesizers employing commercially available protected amino acids, for example, Biosearch [San Rafael, Calif. (USA)] Models 9500 and 9600; Applied Biosystems, Inc. [Foster City, Calif. (USA)] Model 430; Milligen [a division of Millipore Corp.; Bedford, Mass. (USA)] Model 9050; and Du Pont's RAMP (Rapid Automated MultiplePeptide Synthesis) [Du Pont Compass, Wilmington, Del. (USA)]. Generally, however, such synthesis is expensive, and with limitations in the length of the peptides which can be produced ( ^" 50-1 00 amino acid residues), and therefore is not preferred. Allowance is made, however, for advances in the field that might facilitate or promote this means of synthesis in use of the invention.

b. Derivatization

Whether synthesis is performed chemically or making use of recombinant techniques, it may be desirable to further modify the polypeptide backbone prior to use as a diagnostic or therapeutic agent. Covalent modifications of the protein or peptide are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.

For example, cysteinyl residues react with alpha-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, alpha. -bromo- . beta. (5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl d isulf ide, p- chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo- 2-oxa-1 ,3-diazole.

Another amino acid, histidine, is easily derivatized by reaction with diethylprocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Parabromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysine and amino terminal residues may be reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.

Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1 ,2- cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high PK of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.

Tyrosyl residues are well-known targets of modification for introduction of spectral labels by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizol and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R'-N-C-N-R') such as 1 -cyclohexyl-3-(2- morpholinyl(4-ethyl) carbodiimide or 1 -ethyl-3-(4-azonia-4,4-dimethyipentyl) carbodiimide.

Aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions. Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions.

Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains (Creighton, T. E., PROTEINS: STRUCTURE AND MOLECULAR PROPERTIES, W. H. Freeman & Co., San Francisco, pp. 79-86 (1 983)), acetylation of the N-terminal amine, and amidation of the C-terminal carboxyl groups.

Such derivatized moieties may improve the solubility, absorption, biological half life, and the like. The moieties may alternatively eliminate or attenuate any undesirable side effect of the protein. For additional useful discussion, the reader is directed to Remington's Pharmaceutical Sciences, 1 6th ed., Mack Publishing Co., Easton, Pa. (1 980), herein incorporated by reference.

3. Transgenic Organisms

One particularly useful application of the cellular nuclear export proteins of the invention is that they can be used in transgenic animals, e.g., mice, to model different retroviral-mediated diseases, e.g., HIV-1 mediated AIDS, and to otherwise employ the general inventive aspects of the invention, i.e., developing specific and general therapies and enhancing retroviral delivery and expression means for transgenes.

Transgenic mice are achieved routinely in the art using the technique of microinjection, as described in U.S. Patent No. 4,736,866 issued to Leder et al., and as provided by B. Hogan et al. entitled "Manipulating the Mouse Embryo: A Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., USA ( 1 986). U.S. Patent No. 5,574,206 issued to Jolicoeur particularly describes the creation of transgenic mice bearing functional HIV genes and their use in the modeling and study of HIV-mediated diseases. These references are herein incorporated by reference. Species-specific barriers currently impede successful human HIV infection, reproduction, and hence modeling, in the murine system. It is thought that these impediments occur at three levels: infection ( via CD4, CXCR4 and CCR5 receptor differences), transcription (via Cyclin T), and post-transcription. As discussed in a recent review by Tang et al., Lentivirus Replication and Regulation, Annu. Rev. Genet. 33: 1 33-1 70 ( 1 999), significant progress has been made respecting the first two levels. The Applicants believe that cellular nuclear export proteins such as Sam68 hold the key to solving the third level of impediment, and cite as support their in vitro data depicted in Figures 8 and 9 showing enhanced simulated retrovirus expression in murine cells co-transfected with human Sam68.

In light of these findings, the feasibility of generating improved transgenic mouse models for the study of HIV and retrovirus life cycles, etiology, disease pathology, and treatment is self evident.

Improved Transgene Expression Using Retroviral Vectors

Retroviruses are finding increasing importance as vectors because of their unique ability to infect and transduce a broad spectrum of non-dividing cell types, e.g., brain, liver, and hematopoietic cells. The ability to enhance retroviral protein production via the co-supply of cellular nuclear export proteins like Sam68 therefore bodes large for the fields of transgenics, i.e., gene therapy, and commercial retrovirus vector development and production. Examples of the use of retroviruses for delivery of transgenes include

Miyoshi et al., Science 283: 682-86 (1 999); Naldini et al., Science 272: 263-67 (1 996); Poeschla et al., Proc. Natl. Acad. Sci. USA 93: 1 1 395-99 (1 996); Poeschla et al., Nat. Med. 4:354-57 ( 1 998); and Poeschla et al., J. Virol. 72: 6527-36 ( 1 998). Using the general direction provided in such references, and of which those of ordinary skill in the art are aware, coupled with the novel features of Sam68 described herein, improved retroviral vectors, transgenic strategies, methodologies, and results can be achieved.

5. Experimental Uses Employing Attachment to Solid-Supports ln certain applications of the invention, e.g., kits, the cellular nuclear export proteins of the invention may be useful in an affinity screening approach in which they are affixed to a solid support matrix for diagnostic or other experimental purposes. This has already been described in the specific examples above, but is further elaborated here.

Derivatization with bifunctional agents is useful for cross-linking polypeptides to a water-insoluble support matrix or to other macromolecular carriers in preparation for affinity chromatography and other diagnostic and/or purification procedures. In this general manner, and in complementation to the 2- hybrid in vivo systems already described, additional cellular and viral entities important for retrovirus replication may also be identified.

Commonly used cross-linking agents include, for example, 1 , 1 - bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3, 3'- dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N- maleimido-1 ,8-octane. Derivatizing agents such as methyl-3-[p-azidophenyl) dithiolpropioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water- insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691 ,01 6; 4, 1 95, 1 28; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization. The linkages need not be mediated by covalent bonding, but may alternatively or conjunctively employ strong noncovalent bonding means (e.g., streptavidin-biotin) known to those of skill in the art.

6. Use Of Sam68 To Identify Other Useful Pathway Components

It is anticipated that other cellular and viral components that impinge on Sam68 biochemical pathways may be identified and manipulated in similar fashion to what has been described herein.

For example, the very manner in which the claimed utility of Sam68 was first discovered by the Applicants may itself be revisited using the various functional domains of Sam68 in the very yeast-2-hybrid studies that identified this utility in the first place. Yeast 2 hybrid screening is a very effective in vivo screening technique that allows functional assay complementation to identify or confirm protein:protein interactions. The technique was first reported by Fields et al., ( 1 989) Nature 340:245-247, and has since burgeoned as a tool of choice in the art. Numerous improvements and variations have also been made and are widely observed and understood by those of skill in the art. Using Sam68 in this general way, one can identify other useful cellular or viral factors that might be used to arrest or enhance splice-dependent processes, e.g., retroviral expression and replication.

All references cited in this application are herein incorporated by reference in their entireties including all drawings.

Preferred embodiments of the invention have been described above by example and by prophetic discussion understood by one of ordinary skill. It will be understood by those skilled in the field that additional modifications may be made to the disclosed embodiment without departing from the scope of the invention, which is defined by the appended claims.

WE CLAIM:

Claims

1 . A method of altering the expression of or from a retrovirus in a eukaryotic host cell, comprising: a) providing a eukaryotic host cell infected with a retrovirus, said retrovirus capable of producing an RNA transcript within said host cell, said transcript comprising a sequence capable of association with the protein corresponding to Seq. ID. No. 1 or a homolog thereof to promote expression in said host cell; and b) manipulating the activity of said protein or homolog thereof within said host cell to thereby alter said expression.

2. The method of claim 1 wherein said manipulating increases said expression within said host cell.

3. The method of claim 1 wherein said manipulating decreases said expression within said host cell.

4. The method of claim 2 wherein said manipulating raises the amount or activity of said protein or homolog thereof, or analog thereof, within said host cell.

5. The method of any of claims 1 - 4 accomplished by transfecting said host cell with a nucleic acid encoding said protein, homolog thereof, or analog thereof.

6. The method of claim 5 wherein said sequence capable of association comprises RRE or a functional equivalent thereof.

7. The method of any of claims 1 -6 wherein said manipulation has a dominant negative effect on said expression of or from said retrovirus within said host cell.

8. The method of claim 7 wherein said manipulation comprises modifying the carboxy terminal portion of said protein or homolog thereof, and wherein said manipulation results in a reduced ability to transport incompletely spliced retrovirus transcripts from the nucleus of said host cell to the cytoplasm of said host cell.

9. The method of claim 8 wherein said manipulation impedes the nuclear localization or shuttling ability of said protein or homolog thereof thereby inhibiting the expression of or from said retrovirus within said host cell.

1 0. The method of claim 9 wherein said manipulation is further capable of impeding the nuclear localization or shuttling of a viral nuclear export protein.

1 1 . The method of any of claims 8-1 0 wherein said manipulation comprises eliminating one or more amino acids from within the terminal span of amino acid residues beginning at about residue 330 in said protein, homolog, or analog thereof.

1 2. The method of any of claim 8-10 wherein said manipulation comprises eliminating one or more amino acids from within the terminal span of amino acid residues beginning at about residue 410 in said protein, homolog, or analog thereof.

1 3. The method of any of claims 8-10 wherein said manipulation comprises eliminating one or more residues from about the C-terminal one third of said protein, homolog thereof, or analog thereof.

1 4. The method of any of claims 1 -1 3 wherein said protein or homolog thereof possesses a KH domain or equivalent thereof, and wherein the function of said domain is substantially preserved in any analog of said protein or homolog thereof that is used according to said method.

1 5. The method of claim 1 3 wherein at least one residue that is eliminated is proline 439.

1 6. The method of claim 1 5 wherein proline 439 is eliminated through a point mutation made to a nucleic acid sequence that encodes said protein, homolog thereof, or analog thereof.

1 7. The method of any of claims 1 -1 6 wherein said host cell is selected from the group consisting of human and murine, and wherein said retrovirus is selected or derived from the group consisting of lentiviruses and oncoviruses.

1 8. The method of claim 1 7 wherein said retrovirus is a human immunodeficiency virus.

1 9. The method of claim 1 8 wherein said human immunodeficiency virus is HIV-1 .

20. The method of claim 1 7 wherein said host cells are part of a transgenic organism.

21 . A eukaryotic host cell transgenic for the protein of Seq. ID. No. 1 , homolog thereof, or analog thereof, and wherein said cell is used for determining or altering the expression of or from a splice-dependent mechanism in said cell.

22. The transgenic host cell of claim 21 wherein said mechanism comprises expression of or from a retrovirus.

23. The transgenic host cell of claim 21 or 22 wherein said cell is a mammalian cell.

24. A transgenic organism bearing the cell of any of claims 21 -23.

25. The transgenic organism of claim 24 wherein said organism is a mouse.

26. A recombinant nucleic acid capable of expression in a eukaryotic cell and encoding the protein of Seq. ID. No. 1 , or a homolog or analog thereof, and wherein said nucleic acid is used to determine or alter the expression of or from a splice-dependent mechanism in said cell.

27. A purified, recombinant, or synthetic protein of Seq ID. No. 1 , or a homolog or analog thereof, used to modulate a splice-dependent process in a eukaryotic host cell.

28. The recombinant nucleic acid or protein or homolog or analog thereof of claims 26 or 27 that encodes or comprises a modification relative to the C-terminal one third of Seq ID. No. 1 .

29. The recombinant nucleic acid or protein or homolog or analog thereof of claim 28 wherein said modification comprises elimination of proline 439.

30. The transgenic cell, recombinant nucleic acid or protein, or homolog, analog, or derivative thereof of any of claims 21 -29 that is supplied as part of a kit.