WO2008068765A2

WO2008068765A2 - Compositions and methods for inhibiting hiv-1 replication and integrase activity

Info

Publication number: WO2008068765A2
Application number: PCT/IL2007/001516
Authority: WO
Inventors: Assaf Friedler; Abraham Loyter; Moshe Kotler; Amnon Hizi; Zvi Hayouka; Joseph Rosenbluh; Aviad Levin; Shoshana Loya; Elena Britan
Original assignee: Yissum Research Development Company Of The Hebrew University Of Jerusalem; Ramot At Tel-Aviv University Ltd.
Priority date: 2006-12-07
Filing date: 2007-12-09
Publication date: 2008-06-12
Also published as: WO2008068765A3

Abstract

The present invention provides isolated peptides comprising a fragment of an HIV-1 Rev protein and use of same for treating HIV-1 infection, inhibiting HIV-1 replication and inhibiting DNA binding and 3 '-end processing activity of HIV-1 integrase protein.

Description

COMPOSITIONS AND METHODS FOR INHIBITING HIV-I REPLICATION AND

INTEGRASE ACTIVITY

FIELD OF THE INVENTION

The present invention provides isolated peptides comprising a fragment of an HIV-I Rev protein and use of same for treating HIV-I infection, inhibiting HIV-I replication and inhibiting DNA binding and 3 '-end processing activity of HIV-I integrase protein.

BACKGROUND OF THE INVENTION

Human Immunodeficiency Virus 1 (HIV-I) Rev and Integrase (IN) proteins are required within the nuclei of infected cells in the late and early phases of the viral replication cycle, respectively. HIV-I Rev is a karyophilic protein, which is required at the late phase of the viral life cycle for promoting nuclear export of partially-spliced or un-spliced viral RNA.

Following penetration of HIV-I into the host cell, reverse transcription of the viral RNA produces cDNA, which is then integrated into the host chromosome. The integration of the viral cDNA is an essential early step in the HIV-I life cycle. This reaction is catalyzed by the viral integrase (IN), a 32-kDa protein that is an integral part of the viral pre-integration complex (PIC). The IN protein is encoded by the viral pol gene and is translated as part of a large Gag-Pol polyprotein, which is processed by the viral protease (PR).

For integration to occur, the viral IN must recognize specific sequences in the viral cDNA, at the termini of the long terminal repeat (LTR) elements. Retroviral integration proceeds in two steps: in the first, 3 '-end processing, a dinucleotide is removed from the 3' end. This reaction occurs in the cytoplasm, within the PIC. In the next step, after entering the nucleus, the processed viral double-stranded DNA is joined to the host target DNA by an IN-mediated strand-transfer reaction.

Due to its central role in HIV replication, the IN protein is an attractive target for antiviral therapy. Moreover, probably no cellular counterpart of IN exists in human cells and therefore, IN inhibitors will not interfere with normal cellular processes. However, only a few IN inhibitors have been identified to date. Specific domains within viral proteins are responsible for their interaction with host-cell receptors and with other viral and cellular proteins enabling the completion of the viral propagation cycle within the host cell (1,2). Peptides derived from these binding domains may interfere with virus-host and virus-virus protein interactions and as such are excellent candidates as therapeutic agents. Using this approach, short peptides that inhibit IN enzymatic activity were obtained following analysis of the interaction between two of the HIV-I proteins, RT and IN. Screening a complete library of RT-derived peptides demonstrated that two domains of about 20 amino acids mediate this interaction. Peptides bearing these amino acid sequences blocked IN enzymatic activities in vitro (3).

A limited number of IN inhibitory peptides have already been described. Using a combinatorial peptide library, a hexapeptide was selected bearing the sequence HCKFWW (SEQ ID NO: 18) that inhibited the 3 '-processing and integration activity of IN (11). Based on the observation that this peptide also inhibited the IN from HIV-2, FIV, and MLV, it was suggested that a conserved region around the catalytic domain of IN is being targeted. An IN inhibitory peptide was also selected using a phage-display library (12). IN-derived peptides that interfered with its oligomerization also blocked its enzymatic activity (13). Several other inhibitory peptides have been described in the last few years. Other studies described IN inhibitory peptides with anti- HIV-I activity in some cell types (9, 14, 15). PCT7IL2007/001321 describes IN inhibitory peptides derived from LEDGF/p75 protein. None of these references describes the Rev protein derived IN inhibitory peptides of the present invention.

The inclusion or description of literary references in this section or any other part of this application does not constitute an admission that the references are regarded as prior art to this invention.

SUMMARY OF THE INVENTION

The present invention provides isolated peptides comprising a fragment of an HIV-I Rev protein and use of same for treating HIV-I infection, inhibiting HIV-I replication and inhibiting DNA binding and 3 '-end processing activity of HIV-I integrase protein. Specific examples of Rev-derived peptides having integrase-inhibiting activities in accordance with the invention are: SGDSDEELLKTVRLI (SEQ ID NO: 10); DEELLKTVRLIKFLY (SEQ ID NO: 11); LKTVRLIKFLYQSNP (SEQ ID NO: 12); QRQIRSISGWILSTY (SEQ ID NO: 15); RSISGWILSTYLGRP (SEQ ID NO: 7); GWILSTYLGRPAEPV (SEQ ID NO: 16); LKTVRLIKFLY (SEQ ID NO: 6), and derivatives of any of the above

In one embodiment, the present invention provides an isolated fragment of an HIV-I Rev protein, wherein the fragment of an HIV-I Rev protein is 15-25 amino acids in length, and the isolated sequence of the fragment of an HIV-I Rev protein comprises the sequence DEELLKTVRLIKFLY (SEQ IDNO: 11).

In another embodiment, the present invention provides an isolated fragment of an HIV-I Rev protein, wherein the isolated fragment of an HIV-I Rev protein is 6-25 amino acids in length, and the sequence of the isolated fragment of an HIV-I Rev protein comprises a portion of the sequence DEELLKTVRLIKFLY (SEQ ID NO: 11). In another embodiment, the portion is 6-14 amino acids in length. In another embodiment, the portion of SEQ ID NO: 11 comprises LKTVRLIKFLY (SEQ ID NO: 6). In another embodiment, the portion of SEQ ID NO: 11 is another portion of SEQ ID NO: 11. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a mutated fragment of an HIV-I Rev protein, wherein the mutated fragment of an HIV-I Rev protein is 15-25 amino acids in length, and the sequence of the mutated fragment of an HIV-I Rev protein comprises a mutated version) of the sequence DEELLKTVRLIKFLY (SEQ ID NO: 11), wherein the mutated version comprises 1-3 amino acid modifications relative to SEQ ID NO: 11. Preferably, each of the amino acid modifications is independently selected from the group consisting of a substitution, an insertion, and a deletion. In another embodiment, the mutated fragment of an HIV-I Rev protein is an isolated mutated fragment of an HIV-I Rev protein. In another embodiment, the length of the mutated fragment of an HIV-I Rev protein is 13- 25AA. In another embodiment, the mutated fragment of an HIV-I Rev protein comprises a mutated version of a portion of SEQ ID NO: 11; e.g. a portion 6-14 amino acids in length. Each possibility represents a separate embodiment of the present invention.

As provided herein, the present invention shows that the 3 negatively charged residues of SEQ ID NO: 11 can be eliminated, in order to increase the peptide's cell permeability, without compromising its activity. In the present case, this was accomplished by truncating the 4 amino- terminal residues of the peptide. Those skilled in the art will recognize that this could have be accomplished equally effectively by introducing point mutations that mutated these residues to neutral ones, e.g. alanine, leucine, isoleucine, etc.

In another embodiment, the present invention provides an isolated fragment of an HIV-I Rev protein, wherein the fragment of an HIV-I Rev protein is 12-25 amino acids in length, and the isolated sequence of the fragment of an HIV-I Rev protein comprises the sequence LKTVRLIKFLY (SEQ ID NO: 6). In another embodiment, the isolated fragment of an HIV-I Rev protein consists of LKTVRLIKFLY (SEQ ID NO: 6). Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides an isolated fragment of an HIV-I Rev protein, wherein the fragment of an HIV-I Rev protein is 6-25 amino acids in length, and the isolated sequence of the fragment of an HIV-I Rev protein comprises a portion of the sequence LKTVRLIKFLY (SEQ ID NO: 6). In another embodiment, the portion is 6-10 amino acids in length. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a mutated fragment of an HIV-I Rev protein, wherein the mutated fragment of an HIV-I Rev protein is 11-25 amino acids in length, and the sequence of the mutated fragment of an HIV-I Rev protein comprises a mutated version of the sequence LKTVRLIKFLY (SEQ ID NO: 6), wherein the mutated version comprises 1-3 amino acid modifications relative to SEQ ID NO: 6. Preferably, each of the amino acid modifications is independently selected from the group consisting of a substitution, an insertion, and a deletion. In another embodiment, the mutated fragment of an HIV-I Rev protein comprises a mutated version of a portion of SEQ ID NO: 6; e.g. a portion 6-10 amino acids in length. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides an isolated fragment of an HIV-I Rev protein, wherein the fragment of an HIV-I Rev protein is 15-25 amino acids in length, and the sequence of the fragment of an HIV-I Rev protein comprises the sequence RSISG WILSTYLGRP (SEQ ID NO: 7). In another embodiment, the present invention provides an isolated fragment of an HIV-I Rev protein, wherein the fragment of an HIV-I Rev protein is 6-25 amino acids in length, and the sequence of the fragment of an HIV-I Rev protein comprises a portion of the sequence RSISGWILSTYLGRP (SEQ ID NO: 7). In another embodiment, the portion is 6-14 amino acids in length. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a mutated fragment of an HIV-I Rev protein, wherein (a) the mutated fragment of an HIV-I Rev protein is 15-25 amino acids in length; (b) the sequence of the mutated fragment of an HIV-I Rev protein comprises a mutated version of the sequence RSISGWILSTYLGRP (SEQ ID NO: 7); and (c) the mutated version of SEQ ID NO: 7 comprises 1-3 amino acid modifications relative to SEQ ID NO: 7. Preferably, each of the amino acid modifications is independently selected from the group consisting of a substitution, an insertion, and a deletion. In another embodiment, the mutated fragment of an HIV-I Rev protein comprises a mutated version of a portion of SEQ ID NO: 7; e.g. a portion 6-14 amino acids in length. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides an isolated fragment of an HIV-I Rev protein, wherein the fragment of an HIV-I Rev protein is 15-25 amino acids in length, and the sequence of the fragment of an HIV-I Rev protein comprises the sequence LKTVRLIKFL YQSNP (SEQ IDNO: 12).

In another embodiment, the present invention provides an isolated fragment of an HIV-I Rev protein, wherein the fragment of an HIV-I Rev protein is 6-25 amino acids in length, and the sequence of the fragment of an HIV-I Rev protein comprises a portion of the sequence LKTVRLIKFLYQSNP (SEQ ID NO: 12). In another embodiment, the portion is 6-14 amino acids in length. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides an isolated fragment of an HIV-I Rev protein, wherein the fragment of an HIV-I Rev protein is 15-25 amino acids in length, and the sequence of the fragment of an HIV-I Rev protein comprises the sequence QRQIRSISG WlLSTY (SEQ lD NO: 15). In another embodiment, the present invention provides an isolated fragment of an HIV-I Rev protein, wherein the fragment of an HIV-I Rev protein is 6-25 amino acids in length, and the sequence of the fragment of an HIV-I Rev protein comprises a portion of the sequence QRQIRSISGWILSTY (SEQ ID NO: 15). In another embodiment, the portion is 6-14 amino acids in length. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a mutant fragment of an HIV-I Rev protein, wherein (a) the mutant fragment of an HIV-I Rev protein is 15-25 amino acids in length; (b) the sequence of the mutant fragment of an HIV-I Rev protein comprises a mutant version of the sequence LKTVRLIKFLYQSNP (SEQ ID NO: 12); and (c) the mutant version of SEQ ID NO: 12 comprises 1-3 amino acid modifications relative to SEQ ID NO: 12. Preferably, each of the amino acid modifications is independently selected from the group consisting of a substitution, an insertion, and a deletion. In another embodiment, the mutant fragment of an HIV-I Rev protein comprises a mutant version of a portion of SEQ ID NO: 12; e.g. a portion 6-14 amino acids in length. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a mutant fragment of an HIV- 1 Rev protein, wherein (a) the mutant fragment of an HIV-I Rev protein is 15-25 amino acids in length; (b) the sequence of the mutant fragment of an HIV-I Rev protein comprises a mutant version of the sequence QRQIRSISGWILSTY (SEQ ID NO: 15); and (c) the mutant version of SEQ ID NO: 15 comprises 1-3 amino acid modifications relative to SEQ ID NO: 15. Preferably, each of the amino acid modifications is independently selected from the group consisting of a substitution, an insertion, and a deletion. In another embodiment, the mutant fragment of an HIV-I Rev protein comprises a mutant version of a portion of SEQ ID NO: 15; e.g. a portion 6-14 amino acids in length. Each possibility represents a separate embodiment of the present invention.

Preferably, the Rev derived peptides are selected from DEELLKTVRLIKFLY, LKTVRLIKFLY, or RSISGW1LSTYLGRP; and derivatives thereof.

In another embodiment, the present invention provides a peptide having a sequence selected from SGDSDEELLKTVRLI (SEQ ID NO: 10); DEELLKTVRLIKFLY (SEQ ID NO: 11); LKTVRLIKFLYQSNP (SEQ ID NO: 12); QRQIRSISGWILSTY (SEQ ID NO: 15); RSISGWILSTYLGRP (SEQ ID NO: 7); GWILSTYLGRPAEPV (SEQ ID NO: 16); LKTVRLIKFLY (SEQ ID NO: 6), and one of the following derivatives thereof, wherein the derivative exhibits integrase inhibiting properties:

(1) a sequence of any of the above peptides, wherein at least one amino acid has been replaced by a naturally or non-naturally occurring amino acid, or by an organic peptidomemetic moiety;

(2) a sequence of any of the above peptides, wherein at least one side chain of the amino acid has been chemically modified;

(3) a sequence of any of the above peptides, wherein at least one terminal or non terminal amino acid has been deleted;

(4) a sequence of any of the above peptides, wherein at least one peptidic bond has been replaced by a non- naturally occurring peptidic bond;

(5) a sequence of any of the above peptides, wherein at least one of the amino acids is replaced by the corresponding D- amino acid;

(6) a sequence being the sequence of any of the above peptides, in reverse order, preferably having all D-amino acids (retro inverso) ; and

(7) a derivative having a combination of two or more of the derivations described in (l)-(6);

(8) a fragment of any one of (1-7) above, having at least 5 amino acids

The present invention further concerns pharmaceutical compositions comprising a pharmaceutically acceptable carrier and as an active ingredient at least one of the peptides, or the compounds as defined above.

Preferably the pharmaceutical compositions are for the inhibition of the replication of or treatment of HIV.

Other objects, features and advantages of the present invention will become clear from the following description and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: In vivo interaction between HIV-I Rev and IN as visualized by BiFC assay in yeast. A-L. Yeast cells were transfected with the indicated plasmids and following incubation as described in Experimental Procedures, were visualized by fluorescent confocal microscopy (A-G and I, K) or by phase confocal microscopy (H, J₃ L). Note: B, D and F are a magnification of A, C and E respectively. M. HIV-I replication in cells over-expressing Rev-GFP. HEK293T cells were transfected with 10 μg Rev-GFP or GFP or were mock-transfected. 36h post-transfection, cells were infected with HIV-1/VSV-G ("mosaic") virus. Media containing newly produced viruses was sampled at 23, 46 and 72 hours post-infection, and samples were analyzed for infectivity by MAGI. Expression of transfected proteins at these time points was verified by fluorescence microscopy of duplicate non-infected cultures. For each series, first bar = Rev-GFP; second bar = GFP; third bar = no plasmid.

Figure 2: Interaction between Rev and IN in mammalian cells. HEK293T cells were transfected with Rev- and IN-encoding plasmids. Following cell lysis, a mabRev was added (A) or not (B) to the cell lysate. After immunoprecipitation and western blotting, membranes were stained with a polyclonal anti-IN antibodies.

Figure 3: In vitro binding of Rev and IN. Plates coated with Rev-GFP (A and B) were blocked with 5% BSA. Following washing, Bb-IN or Bb (biotin-labeled BSA) at the indicated concentrations (A) or 10 μM Bb-IN pre-incubated with various molar ratios of Rev-GFP or IN (B) were added. All other experimental conditions, including the estimation of bound biotin molecules, were as described in Experimental Procedures. (C) Purified GST-IN or GST were incubated with histidine-tagged Rev-GFP or GFP and after precipitation with glutathione beads and washing, were analyzed by Western blotting using a monoclonal anti-His antibody.

Figure 4: Peptide mapping of Rev-IN interaction. Binding of peptides derived from the Rev protein to IN. (A) Binding of long peptides: (o) Revl-30, (x) Rev31-48, (A) Rev49-74, (+) Rev74-93, (Δ) Rev94-116. (B) Binding of short peptides: (■) Revl3-23, (A) Rev53-67. Figure 5: Analysis of the effect of Rev-derived peptides on IN strand-transfer activity. IN

(0.8 μM) was incubated with the indicated peptides and its strand-transfer activity was analyzed as described in Experimental Procedures.

Figure 6: Rev-derived short peptides from the NIH library can inhibit IN catalytic activity. IN (0.8 μM) was incubated with the indicated peptides and its 3 '-processing (A) or strand- transfer activities (B) were analyzed as described in Experimental Procedures.

Figure 7: Cell penetration of Rev peptides. Fluorescein-labeled Revl3-23 (A) or Rev53-67 (B) (10 μM in each case) was incubated for 2 h at 37⁰C with HeLa cells. Cells were then washed three times with PBS and visualized with a fluorescence microscope.

Figure 8: Inhibition of HIV-I replication by Revl3-23 and Rev53-67 peptides. (A) TZM-bl cells were incubated with the indicated peptides at the indicated concentrations, HIV-I infected, and tested for β-galactosidase activity. (B) T-lymphoid H9 cells were incubated with the indicated peptides and after infection with HIV-I their P24 content was estimated. (C) SupTl T- lymphoid cells were incubated with the indicated peptides at the indicated concentrations and following HIV-I infection the percentage of integrated viral DNA was assessed. (D) Effect of peptides on cell toxicity, using the MTT assay. All other experimental conditions as described in Experimental Procedures.

Figure 9: Cell penetration and binding to the IN protein of the IN5 peptide, (a) 10 μM fluorescein-labeled IN5 peptide was incubated for 2 h in 37°C with HeLa cells. Cells were washed three times with PBS and visualized with a fluorescent microscope, (b) Anisotropy analysis of binding of IN5 peptide to full-length IN enzyme.

Figure 10: IN-binding Rev-derived peptides shift the IN oligomerization equilibrium towards the tetramer. Oligomerization of IN in the presence of the peptides was studied using analytical gel filtration. IN 1-288 (14 μM) alone eluted as a high order oligomer (leftmost peak). In the presence of 14 μM viral LTR DNA, IN eluted as a dimer (rightmost peak). In the presence of 14 μM Rev 13-23 or Rev 53-67, IN eluted as a tetramer (center peaks). Figure 11: IN-binding Rev-derived peptides inhibit DNA binding of IN. IN was titrated into fluorescein-labeled HIV-I LTR DNA (10 nM) alone (■) and in the presence of 1 μM of Rev 13- 23 (•) and Rev 53-67 (♦). Binding affinities and Hill coefficients are depicted in Table 3B.

Figure 12: Effect of relative order of addition and excess of the Rev derived peptides on IN catalytic activity. (A, B). When the viral LTR DNA was added to a preformed IN - peptide complex, with (A) Rev 13-23 (B) Rev 53-67, IN catalytic activity was significantly inhibited.

However, when Rev derived peptides were added to a pre-formed IN: DNA complex, IN catalytic activity was only slightly inhibited (at 1:1 molar ratio IN: Rev derived peptides). (C,

D). When the Rev-derived peptides (C) Rev 13-23 and (D) Rev 53-67 were added to a preformed IN-DNA complex, inhibition of IN catalytic activity was more pronounced with increasing peptide: IN ratio.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides isolated peptides comprising a fragment of an HIV-I Rev protein and use of same for treating HIV-I infection, inhibiting HIV-I replication and inhibiting DNA binding and 3 ' -end processing activity of HIV- 1 integrase protein.

In one embodiment, the present invention provides an isolated fragment of an HIV-I Rev protein, wherein the fragment of an HIV-I Rev protein is 15-25 amino acids in length, and the isolated sequence of the fragment of an HIV-I Rev protein comprises the sequence DEELLKT VRLIKFLY (SEQ ID NO: 11).

hi another embodiment, the present invention provides an isolated fragment of an HIV-I Rev protein, wherein the isolated fragment of an HIV-I Rev protein is 6-25 amino acids in length, and the sequence of the isolated fragment of an HIV-I Rev protein comprises a portion of the sequence DEELLKTVRLIKFLY (SEQ ID NO: 11). In another embodiment, the portion is 6-14 amino acids in length. In another embodiment, the portion of SEQ ID NO: 11 comprises LKTVRLIKFLY (SEQ ID NO: 6). In another embodiment, the portion of SEQ ID NO: 11 is another portion of SEQ ID NO: 11. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a mutant fragment of an HIV-I Rev protein, wherein the mutant fragment of an HIV-I Rev protein is 15-25 amino acids in length, and the sequence of the mutant fragment of an HIV-I Rev protein comprises a mutant version of the sequence DEELLKTVRLIKFLY (SEQ ED NO: 11), wherein the mutant version comprises 1-3 amino acid modifications relative to SEQ ID NO: 11. Preferably, each of the amino acid modifications is independently selected from the group consisting of a substitution, an insertion, and a deletion. In another embodiment, the mutant fragment of an HIV-I Rev protein is an isolated mutant fragment of an HIV-I Rev protein. In another embodiment, the length of the mutant fragment of an HIV-I Rev protein is 13- 25AA. In another embodiment, the mutated version of SEQ ED NO: 11 contains 1 amino acid modification relative to SEQ ID NO: 11. In another embodiment, 2 amino acid modifications are present. In another embodiment, 3 amino acid modifications are present. In another embodiment, the mutated version of SEQ ED NO: 11 contains no more than 3 amino acid modifications relative to SEQ ID NO: 11. In another embodiment, the mutant fragment of an HIV-I Rev protein comprises a mutant version of a portion of SEQ ID NO: 11 ; e.g. a portion 6-14 amino acids in length. E ach possibility represents a separate embodiment of the present invention.

As provided herein, the present invention shows that the 3 positively charged residues of SEQ ID NO: 11 can be eliminated, in order to increase the peptide's cell permeability, without compromising its activity. In the present case, this was accomplished by truncating the 4 amino- terminal residues of the peptide. Those skilled in the art will recognize that this could have be accomplished equally effectively by introducing point mutations that mutated these residues to neutral ones, e.g. alanine, leucine, isoleucine, etc.

In another embodiment, the present invention provides an isolated fragment of an HIV-I Rev protein, wherein the fragment of an HIV-I Rev protein is 12-25 amino acids in length, and the isolated sequence of the fragment of an HIV-I Rev protein comprises the sequence LKTVRLIKFLY (SEQ IDNO: 6). In another embodiment, the isolated fragment of an HIV-I Rev protein consists of LKTVRLIKFLY (SEQ ID NO: 6). Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides an isolated fragment of an HIV-I Rev protein, wherein the fragment of an HIV-I Rev protein is 6-25 amino acids in length, and the isolated sequence of the fragment of an HIV-I Rev protein comprises a portion of the sequence LKTVRLIKELY (SEQ ID NO: 6). In another embodiment, the portion is 6-10 amino acids in length. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a mutant fragment of an HIV-I Rev protein, wherein the mutant fragment of an HIV-I Rev protein is 11-25 amino acids in length, and the sequence of the mutant fragment of an HIV-I Rev protein comprises a mutant version of the sequence LKT VRLIKFLY (SEQ ID NO: 6), wherein the mutant version comprises 1-3 amino acid modifications relative to SEQ ID NO: 6. Preferably, each of the amino acid modifications is independently selected from the group consisting of a substitution, an insertion, and a deletion. In another embodiment, the mutant fragment of an HIV-I Rev protein is an isolated mutant fragment of an HIV-I Rev protein. In another embodiment, the mutated version of SEQ ID NO: 6 contains 1 amino acid modification relative to SEQ ID NO: 6. In another embodiment, 2 amino acid modifications are present. In another embodiment, 3 amino acid modifications are present. In another embodiment, the mutated version of SEQ ID NO: 6 contains no more than 3 amino acid modifications relative to SEQ ID NO: 6. In another embodiment, the mutant fragment of an HIV-I Rev protein comprises a mutant version of a portion of SEQ ID NO: 6; e.g. a portion 6-10 amino acids in length. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides an isolated fragment of an HIV-I Rev protein, wherein the fragment of an HIV-I Rev protein is 15-25 amino acids in length, and the sequence of the fragment of an HIV-I Rev protein comprises the sequence RSISGWILSTYLGRP (SEQ ID NO: 7).

In another embodiment, the present invention provides an isolated fragment of an HIV-I Rev protein, wherein the fragment of an HIV-I Rev protein is 6-25 amino acids in length, and the sequence of the fragment of an HIV-I Rev protein comprises a portion of the sequence RSISGWILSTYLGRP (SEQ ID NO: 7). In another embodiment, the portion is 6-14 amino acids in length. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a mutated fragment of an HIV-I Rev protein, wherein (a) the mutated fragment of an HIV-I Rev protein is 15-25 amino acids in length; (b) the sequence of the mutated fragment of an HIV-I Rev protein comprises a mutated version of the sequence RSISGWILSTYLGRP (SEQ ID NO: 7); and (c) the mutated version of SEQ ID NO: 7 comprises 1-3 amino acid modifications relative to SEQ ID NO: 7. Preferably, each of the amino acid modifications is independently selected from the group consisting of a substitution;, an insertion, and a deletion. In another embodiment, the mutated fragment of an HIV-I Rev protein is an isolated mutated fragment of an HIV-I Rev protein. In another embodiment, the mutated version of SEQ ID NO: 7 contains 1 amino acid modification relative to SEQ ID NO: 7. In another embodiment, 2 amino acid modifications are present. In another embodiment, 3 amino acid modifications are present. In another embodiment, the mutated version of SEQ ID NO: 7 contains no more than 3 amino acid modifications relative to SEQ ID NO: 7. In another embodiment, the mutant fragment of an HIV-I Rev protein comprises a mutant version of a portion of SEQ ID NO: 7; e.g. a portion 6-14 amino acids in length. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides an isolated fragment of an HIV-I Rev protein, wherein the fragment of an HIV-I Rev protein is 15-25 amino acids in length, and the sequence of the fragment of an HIV-I Rev protein comprises the sequence LKTVRLIKFL YQSNP (SEQ ID NO: 12).

In another embodiment, the present invention provides an isolated fragment of an HQV-I Rev protein, wherein the fragment of an HIV-I Rev protein is 6-25 amino acids in length, and the sequence of the fragment of an HIV-I Rev protein comprises a portion of the sequence LKTVRLIKFLYQSNP (SEQ ID NO: 12). In another embodiment, the portion is 6-14 amino acids in length. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides an isolated fragment of an HIV-I Rev protein, wherein the fragment of an HIV-I Rev protein is 15-25 amino acids in length, and the sequence of the fragment of an HIV-I Rev protein comprises the sequence QRQIRSISGWILSTY (SEQ ID NO: 15).

In another embodiment, the present invention provides an isolated fragment of an HIV-I Rev protein, wherein the fragment of an HTV-I Rev protein is 6-25 amino acids in length, and the sequence of the fragment of an HIV-I Rev protein comprises a portion of the sequence QRQIRSISGWILSTY (SEQ ID NO: 15). In another embodiment, the portion is 6-14 amino acids in length. Each possibility represents a separate embodiment of the present invention. In another embodiment, the present invention provides a mutated fragment of an HIV-I Rev protein, wherein (a) the mutated fragment of an HIV-I Rev protein is 15-25 amino acids in length; (b) the sequence of the mutated fragment of an HIV-I Rev protein comprises a mutated version of the sequence LKTVRLIKFLYQSNP (SEQ ID NO: 12); and (c) the mutated version of SEQ ID NO: 12 comprises 1-3 amino acid modifications relative to SEQ ID NO: 12. Preferably, each of the amino acid modifications is independently selected from the group consisting of a substitution, an insertion, and a deletion. In another embodiment, the mutated fragment of an HIV-I Rev protein is an isolated mutated fragment of an HIV-I Rev protein. In another embodiment, the mutated version of SEQ ID NO: 12 contains 1 amino acid modification relative to SEQ ID NO: 12. In another embodiment, 2 amino acid modifications are present. In another embodiment, 3 amino acid modifications are present. In another embodiment, the mutated version of SEQ ID NO: 12 contains no more than 3 amino acid modifications relative to SEQ ID NO: 12. In another embodiment, the mutant fragment of an HIV-I Rev protein comprises a mutant version of a portion of SEQ ID NO: 12; e.g. a portion 6-14 amino acids in length. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a mutated fragment of an HIV-I Rev protein, wherein (a) the mutated fragment of an HIV-I Rev protein is 15-25 amino acids in length; (b) the sequence of the mutated fragment of an HIV-I Rev protein comprises a mutated version of the sequence QRQIRSISGWILSTY (SEQ ID NO: 15); and (c) the mutated version of SEQ ID NO: 15 comprises 1-3 amino acid modifications relative to SEQ ID NO: 15. Preferably, each of the amino acid modifications is independently selected from the group consisting of a substitution, an insertion, and a deletion. In another embodiment, the mutated fragment of an HIV-I Rev protein is an isolated mutated fragment of an HIV-I Rev protein. In another embodiment, the mutated version of SEQ ID NO: 15 contains 1 amino acid modification relative to SEQ ID NO: 15. In another embodiment, 2 amino acid modifications are present. In another embodiment, 3 amino acid modifications are present. In another embodiment, the mutated version of SEQ ID NO: 15 contains no more than 3 amino acid modifications relative to SEQ ID NO: 15. In another embodiment, the mutant fragment of an HIV-I Rev protein comprises a mutant version of a portion of SEQ ID NO: 15; e.g. a portion 6-14 amino acids in length. Each possibility represents a separate embodiment of the present invention. It should be appreciated that only those Rev-derived peptides that can inhibit the integrase catalytic activity fall within the scope of the invention. Peptides than fall within the invention can be discovered by preparing a series of overlapping short sequences from the Rev protein, and screening for those that inhibit integrase, or those that inhibit HIV-I replication for example by using the assays disclosed herein.

In another embodiment, the length of the portion of SEQ ID NO: 7, 11, 12, or 15 or a mutated version of SEQ ID NO: 7, 11, 12, or 15 contained in the fragment of an HIV-I Rev protein of the present invention is 7-14 amino acids (AA). In another embodiment, the length is 7-13 AA. In another embodiment, the length is 7-12 AA. In another embodiment, the length is 7-11 AA. In another embodiment, the length is 7-10 AA. In another embodiment, the length is 7-9 AA. In another embodiment, the length is 7-8 AA. In another embodiment, the length is 6-13 AA. In another embodiment, the length is 6-12 AA. In another embodiment, the length is 6-11 AA. In another embodiment, the length is 6-10 AA. In another embodiment, the length is 6-9 AA. In another embodiment, the length is 6-8 AA. In another embodiment, the length is 6-7 AA. In another embodiment, the length is 8-12 AA. In another embodiment, the length is 8-11 AA. In another embodiment, the length is 8-10 AA. In another embodiment, the length is 8-9 AA. In another embodiment, the length is 9-12 AA. In another embodiment, the length is 9-11 AA. In another embodiment, the length is 9-10 AA. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the length of the portion of SEQ ID NO: 6 or a mutated version of SEQ ID NO: 6 contained in the fragment of an HIV-I Rev protein of the present invention is 6-9 amino acids. In another embodiment, the length is 6-8 AA. In another embodiment, the length is 6-7 AA. In another embodiment, the length is 7-10 AA. In another embodiment, the length is 7-9 AA. In another embodiment, the length is 7-8 AA. In another embodiment, the length is 8-10 AA. In another embodiment, the length is 8-9 AA. In another embodiment, the length is 9-10 AA. Each possibility represents a separate embodiment of the present invention.

In another embodiment, an amino acid modification in a mutated version of an HIV-I Rev sequence eliminates a negatively charged amino acid from the native HIV-I Rev sequence corresponding to the isolated fragment of an HIV-I Rev protein. In another embodiment, the modification eliminates a negatively charged amino acid from the isolated fragment of an HIV-I Rev protein. In another embodiment, 2 of the amino acid modifications in a mutated version of an HIV-I Rev sequence each eliminates a negatively charged amino acid from the native HIV-I Rev sequence. In another embodiment, all 3 of the amino acid modifications in a mutated version of an HIV-I Rev sequence eliminate a negatively charged amino acid from the native HIV-I Rev sequence. In another embodiment, the negatively charged amino acid is selected from the group consisting of aspartate and glutamate. Each possibility represents a separate embodiment of the present invention.

hi another embodiment, the present invention provides an isolated peptide comprising an isolated fragment of an HIV-I Rev protein of the present invention. Preferably, the isolated peptide is 12-100 amino acids in length. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides an isolated peptide comprising a mutant fragment of an HIV-I Rev protein of the present invention. Preferably, the isolated peptide is 12-100 amino acids in length. Each possibility represents a separate embodiment of the present invention.

"Isolated fragment of an HIV-I Rev protein" preferably refers to an HIV-I Rev fragment that is isolated from contiguous HIV-I Rev protein sequences. In another embodiment, the term refers to an HIV-I Rev fragment that is isolated from additional HIV-I Rev protein sequence other than the recited sequence. The term is not intended to exclude peptides that comprise, in addition to the recited HIV-I Rev fragment, additional non-HIV-1 Rev amino acid residues, either naturally occurring or non-naturally occurring.

In another embodiment, the present invention provides an isolated peptide comprising a fragment of a HIV-I Rev protein, wherein the fragment of a HIV-I Rev protein is 15-25 amino acids in length, and the sequence of the fragment of a HIV-I Rev protein comprises the sequence

DEELLKTVRLIKFLY (SEQ ID NO: 11). Preferably, the length of the isolated peptide is 15-100 amino acids. In another embodiment, the isolated peptide consists of the above-described fragment of a HIV-I Rev protein. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides an isolated peptide comprising a fragment of a HIV-I Rev protein, wherein the fragment of a HIV-I Rev protein is 11-25 amino acids in length, and the sequence of the fragment of a HIV-I Rev protein comprises the sequence LKTVRLIKFLY (SEQ ID NO: 6). Preferably, the length of the isolated peptide is 11-100 amino acids. In another embodiment, the isolated peptide consists of the above-described fragment of a HIV-I Rev protein. Each possibility represents a separate embodiment of the present invention.

RSISGWILSTYLGRP (SEQ ID NO: 7). Preferably, the length of the isolated peptide is 15-100 amino acids. In another embodiment, the isolated peptide consists of the above-described fragment of a HIV-I Rev protein. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides an isolated peptide comprising a fragment of a HIV-I Rev protein, wherein the fragment of a HIV-I Rev protein is 15-25 amino acids in length, and the sequence of the fragment of a HIV-I Rev protein comprises the sequence LKTVRLIKFLYQSNP (SEQ ID NO: 12). Preferably, the length of the isolated peptide is 15-100 amino acids. In another embodiment, the isolated peptide consists of the above-described fragment of a HIV-I Rev protein. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides an isolated peptide comprising a fragment of a HIV-I Rev protein, wherein the fragment of a HIV-I Rev protein is 15-25 amino acids in length, and the sequence of the fragment of a HIV-I Rev protein comprises the sequence QRQIRSISGWILSTY (SEQ ID NO: 15). Preferably, the length of the isolated peptide is 15-100 amino acids. In another embodiment, the isolated peptide consists of the above-described fragment of a HIV-I Rev protein. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides an isolated peptide comprising a fragment of a HIV-I Rev protein, wherein the fragment of a HIV-I Rev protein consists of residues 9-23 from SEQ ID NO: 31 or a corresponding fragment from a homologous Rev protein. Preferably, the length of the isolated peptide is 15-100 amino acids. In another embodiment, the isolated peptide consists of the above-described fragment of a HIV-I Rev protein. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides an isolated peptide comprising a fragment of a HIV-I Rev protein, wherein the fragment of a HIV-I Rev protein consists of residues 13-23 from SEQ ID NO: 31 or a corresponding fragment from a homologous Rev protein. Preferably, the length of the isolated peptide is 11-100 amino acids. In another embodiment, the isolated peptide consists of the above-described fragment of a HIV-I Rev protein. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides an isolated peptide comprising a fragment of a HIV-I Rev protein, wherein the fragment of a HIV-I Rev protein consists of residues 53-67 from SEQ ID NO: 31 or a corresponding fragment from a homologous Rev protein. Preferably, the length of the isolated peptide is 15-100 amino acids. In another embodiment, the isolated peptide consists of the above-described fragment of a HIV-I Rev protein. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides an isolated peptide comprising a fragment of a HIV-I Rev protein, wherein the fragment of a HIV-I Rev protein consists of residues 13-27 from SEQ ID NO: 31 or a corresponding fragment from a homologous Rev protein. Preferably, the length of the isolated peptide is 15-100 amino acids. In another embodiment, the isolated peptide consists of the above-described fragment of a HIV-I Rev protein. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides an isolated peptide comprising a fragment of a HIV- 1 Rev protein, wherein the fragment of a HIV- 1 Rev protein consists of residues 49-63 from SEQ ID NO: 31 or a corresponding fragment from a homologous Rev protein. Preferably, the length of the isolated peptide is 15-100 amino acids. In another embodiment, the isolated peptide consists of the above-described fragment of a HIV-I Rev protein. Each possibility represents a separate embodiment of the present invention.

As demonstrated herein, Rev fragments corresponding to residues 9-23, 13-23, 13-27, 49-63, and 53-67 inhibit HIV-I IN activity and HIV-I replication. In another embodiment, the present invention provides a peptide comprising a mutated fragment of an HIV-I Rev protein, wherein (a) the fragment of an HIV-I Rev protein is 15-25 amino acids in length; (b) the wild-type sequence of the fragment of an HIV-I Rev protein comprises the sequence DEELLKTVRLIKFLY (SEQ ID NO: 11); and (c) the mutated fragment of an HIV-I 5 Rev protein comprises 1-3 single amino acid modifications relative to SEQ ID NO: 11. The single amino acid modification preferably are independently selected from the group consisting of a substitution, an insertion, and a deletion. In another embodiment, the peptide is an isolated peptide. In another embodiment, the peptide consists of the above-described mutated fragment of an HIV-I Rev protein. In another embodiment, the fragment of an HIV-I Rev protein contains

10 residues other than those set forth in SEQ ID NO: 11. In another embodiment, if residues other than those set forth in SEQ ID NO: 11 are present, then the isolated peptide optionally comprises 1-2 additional single amino acid mutations in the residues other than those set forth in SEQ ID NO: 11. In another embodiment, the additional single amino acid mutations are independently selected from the group consisting of a substitution, an insertion, and a deletion. In another

15 embodiment, the wild-type sequence of the fragment of an HIV-I Rev protein comprises a portion of SEQ ID NO: 11; in this case, the fragment of an HIV-I Rev protein can be 6-25 amino acids in length. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a peptide comprising a mutated fragment of an HIV-I Rev protein, wherein (a) the fragment of an HIV-I Rev protein is 10-25 amino acids 0 in length; (b) the wild-type sequence of the fragment of an HIV-I Rev protein comprises the sequence LKTVRLIKFLY (SEQ ID NO: 6); and (c) the mutated fragment of an HIV-I Rev protein comprises 1-3 single amino acid modifications relative to SEQ ID NO: 6. The single amino acid modification preferably are independently selected from the group consisting of a substitution, an insertion, and a deletion. In another embodiment, the peptide is an isolated

-5 peptide. In another embodiment, the peptide consists of the above-described mutated fragment of an HIV-I Rev protein. In another embodiment, the fragment of an HIV-I Rev protein contains residues other than those set forth in SEQ ID NO: 6. In another embodiment, if residues other than those set forth in SEQ ID NO: 6 are present, then the isolated peptide optionally comprises 1-2 additional single amino acid mutations in the residues other than those set forth in SEQ ID

O NO: 6. In another embodiment, the additional single amino acid mutations are independently selected from the group consisting of a substitution, an insertion, and a deletion. In another embodiment, the wild-type sequence of the fragment of an HIV-I Rev protein comprises a portion of SEQ ID NO: 6; in this case, the fragment of an HIV-I Rev protein can be 6-25 amino acids in length. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a peptide comprising a mutated fragment of an HIV-I Rev protein, wherein (a) the fragment of an HIV-I Rev protein is 15-25 amino acids in length; (b) the wild-type sequence of the fragment of an HIV-I Rev protein comprises the sequence RSISGWILSTYLGRP (SEQ ID NO: 7); and (c) the mutated fragment of an HIV-I Rev protein comprises 1-3 single amino acid modifications relative to SEQ ID NO: 7. The single amino acid modification preferably are independently selected from the group consisting of a substitution, an insertion, and a deletion. In another embodiment, the peptide is an isolated peptide. In another embodiment, the peptide consists of the above-described mutated fragment of an HIV-I Rev protein. In another embodiment, the fragment of an HIV-I Rev protein contains residues other than those set forth in SEQ ID NO: 7. In another embodiment, if residues other than those set forth in SEQ ID NO: 7 are present, then the isolated peptide optionally comprises 1-2 additional single amino acid mutations in the residues other than those set forth in SEQ ID NO: 7. In another embodiment, the additional single amino acid mutations are independently selected from the group consisting of a substitution, an insertion, and a deletion. In another embodiment, the wild-type sequence of the fragment of an HIV-I Rev protein comprises a portion of SEQ ID NO: 7; in this case, the fragment of an HIV-I Rev protein can be 6-25 amino acids in length. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a peptide comprising a mutated fragment of an HIV-I Rev protein, wherein (a) the fragment of an HIV-I Rev protein is 15-25 amino acids in length; (b) the wild-type sequence of the fragment of an HIV-I Rev protein comprises the sequence LKTVRLIKFL YQSNP (SEQ ID NO: 12); and (c) the mutated fragment of an HIV-I Rev protein comprises 1-3 single amino acid modifications relative to SEQ ID NO: 12. The single amino acid modification preferably are independently selected from the group consisting of a substitution, an insertion, and a deletion. In another embodiment, the peptide is an isolated peptide. In another embodiment, the peptide consists of the above-described mutated fragment of an HIV-I Rev protein. In another embodiment, the fragment of an HIV-I Rev protein contains residues other than those set forth in SEQ ID NO: 12. In another embodiment, if residues other than those set forth in SEQ ID NO: 12 are present, then the isolated peptide optionally comprises 1-2 additional single amino acid mutations in the residues other than those set forth in SEQ ID NO: 12. In another embodiment, the additional single amino acid mutations are independently selected from the group consisting of a substitution, an insertion, and a deletion. In another 5 embodiment, the wild-type sequence of the fragment of an HIV-I Rev protein comprises a portion of SEQ ID NO: 12; in this case, the fragment of an HIV-I Rev protein can be 6-25 amino acids in length. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a peptide comprising a mutated fragment of an HIV-I Rev protein, wherein (a) the fragment of an HIV-I Rev protein is 15-25 amino acids0 in length; (b) the wild-type sequence of the fragment of an HIV-I Rev protein comprises the sequence QRQIRSISGWILSTY (SEQ ID NO: 15); and (c) the mutated fragment of an HIV-I Rev protein comprises 1-3 single amino acid modifications relative to SEQ ID NO: 15. The single amino acid modification preferably are independently selected from the group consisting of a substitution, an insertion, and a deletion. In another embodiment, the peptide is an isolated5 peptide. In another embodiment, the peptide consists of the above-described mutated fragment of an HIV-I Rev protein. In another embodiment, the fragment of an HIV-I Rev protein contains residues other than those set forth in SEQ ID NO: 15. In another embodiment, if residues other than those set forth in SEQ ID NO: 15 are present, then the isolated peptide optionally comprises 1-2 additional single amino acid mutations in the residues other than those set forth in SEQ ID NO: 15. In another embodiment, the additional single amino acid mutations are independently selected from the group consisting of a substitution, an insertion, and a deletion. In another embodiment, the wild-type sequence of the fragment of an HIV-I Rev protein comprises a portion of SEQ ID NO: 15; in this case, the fragment of an HIV-I Rev protein can be 6-25 amino acids in length. Each possibility represents a separate embodiment of the present invention.

The length of the fragment of a HIV-I Rev protein contained in peptides of the present invention (e.g. the fragment of a HIV-I Rev protein that comprises SEQ ID NO: 6, 7, 11, 12, or 15 or a fragment thereof) is, in another embodiment, 7-25 amino acids (AA). In another embodiment, the length of the HIV-I Rev fragment is 8-25 AA. In another embodiment, the length is 9-25 AA. In another embodiment, the length is 10-25 AA. In another embodiment, the length is 11-25 AA. In

) another embodiment, the length is 12-25 AA. In another embodiment, the length is 13-25 AA. In another embodiment, the length is 14-25 AA. In another embodiment, the length is 15-25 AA. In another embodiment, the length is 6-24 AA. In another embodiment, the length is 6-23 AA. In another embodiment, the length is 6-22 AA. In another embodiment, the length is 6-21 AA. In another embodiment, the length is 6-20 AA. In another embodiment, the length is 6-19 AA. In another embodiment, the length is 6-18 AA. In another embodiment, the length is 6-17 AA. In another embodiment, the length is 6-16 AA. In another embodiment, the length is 6-16 AA. In another embodiment, the length is 6-14 AA. In another embodiment, the length is 6-13 AA. In another embodiment, the length is 6-12 AA. In another embodiment, the length is 7-24 AA. In another embodiment, the length is 7-23 AA. In another embodiment, the length is 7-22 AA. In another embodiment, the length is 7-21 AA. In another embodiment, the length is 7-20 AA. In another embodiment, the length is 7-19 AA. In another embodiment, the length is 7-18 AA. In another embodiment, the length is 7-17 AA. In another embodiment, the length is 7-16 AA. In another embodiment, the length is 7-16 AA. In another embodiment, the length is 7-14 AA. In another embodiment, the length is 7-13 AA. In another embodiment, the length is 7-12 AA. In another embodiment, the length is 8-24 AA. In another embodiment, the length is 8-23 AA. In another embodiment, the length is 8-22 AA. In another embodiment, the length is 8-21 AA. In another embodiment, the length is 8-20 AA. In another embodiment, the length is 8-19 AA. In another embodiment, the length is 8-18 AA. In another embodiment, the length is 8-17 AA. In another embodiment, the length is 8-16 AA. In another embodiment, the length is 8-16 AA. In another embodiment, the length is 8-14 AA. In another embodiment, the length is 8-13 AA. In another embodiment, the length is 8-12 AA. In another embodiment, the length is 9-24 AA. In another embodiment, the length is 9-23 AA. In another embodiment, the length is 9-22 AA. In another embodiment, the length is 9-21 AA. In another embodiment, the length is 9-20 AA. In another embodiment, the length is 9-19 AA. In another embodiment, the length is 9-18 AA. In another embodiment, the length is 9-17 AA. In another embodiment, the length is 9-16 AA. In another embodiment, the length is 9-16 AA. In another embodiment, the length is 9-14 AA. In another embodiment, the length is 9-13 AA. In another embodiment, the length is 9-12 AA. In another embodiment, the length is 10-24 AA. In another embodiment, the length is 10-23 AA. In another embodiment, the length is 10-22 AA. In another embodiment, the length is 10-21 AA. In another embodiment, the length is 10-20 AA. In another embodiment, the length is 10-19 AA. In another embodiment, the length is 10-18 AA. In another embodiment, the length is 10-17 AA. In another embodiment, the length is 10-16 AA. In another embodiment, the length is 10-16 AA. In another embodiment, the length is 10-14 AA. In another embodiment, the length is 10-13 AA. In another embodiment, the length is 10-12 AA. Each possibility represents a separate embodiment of the present invention.

Defining herein a "length" in terms of a number of "amino acids" or "AA" preferably refers to peptide or peptidomimetic containing the number of total AA specified, including both naturally occurring AA and AA modified in any manner disclosed herein. In another embodiment, the term refers only to the number of L-amino acids (i.e. AA having the naturally occurring stereo configuration) in the peptide or peptidomimetic. In another embodiment, the term refers only to the number of unmodified AA in the peptide or peptidomimetic. In another embodiment, the term refers only to the number of AA in the peptide or peptidomimetic wherein the side chain is unmodified (i.e. AA preceded by or following a modified peptide bond would be included in the count). Each possibility represents a separate embodiment of the present invention.

In some embodiments, those mutations and fragments of SEQ ID NO: 6, 7, 11, 12, or 15 are those that exhibit substantially similar activity to the peptide from which it was derived (e.g. SEQ ID NO: 6, 7, 11, 12, or 15) in one or more of the following: inhibiting binding of an HIV-I integrase protein to an HIV-I long terminal repeat DNA terminus, inhibiting replication of an HIV-I in a target cell, treating HIV-I infection in a subject in need thereof, or inhibiting 3 '-end processing of an HIV-I integrase protein.

The length of an isolated peptide of methods and compositions of the present invention (e.g. an isolated peptide comprising a native or mutated version of a fragment of a HIV-I Rev protein) is, in another embodiment, 13-100 AA. In another embodiment, the length is 14-100 AA. In another embodiment, the length is 15-100 AA. In another embodiment, the length is 20-100 AA. In another embodiment, the length is 25-100 AA. In another embodiment, the length is 30-100 AA. In another embodiment, the length is 40-100 AA. In another embodiment, the length is 50-100 AA. In another embodiment, the length is 12-90 AA. In another embodiment, the length is 13-90 AA. In another embodiment, the length is 14-90 AA. In another embodiment, the length is 15-90 AA. In another embodiment, the length is 20-90 AA. In another embodiment, the length is 25-90 AA. In another embodiment, the length is 30-90 AA. In another embodiment, the length is 40-90 AA. In another embodiment, the length is 50-80 AA. In another embodiment, the length is 12-80 AA. In another embodiment, the length is 13-80 AA. In another embodiment, the length is 14-80 AA. In another embodiment, the length is 15-80 AA. In another embodiment, the length is 20-80 AA. In another embodiment, the length is 25-80 AA. In another embodiment, the length is 30-80 AA. In another embodiment, the length is 40-80 AA. In another embodiment, the length is 50-80 AA. In another embodiment, the length is 12-70 AA. In another embodiment, the length is 13-70 AA. In another embodiment, the length is 14-70 AA. In another embodiment, the length is 15-70 AA. In another embodiment, the length is 20-70 AA. In another embodiment, the length is 25-70 AA. In another embodiment, the length is 30-70 AA. In another embodiment, the length is 40-70 AA. In another embodiment, the length is 50-70 AA. In another embodiment, the length is 12-90 AA. In another embodiment, the length is 13-60 AA. In another embodiment, the length is 14-60 AA. In another embodiment, the length is 15-60 AA. In another embodiment, the length is 20-60 AA. In another embodiment, the length is 25-60 AA. In another embodiment, the length is 30-60 AA. In another embodiment, the length is 40-60 AA. In another embodiment, the length is 50-60 AA. Each possibility represents a separate embodiment of the present invention.

A "mutation" of methods and compositions of the present invention is, in another embodiment, a substitution. In another embodiment, the mutation is an insertion. In another embodiment, the mutation is a deletion. In another embodiment, the mutation is an internal deletion. In another embodiment, the mutation is a truncation. Preferably, the term "mutation" refers to an alteration or modification in the sequence of either a peptide or a nucleotide molecule encoding same. In another embodiment, a peptide of methods and compositions of the present invention comprises multiple AA mutations, in some cases multiple AA mutations relative to the fragment of a HIV-I Rev protein. Each possibility represents a separate embodiment of the present invention.

The term "peptide," as used herein, refers to either a peptide or a peptidomimetic. "Peptidomimetic" refers to a moiety derived from a peptide and having any of the modifications described herein, either singly or in combination. Each possibility represents a separate embodiment of the present invention.

Preferably, the isolated peptide or fragment of a HIV-I Rev protein of methods and compositions of the present invention binds, in a physiological solution, a tetramer of an HIV-I integrase protein with a greater affinity than the isolated peptide or fragment binds a dimer of the HIV-I integrase protein, thereby increasing the ratio of the tetramer to the dimer in the physiological solution. In another embodiment, the isolated peptide or fragment binds a tetramer of an HIV-I integrase protein under physiological conditions with a greater affinity than the isolated peptide or fragment binds a dimer of the HIV-I integrase protein under the same conditions. Each possibility represents a separate embodiment of the present invention.

Methods for measuring the oligomeric state of HIV-I IN are well known in the art, and include the methods disclosed herein (see, inter alia, Examples 3, 5, 1, and 9 and the sections entitled "fluorescence anisotropy," "analytical gel filtration," and "analytical ultracentrifugation" hereinbelow). Each method represents a separate embodiment of the present invention.

More preferably, the isolated peptide or fragment of a HIV-I Rev protein of methods and compositions of the present invention is capable of inhibiting 3 '-end processing activity of an HIV-I integrase protein. In another embodiment, the inhibition is measured in a physiological solution. Each possibility represents a separate embodiment of the present invention.

Methods for measuring the 3 '-end processing activity of HIV IN are well known in the art, and include the methods disclosed herein (see, inter alia, the sections entitled "In- vitro 3 '-end processing and strand transfer assays" and "Quantitative estimation of integrase catalytic activity in vitro" hereinbelow) and methods known in the art, e.g. those described in Craigie (1991) Nucl Acid Res. 19:2729-34; and Hwang (2000) Nucl Acid Res. 28:4884-92. Each method represents a separate embodiment of the present invention.

More preferably, the isolated peptide or fragment of a HIV-I Rev protein of methods and compositions of the present invention is capable of inhibiting binding of an HIV-I integrase protein to an HIV-I long terminal repeat (LTR) DNA terminus. In another embodiment, the inhibition is measured in a physiological solution. Each possibility represents a separate embodiment of the present invention.

Methods for measuring binding of an HIV-I integrase protein to an HIV-I LTR DNA terminus are well known in the art, and include the methods disclosed herein (see, inter alia, Example 9 and the section entitled "fluorescence anisotropy" hereinbelow). Each method represents a separate embodiment of the present invention. "Inhibiting binding" refers, preferably, to ability to inhibit IN binding to LTR DNA with an IC₅₀ of 1 nM (nanomolar)-5 mcM (micromolar). In another embodiment, the term refers to ability to inhibit binding with an IC₅₀ of 1 nM-4 mcM. In another embodiment, the term refers to an ICs₀ range of 1 nM-10 mcM. In another embodiment, the term refers to an ICs₀ range of 1 nM-8 mcM. In another embodiment, the term refers to an IC₅₀ range of 1 nM-3 mcM. In another embodiment, the term refers to an IC50 range of 1 nM-2.5 mcM. In another embodiment, the term refers to an IC₅₀ range of 1 nM-2 mcM. In another embodiment, the term refers to an IC₅₀ range of 1 nM-1.5 mcM. In another embodiment, the term refers to an IC₅₀ range of 1 nM-1 mcM. In another embodiment, the term refers to an IC₅₀ range of 1-700 nM. In another embodiment, the term refers to an IC₅₀ range of 1-500 nM. In another embodiment, the term refers to an IC₅₀ range of 1- 300 nM. In another embodiment, the term refers to an IC_5O range of 1-200 nM. In another embodiment, the term refers to an IC_5O range of 1-100 nM. In another embodiment, the term refers to an IC₅₀ range of 1-70 nM. In another embodiment, the term refers to an ICs₀ range of 1- 50 nM. In another embodiment, the term refers to an IC₅₀ range of 1 nM-30 nM. In another embodiment, the term refers to an IC₅₀ range of 2 nM-10 mcM. In another embodiment, the term refers to an IC₅Q range of 2 nM-7 mcM. In another embodiment, the term refers to an IC₅₀ range of 2 nM-5 mcM. In another embodiment, the term refers to an IC₅₀ range of 2 nM-3 mcM. In another embodiment, the term refers to an IC₅₀ range of 2 nM-2.5 mcM. In another embodiment, the term refers to an IC5₀ range of 2 nM-2 mcM. In another embodiment, the term refers to an IC₅₀ range of 2 nM-1.5 mcM. In another embodiment, the term refers to an IC₅₀ range of 2 nM-1 mcM. In another embodiment, the term refers to an IC₅₀ range of 2-700 nM. In another embodiment, the term refers to an IC₅₀ range of 2-500 nM. In another embodiment, the term refers to an IC₅₀ range of 2-300 nM. In another embodiment, the term refers to an IC₅₀ range of 2- 200 nM. In another embodiment, the term refers to an IC₅₀ range of 2-100 nM. In another embodiment, the term refers to an IC₅₀ range of 2-70 nM. In another embodiment, the term refers to an IC₅₀ range of 2-50 nM. In another embodiment, the term refers to an IC₅₀ range of 2-30 nM. In another embodiment, the teπn refers to an IC₅₀ range of 3nM-10mcM. In another embodiment, the term refers to an IC₅₀ range of 3nM-7mcM. In another embodiment, the term refers to an IC₅₀ range of 3nM-5mcM. In another embodiment, the term refers to an IC₅₀ range of 3 nM-3mcM. In another embodiment, the term refers to an IC₅₀ range of 3nM-2.5mcM. In another embodiment, the term refers to an IC₅₀ range of 3 nM-2mcM. In another embodiment, the term refers to an IC₅₀ range of 3nM-lmcM. In another embodiment, the term refers to an IC₅₀ range of 3-700 nM. In another embodiment, the term refers to an IC₅₀ range of 3-500 nM. In another embodiment, the term refers to an ICs₀ range of 3-300 nM. In another embodiment, the term refers to an IC₅₀ range of 3-200 nM. In another embodiment, the term refers to an IC₅₀ range of 3-100 nM. In another embodiment, the term refers to an ICs₀ range of 3-70 nM. In another embodiment, the term refers to an IC_5O range of 3-50 nM. In another embodiment, the term refers to an IC₅₀ range of 5nM- lOmcM. In another embodiment, the term refers to an IC_5O range of 5nM-7mcM. In another embodiment, the term refers to an IC₅₀ range of 5nM-5mcM. In another embodiment, the term refers to an IC₅₀ range of 5nM-3mcM. In another embodiment, the term refers to an IC₅₀ range of 5nM-2mcM. In another embodiment, the term refers to an IC₅₀ range of 5nM-lmcM. In another embodiment, the term refers to an IC₅₀ range of 5-700 nM. In another embodiment, the term refers to an IC₅₀ range of 5-500 nM. In another embodiment, the term refers to an IC₅₀ range of 5- 300 nM. In another embodiment, the term refers to an IC₅₀ range of 5-200 nM. In another embodiment, the term refers to an IC₅₀ range of 5-100 nM. In another embodiment, the term refers to an IC₅₀ range of 5-50 nM. In another embodiment, the term refers to an IC₅₀ range of 5- 30 nM. In another embodiment, the term refers to an IC₅₀ range of IOnM-lOmcM. In another embodiment, the term refers to an IC₅₀ range of 10nM-7mcM. In another embodiment, the term refers to an IC₅₀ range of 10nM-5mcM. In another embodiment, the term refers to an IC₅₀ range of 10nM-3mcM. In another embodiment, the term refers to an IC₅₀ range of 10nM-2mcM. In another embodiment, the term refers to an IC₅₀ range of lOnM-lmcM. In another embodiment, the term refers to an IC_5O range of 10-700 nM. In another embodiment, the term refers to an IC₅₀ range of 10-500 nM. In another embodiment, the term refers to an IC₅₀ range of 10-300 nM. In another embodiment, the term refers to an IC₅₀ range of 10-200 nM. In another embodiment, the term refers to an IC₅₀ range of 10-100 nM. In another embodiment, the term refers to an IC₅₀ range of 10-70 nM. In another embodiment, the term refers to an IC₅₀ range of 10-50 nM. In another embodiment, the term refers to an IC₅₀ range of 10-30 nM. In another embodiment, the term refers to an IC₅₀ range of 2OnM-I OmcM. In another embodiment, the term refers to an IC₅₀ range of 20nM-7mcM. In another embodiment, the term refers to an IC₅₀ range of 20nM-5mcM. In another embodiment, the term refers to an IC₅₀ range of 20nM-3mcM. In another embodiment, the term refers to an IC5₀ range of 20nM-2mcM. In another embodiment, the term refers to an IC₅₀ range of 2OnM-I mcM. In another embodiment, the term refers to an IC₅₀ range of 20-700 nM. In another embodiment, the term refers to an IC₅₀ range of 20-500 nM. In another embodiment, the term refers to an IC₅₀ range of 20-300 nM. In another embodiment, the term refers to an IC₅₀ range of 20-200 nM. In another embodiment, the term refers to an IC₅₀ range of 20-100 nM. In another embodiment, the term refers to an IC50 range of 20-70 nM. In another embodiment, the term refers to an IC₅₀ range of 20-50 nM. In another embodiment, the term refers to an IC₅₀ range of 3OnM-I OmcM. In another embodiment, the term refers to an IC₅₀ range of 30nM-7mcM. In another embodiment, the term refers to an IC₅₀ range of 30nM-5mcM. In another embodiment, the term refers to an IC₅₀ range of 30nM-3mcM. In another embodiment, the term refers to an IC_5O range of 30nM-2mcM. In another embodiment, the term refers to an IC₅₀ range of 30nM-lmcM. In another embodiment, the term refers to an IC_5O range of 30-700 nM. In another embodiment, the term refers to an IC₅0 range of 30-500 nM. In another embodiment, the term refers to an IC₅₀ range of 30-300 nM. In another embodiment, the term refers to an IC₅₀ range of 30-200 nM. In another embodiment, the term refers to an IC₅₀ range of 30-100 nM. In another embodiment, the term refers to an IC5₀ range of 30-70 nM. In another embodiment, the term refers to an IC₅₀ range of 30-50 nM. In another embodiment, the term refers to an IC₅₀ range of 5OnM-I OmcM. In another embodiment, the term refers to an IC₅₀ range of 50nM-7mcM. In another embodiment, the term refers to an IC₅₀ range of 50nM-5mcM. In another embodiment, the term refers to an IC₅₀ range of 50nM-3mcM. In another embodiment, the term refers to an IC₅₀ range of 50nM-2mcM. In another embodiment, the term refers to an IC₅₀ range of 50nM-lmcM. In another embodiment, the term refers to an IC₅₀ range of 50-700 nM. In another embodiment, the term refers to an IC₅0 range of 50-500 nM. In another embodiment, the term refers to an IC₅₀ range of 50-300 nM. In another embodiment, the term refers to an IC₅₀ range of 50-200 nM. In another embodiment, the term refers to an IC_5O range of 50-100 nM. In another embodiment, the term refers to an IC₅₀ range of 50-70 nM. In another embodiment, the term refers to an IC₅₀ range of 7OnM-I OmcM. In another embodiment, the term refers to an IC₅₀ range of 70nM-7mcM. In another embodiment, the term refers to an IC₅₀ range of 70nM-5nicM. In another embodiment, the term refers to an IC₅₀ range of 70nM-3mcM. In another embodiment, the term refers to an IC5₀ range of 70nM-2mcM. In another embodiment, the term refers to an IC₅₀ range of 7OnM- lmcM. In another embodiment, the term refers to an IC_5O range of 70-700 nM. In another embodiment, the term refers to an IC₅₀ range of 70-500 nM. In another embodiment, the term refers to an IC50 range of 70-300 nM. In another embodiment, the term refers to an IC_5O range of 70-200 nM. In another embodiment, the term refers to an IC₅₀ range of 70-100 nM. In another embodiment, the term refers to an IC₅₀ range of lOOnM-lOmcM. In another embodiment, the term refers to an IC₅₀ range of 100nM-7mcM. In another embodiment, the term refers to an IC₅₀ range of 100nM-5mcM. In another embodiment, the term refers to an IC₅₀ range of 100nM-3mcM. In another embodiment, the term refers to an IC₅₀ range of 100nM-2mcM. In another embodiment, the term refers to an IC₅₀ range of lOOnM-lmcM. In another embodiment, the term refers to an IC₅₀ range of 100-700 nM. In another embodiment, the term refers to an IC₅₀ range of 100-500 nM. In another embodiment, the term refers to an IC₅₀ range of 100-300 nM. In another embodiment, the term refers to an IC_5O range of 100-200 nM. In another embodiment, the term refers to an IC₅₀ range of 100- 150 nM. In another embodiment, the term refers to an IC₅₀ range of 15OnM-I OmcM. In another embodiment, the term refers to an IC₅Q range of 150nM-7mcM. In another embodiment, the term refers to an IC₅₀ range of 15 OnM- 5mcM. In another embodiment, the term refers to an IC₅₀ range of 150nM-3mcM. In another embodiment, the term refers to an IC₅₀ range of 150nM-2mcM. In another embodiment, the term refers to an IC₅₀ range of 150nM-lmcM. In another embodiment, the term refers to an IC₅₀ range of 150-700 nM. In another embodiment, the term refers to an IC₅₀ range of 150-500 nM. In another embodiment, the term refers to an IC₅₀ range of 150-300 nM. In another embodiment, the term refers to an IC₅₀ range of 150-200 nM. In another embodiment, the term refers to an IC_5O range of 20OnM-I OmcM. In another embodiment, the term refers to an IC_5O range of 20OnM- 7mcM. In another embodiment, the term refers to an IC₅₀ range of 200nM-5mcM. In another embodiment, the term refers to an IC₅₀ range of 200nM-3mcM. In another embodiment, the term refers to an IC₅₀ range of 200nM-2mcM. In another embodiment, the term refers to an IC₅₀ range of 20OnM-I mcM. In another embodiment, the term refers to an IC₅₀ range of 200-700 nM. In another embodiment, the term refers to an IC₅₀ range of 200-500 nM. In another embodiment, the term refers to an IC₅₀ range of 200-300 nM. In another embodiment, the term refers to an IC₅₀ range of 300nM-10mcM. In another embodiment, the term refers to an IC_5O range of 30OnM- 7mcM. In another embodiment, the term refers to an IC5₀ range of 300nM-5mcM. In another embodiment, the term refers to an IC₅₀ range of 300nM-3mcM. In another embodiment, the term refers to an IC₅₀ range of 300nM-2mcM. In another embodiment, the term refers to an IC₅₀ range of 30OnM- lmcM. In another embodiment, the term refers to an IC₅Q range of 300-700 nM. In another embodiment, the term refers to an IC₅₀ range of 300-500 nM. In another embodiment, the term refers to an IC₅₀ range of 500nM-10mcM. In another embodiment, the term refers to an IC₅O range of 500nM-7mcM. In another embodiment, the term refers to an IC₅₀ range of 50OnM- 5mcM. In another embodiment, the term refers to an I C₅₀ range of 500nM-3mcM. In another embodiment, the term refers to an IC₅₀ range of 500nM-2mcM. In another embodiment, the term refers to an IC₅₀ range of 500nM-lmcM. In another embodiment, the term refers to an IC₅₀ range of 500-700 nM. In another embodiment, the term refers to an IC₅₀ range of 70OnM-I OmcM. In another embodiment, the term refers to an IC₅₀ range of 700nM-7mcM. In another embodiment, the term refers to an IC_5O range of 700nM-5mcM. In another embodiment, the term refers to an ICso range of 700nM-3mcM. In another embodiment, the term refers to an IC₅₀ range of 70OnM- 2mcM. In another embodiment, the term refers to an IC_5O range of 70OnM- lmcM. In another embodiment, the term refers to an IC₅₀ range of 1-1 OmcM. In another embodiment, the term refers to an IC₅₀ range of l-7mcM. In another embodiment, the term refers to an IC₅₀ range of 1- 5mcM. In another embodiment, the term refers to an IC₅₀ range of l-3mcM. In another embodiment, the term refers to an IC₅₀ range of l-2mcM. In another embodiment, the term refers to an IC_5O range of 1.5-lOmcM. In another embodiment, the term refers to an IC₅₀ range of 1.5- 7mcM. In another embodiment, the term refers to an IC_5O range of 1.5-5mcM. In another embodiment, the term refers to an IC₅₀ range of 1.5-3mcM. In another embodiment, the term refers to an IC₅₀ range of 2-1 OmcM. In another embodiment, the term refers to an IC_5O range of 2- 7mcM. In another embodiment, the term refers to an IC₅₀ range of 2-5mcM. In another embodiment, the term refers to an IC₅₀ range of 2-3mcM. In another embodiment, the term refers to an IC₅₀ range of 3-1 OmcM. In another embodiment, the term refers to an IC₅₀ range of 3- 7mcM. In another embodiment, the term refers to an IC_5O range of 3-5mcM. In another embodiment, the term refers to an IC_5O range of 5-1 OmcM. In another embodiment, the term refers to an IC₅₀ range of 5-7mcM. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the term refers to ability to inhibit DNA binding by at least 3 -fold, when pre-incubated with IN at a concentration of 500 nM peptide under physiological conditions (e.g. with IN at a concentration of 4 μM) and DNA at a concentration of 1OnM. In another embodiment, the term refers to ability to inhibit DNA binding by at least 6-fold under the above conditions. In another embodiment, the term refers to ability to inhibit DNA binding by at least 2- fold under the above conditions. Each possibility represents a separate embodiment of the present invention.

"Physiological conditions" are well known to those skilled in the art, and include, for example, 0.2 M Tris, pH 7.4, with 0.15 M NaCl. Those skilled in the art will be readily able to discern physiological conditions appropriate for DNA binding assays, 3 '-end processing activity, etc. Each possibility represents a separate embodiment of the present invention.

Most preferably, the isolated peptide or fragment of a HIV-I Rev protein of methods and compositions of the present invention is capable of inhibiting HIV-I replication in a target cell. "Target cell," as used herein, may refer to any cell wherein HIV-I is capable of replicating. Preferably, the target cell is a human cell or an immortalized cell line derived from a human cell. Each possibility represents a separate embodiment of the present invention.

Methods for measuring HIV-I replication in a target cell are well known in the art, and include the use of HeLa MAGI cells (see, inter alia, Example 6 and the section entitled "HIV-I titration multinuclear activation..." hereinbelow). Each method represents a separate embodiment of the present invention.

In another embodiment, a composition of the present invention further comprises a non-naturally occurring amino acid, in addition to the fragment of a HIV-I Rev protein. In another embodiment, a composition of the present invention further comprises an organic peptidomimetic moiety, in addition to the fragment of a HIV-I Rev protein. In another embodiment, a side chain of an amino acid of the fragment of a HIV-I Rev protein has been chemically modified. In another embodiment, a peptidic bond has been replaced by a non- naturally occurring peptidic bond. In another embodiment, one of the amino acids is replaced by the corresponding D- amino acid. In another embodiment, the present invention encompasses an N-methyl variant of the sequence. In another embodiment, the present invention provides a sequence disclosed herein in reverse order, preferably having all D-amino acids (refro inversό). In another embodiment, a derivative of the present invention possesses one of the above modifications at a plurality of locations (e.g. a plurality of residues). In another embodiment, a derivative of the present invention possesses two of the above modifications. In another embodiment, a derivative of the present invention possesses more than 2 of the above modifications. In another embodiment, one of the above modifications is introduced at a location outside the fragment of a HIV-I Rev protein; i.e. in the surrounding sequence. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a pharmaceutical composition, comprising an isolated peptide isolated peptide or fragment of a HIV-I Rev protein of the present invention and a carrier, diluent, or additive.

In another embodiment, the present invention provides a pharmaceutical composition, comprising: (a) an isolated HIV-I Rev fragment with a sequence selected from SEQ ID NO: 11 and SEQ ID NO: 6, or a peptide comprising a fragment selected from SEQ ID NO: 11 and SEQ ID NO: 6; (b) an isolated HIV-I Rev fragment with a sequence set forth in SEQ ID NO: 7, or a peptide comprising SEQ ID NO: 7; and (c) a pharmaceutically acceptable carrier, diluent, or additive. In another embodiment, HIV-I Rev fragment (a) comprises a fragment of SEQ ID NO: 7. In another embodiment, HIV-I Rev fragment (b) comprises a fragment of a sequence selected from SEQ ID NO: 11 and SEQ ID NO: 6. In another embodiment, both (a) and (b) contain fragments of the respective sequences set forth above. In another embodiment, HIV-I Rev fragment (b) comprises a mutated version of a sequence selected from SEQ ID NO: 11 and SEQ ID NO: 6. In another embodiment, HIV-I Rev fragment (a) comprises a mutated version of SEQ ID NO: 7. In another embodiment, both (a) and (b) contain mutated versions of the respective sequences set forth above. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a pharmaceutical composition, comprising: (a) an isolated HIV-I Rev fragment with a sequence selected from SEQ ID NO: 11 and SEQ ID NO: 6 or a peptide comprising the HIV-I Rev fragment; (b) an isolated HIV-I Rev fragment with a sequence selected from the sequences set forth in a sequence selected from SEQ ID NO: 12 and SEQ ID NO: 15, or a peptide comprising the HIV-I Rev fragment; and (c) a pharmaceutically acceptable carrier, diluent, or additive. In another embodiment, HIV-I Rev fragment (a) comprises a fragment of SEQ ID NO: 11 or SEQ ID NO: 6. In another embodiment, HIV-I Rev fragment (b) comprises a fragment of a sequence selected from SEQ ID NO: 12 or SEQ ID NO: 15. In another embodiment, both (a) and (b) contain fragments of the respective sequences set forth above. In another embodiment, HIV-I Rev fragment (a) comprises a mutated version of SEQ ID NO: 11 or SEQ ID NO: 6. In another embodiment, HIV-I Rev fragment (b) comprises a mutated version of SEQ ID NO: 12 or SEQ ID NO: 15. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a pharmaceutical composition, comprising: (a) an isolated HIV-I Rev fragment with a sequence set forth in SEQ ID NO: 7 or a peptide comprising the HIV-I Rev fragment; (b) an isolated HIV-I Rev fragment with a sequence selected from the sequences set forth in a sequence selected from SEQ ID NO: 12 and SEQ ID NO: 15, or a peptide comprising the HIV-I Rev fragment; and (c) a pharmaceutically acceptable carrier, diluent, or additive. In another embodiment, HIV-I Rev fragment (a) comprises a fragment of SEQ ID NO: 7. In another embodiment, HIV-I Rev fragment (b) comprises a fragment of a sequence selected from SEQ ID NO: 12 or SEQ ID NO: 15. In another embodiment, both (a) and (b) contain fragments of the respective sequences set forth above. In another embodiment, HIV-I Rev fragment (a) comprises a mutated version of SEQ ID NO: 7. In another embodiment, HIV-I Rev fragment (b) comprises a mutated version of SEQ ID NO: 12 or SEQ ID NO: 15. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a pharmaceutical composition of the present invention for inhibiting replication of an HIV-I in a target cell.

In another embodiment, the present invention provides a pharmaceutical composition of the present invention for treating HIV-I infection in a subject in need thereof.

In another embodiment, the present invention provides a pharmaceutical composition of the present invention for inhibiting binding of an HIV-I integrase protein to an HIV-I long terminal repeat DNA terminus.

In another embodiment, the present invention provides a pharmaceutical composition of the present invention for inhibiting 3 '-end processing of an HIV-I integrase protein. In another embodiment, the pharmaceutical composition is utilized in an in vitro assay. In another embodiment, the pharmaceutical composition is utilized in a target cell. Each possibility represents a separate embodiment of the present invention. In another embodiment, the present invention provides a use of a peptide of the present invention in the preparation of a medicament for inhibiting replication of an HIV-I in a target cell.

In another embodiment, the present invention provides a use of a peptide of the present invention in the preparation of a medicament for treating HIV-I infection in a subject in need thereof.

In another embodiment, the present invention provides a use of a peptide of the present invention in the preparation of a medicament for inhibiting binding of an HIV-I integrase protein to an HIV-I long terminal repeat DNA terminus.

In another embodiment, the present invention provides a use of a peptide of the present invention in the preparation of a medicament for inhibiting 3 '-end processing of an HIV-I integrase protein. In another embodiment, the pharmaceutical composition is utilized in an in vitro assay. In another embodiment, the pharmaceutical composition is utilized in a target cell. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of inhibiting replication of an HIV-I in a target cell, comprising administering a peptide of the present invention to the target cell, thereby inhibiting replication of an HIV- 1 in a target cell.

In another embodiment, the present invention provides a method of treating HIV-I infection in a subject in need thereof, comprising administering a peptide of the present invention to the subject, thereby treating HIV-I infection in a subject in need thereof.

In another embodiment, the present invention provides a method of inhibiting binding of an HIV- 1 integrase protein to an HIV-I long terminal repeat DNA terminus, comprising contacting the HIV-I integrase protein with a peptide of the present invention, thereby inhibiting binding of an HIV-I integrase protein to an HIV-I long terminal repeat DNA terminus.

In another embodiment, the present invention provides a method of inhibiting 3 '-end processing of an HIV-I integrase protein, comprising contacting the HIV-I integrase protein with a peptide of the present invention, thereby inhibiting 3 '-end processing of an HIV-I integrase protein. In another embodiment, the method is performed in an in vitro assay. In another embodiment, the method is performed in a target cell. Each possibility represents a separate embodiment of the present invention.

"Treatment of HIV infection" refers to improvement in at least one clinical parameter associated with the HIV infection as compared to non-treated control and notably to improve in the viral load count and increase in CD4+ bearing cells. The improvement may be actual reduction in the viral load, but may also be slowing down in the rate of increase of the viral load, or inhibition of complications of the disease.

In another embodiment, a peptide of the present invention is capable of entering a mammalian cell under physiological conditions. In another embodiment, the peptide penetrates the cell membrane of the mammalian cell. In another embodiment, the peptide is actively transported through the cell membrane. In another embodiment, the peptide diffuses through the cell membrane. Each possibility represents a separate embodiment of the present invention.

"HIV-I Rev protein" refers, in another embodiment, to a protein having the sequence set forth in SEQ ID NO: 31 or a homologue, variant, or isoform of this sequence. In another embodiment, the sequence of the HIV-I Rev protein is:

MAGRSGDSDEELLKTVRLIKFLYQSNPPPSPEGTRQARRNRRRRWRERQRQIRSISGWILS TYLGRP AEPVPLQQLPPLERLTLDCNEDCGTSGTQGVGSPQILVESPAVLESGTKE (SEQ ID NO: 31). In another embodiment, the Rev sequence is a homologue of SEQ ID NO: 31. In another embodiment, the sequence is a variant of SEQ ID NO: 31. In another embodiment, the sequence is a fragment of SEQ ID NO: 31. In another embodiment, the sequence is a homologue of a fragment of SEQ ID NO: 31. "Homologue" may refer to any degree of homology disclosed herein. In another embodiment, the sequence is a variant of a fragment of SEQ ID NO: 31. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the Rev protein has the sequence:

MAGRSGDSDEELLKAVRIIKILYQSNPYPKPEGTRQARRNRRRRWRARQRQIHSISERILS TCLGPJ⁵AEPVPLQLPPLERLHLDCSEDCGTSGTQQGTGVGSPQISVESP AVLGSGTKE (SEQ ID NO: 32). In another embodiment, the Rev sequence is a homologue of SEQ ID NO: 32. In another embodiment, the sequence is a variant of SEQ ID NO: 32. In another embodiment, the sequence is a fragment of SEQ ID NO: 32. In another embodiment, the sequence is a homologue of a fragment of SEQ ID NO: 32. "Homologue" may refer to any degree of homology disclosed herein. In another embodiment, the sequence is a variant of a fragment of SEQ ID NO: 32. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the Rev protein has the sequence:

MAGRSGDSDEELLKAVRLIKFLYQSNPPPNPEGTRQARRNRRRRWRERQRQIHSISERILS TYLGRSAEPVPLQLPPLERLTLDCNEDCGTSGTQGVGSPQILVESPTILESGAKE (SEQ ID NO: 33; GenBank Accession No. P04325). In another embodiment, the Rev sequence is a homologue of SEQ ID NO: 33. In another embodiment, the sequence is a variant of SEQ ID NO: 33. In another embodiment, the sequence is a fragment of SEQ ID NO: 33. In another embodiment, the sequence is a homologue of a fragment of SEQ ID NO: 33. "Homologue" may refer to any degree of homology disclosed herein. In another embodiment, the sequence is a variant of a fragment of SEQ ID NO: 33. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the Gag-Pol protein from which the Rev protein is derived has the sequence:

FFRENLAFQQGEARKFSSEQTGTNSPTSRELWDGGRDNLLSEAGTEGQGTISSFNFPQITL WQRPLVTVRIGGQLIEALLDTGADDTVLEEINLPGKWKPKMIGGIGGFIKVRQYDQILIEI CGKKAIGTVLVGPTP VNIIGRNMLTQIGCTLNFPISPIETVP VKLKPGMDGPKVKQWPLTE EKIKALTDICTEMEKEGKISKIGPENPYNTPVF AIKKKDSTKWRKL VDFRELNKRTQDFW EVQLGIPHPAGLKKKKSVTVLDVGDAYFSVPLDKDFRKYTAFTIPSVNNETPGIRYQYNV LPQGWKGSPAIFQASMTKILEPFRTKNPEIVIYQYMDDLYVGSDLEIGQHRAKIEELREHL LRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIQLPEKDSWTVNDIQKLVGKLNW ASQIYAGIKVKQLCKLLRGAKALTDIVTLTEEAELELAENREILKEPVHGVYYDPTKDLI AEIQKQGQDQWTYQIYQEPFKNLKTGKYAKMRSAHTNDVKQLTEVVQKVATESIVIWG KTPKFRLPIQRETWEAWWMEYWQATWIPEWEFVNTPPLVKLWYQLEKDPIVGAETFYV DGAANRETKLGKAGYVTDRGRQKVVSLTETTNQKTELHAIHLALQDSGSEVNIVTDSQ YALGIIQAQPDRSESELVNQIIEKLIEKDKVYLSWVPAHKGIGGNEQVDKLVSNGIRKVLF LDGIDKAQEEHERYHSNWRAMASDFNLPPIVAKEIVASCDKCQLKGEAMHGQ VDCSPGI WQLDCTHLEGKIILVAVHVASGYIEAEVIPAETGQETAYFILKLAGRWPVKVIHTDNGSN FTSAAVKAACWWANVTQEFGIPYNPQSQGVVESMNKELKKIIGQVRDQAEHLKTAVQ MAVFUΓNFKRKGGIGGYSAGERIIDΠASDIQTKELQKQITKIQNFRVYYRDSRDPIWKGPA KLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQMAGDDCVAGRQDED (SEQ ID NO: 34). In another embodiment, the Rev sequence is a homologue of SEQ ID NO: 34. In another embodiment, the sequence is a variant of SEQ ID NO: 34. In another embodiment, the sequence is a fragment of SEQ ID NO: 34. In another embodiment, the sequence is a homologue of a fragment of SEQ ID NO: 34. "Homologue" may refer to any degree of homology disclosed herein. In another embodiment, the sequence is a variant of a fragment of SEQ ID NO: 34. Each possibility represents a separate embodiment of the present invention.

FFRENLAFQQGEAREFSSEQTRANSPTSRELRVRGGDNPLSEAGAERQGTVSLSFPQITL WQRPLVTVKIGGQLKEALLDTGADDTVLEEΓNLPGKWKPKMIGGIGGFIKVRQYDQILIE ICGKKAIGTVLVGPTPVNIIGRNMLTQIGCTLNFPISPIETVP VKLKPGMDGPKVKQ WPLT

EEKIKALTEICTEMEKEGKISKIGPENP YNTPIFAIKKKDSTK WRKLVDFRELNKRTQDFW

EVQLGiPHPAGLKKKKsvTVLDVGDA YFSVPLDEDFRKYTAFTIPSΓNNETPGIRYQYNV LPQGWKGSPAIFQSSMTKILEPFRTQNPEIVIYQYMDDLYVGSDLEIGQHRTKIEELREHL LRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIQLPEKDSWTVNDIQKLVGKLNW ASQIYPGIKVKQLCKLLRGAKALTDIVPLTEEAELELAENREILKEPVHGVYYDPSKDLIA EIQKQGQDQWTYQIYQEPFKNLKTGKYAKMRSAHTNDVKQLTEAVQKIATESIVIWGK TPKFRLPIQKETWETWWTEYWQATWIPEWEFVNTPPLVKLWYQLEKEPIVGAETFYVD GAANRETKLGKAGYVTDRGRQKVVSLTETTNQKTELQAIHLALQDSGSEVNIVTDSQY ALGIIQAQPDKSESELVNQIIEQLIKKEKVYLSWVPAHKGIGGNEQVDKLVSTGIRKVLFL DGIDKAQEEHEKYHSNWRAMASDFNLPPIVAKEIVASCDKCQLKGEAMHGQVDCSPGI

WQLDCTHLEGKIIL VAVHVASGYIEAEVIP AETGQETA YFILKLAGRWPVKVIHTDNGSN FTSAA VKAACWWAGIQQEFGIP YNPQSQGVVESMNKELKKIIGQ VRDQAEHLKTA VQM AVFIHNFKRKGGIGGYSAGERIIDIIATDIQTKELQKQITKIQNFRVYYRDSRDPIWKGPAK LLWKGEGAVVIQDNSEIKVVPRRKAKIIRDYGKQMAGDDCVAGRQDED (SEQ ID NO: 35). In another embodiment, the Rev sequence is a homologue of SEQ ID NO: 35. In another embodiment, the sequence is a variant of SEQ ID NO: 35. In another embodiment, the sequence is a fragment of SEQ ID NO: 35. In another embodiment, the sequence is a homologue of a fragment of SEQ ID NO: 35. "Homologue" may refer to any degree of homology disclosed herein. In another embodiment, the sequence is a variant of a fragment of SEQ ID NO: 35. Each possibility represents a separate embodiment of the present invention.

FFRENLAFQQGEAREFSSEQTRANSPTSRELRVRGGDNPLSEAGAERQGTVSFSFPQITLW QRPLVTIKIGGQLREALLDTGADDTVLEEΓNLPGKWKPKMIGGIGGFIKVRQYDQILIEIC GKKAIGTVLVGPTPVNIIGRNMLTQIGCTLNFPISPIETVPVKLKPGMDGPKVKQ WPLTEE

KIKALTEICTEMEKEGKISKIGPENPYNTP VFAIKKKDSTKWRKLVDFRELNKRTQDFWE

VQLGIPHPAGLKKKKSVTVLDVGDAYFSVPLDEDFRKYTAFTIPSΓNNETPGIRYQYNVLP QGWKGSPAIFQSSMTKILEPFRTKNPEIVIYQYMDDLYVGSDLEIGQHRAKIEELREHLLR WGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIQLPEKDSWTVNDIQKLVGKLNWAS QI YPGIKVKQLCKLLRGAKALTDIVPLTEEAELELAENREILKEPVHGV YYDPSKDLI AEI QKQGQDQWTYQIYQEPFKNLKTGKYAKMRSAHTNDVKQLTEAVQKIATESIVIWGKTP KFRLPIQKETWETWWTEYWQATWIPEWEFVNTPPLVKLWYQLEKEPIVGAETFYVDGA ANRETKLGKAGYVTDRGRQKVVSLTETTNQKTELQAIHLALQDSGSEVNIVTDSQYALG IIQAQPDKSESELVNQIIEQLIKKEKVYLSWVPAHKGIGGNEQVDKL VSSGIRK VLFLDGI DKAQEEHEKYHSNWRAMASDFNLPPVVAKEIVASCDKCQLKGEAMHGQVDCSPGIWQ LDCTHLEGKVILVAVHVASGYIEAEVIPAETGQETAYFILKLAGRWPVKVIHTDNGSNFT SAA VKAACWWAGIQQEFGIP YNPQSQGVVESMNKELKKIIGQ VRDQAEHLKTAVQMA VFIHNFKRKGGIGGYSAGERIIDIIATDIQTKELQKQITKIQNFRVYYRDSRDPIWKGPAKL LWKGEGAVVIQDNSEIKVVPRRKAKIIRDYGKQMAGDDCVAGRQDED (SEQ ID NO: 36). In another embodiment, the Rev sequence is a homologue of SEQ ID NO: 36. In another embodiment, the sequence is a variant of SEQ ID NO: 36. In another embodiment, the sequence is a fragment of SEQ ID NO: 36. In another embodiment, the sequence is a homologue of a fragment of SEQ ID NO: 36. "Homologue" may refer to any degree of homology disclosed herein. In another embodiment, the sequence is a variant of a fragment of SEQ ID NO: 36. Each possibility represents a separate embodiment of the present invention. In another embodiment, the Gag-Pol protein from which the Rev protein is derived has the sequence:

FFRENLAFQQGKAGEFSSEQTRANSPTSRKLGDGGRDNLLTEAGAERQGTSSSFSFPQITL WQRPLVTVKIGGQLKEALLDTGADDTVLEDINLPGKWKPKMIGGIGGFIKVRQYDQILIE ICGKKAIGTVLVGPTPVNIIGRNMLTQIGCTLNFPISPIDTVPVTLKPGMDGPKVKQWPLT EEKIKALTEICKEMEEEGKISKIGPENPYNTP VF AIKKKDSTKWRKLVDFRELNKRTQDF WEVQLGIPHPAGLKKKKSVTVLDVGDAYFSVPLDESFRKYTAFTIPSINNETPGIRYQYN VLPQGWKGSPAIFQSSMTKILEPFRIKNPEMVIYQYMDDLYVGSDLEIGQHRTKIEELRA HLLSWGFTTPDKKHQKEPPFLWMGYELHPDRWTVQPIELPEKDSWTVNDIQKLVGKLN WASQIYAGIKVKQLCKLLRGAKALTDIVPLTEEAELELAENREILKTP VHGVYYDPSKDL

VAEVQKQGQDQWTYQIYQEPFKNLKTGKYARKRSAHTNDVRQLTEVVQKIATESIVIW GKTPKFRLPIQRETWETWWMEYWQATWIPEWEFVNTPPLVKLWYQLEKDPIVGAETFY VDGAASRETKLGKAGYVTDRGRQKVVSLTETTNQKTELHAIHLALQDSGSEVNIVTDSQ YALGIIQAQPDRSESEVVNQIIEELIKKEKVYLSWVPAHKGIGGNEQVDKLVSSGIRKVLF LDGIDKAQEEHERYHSNWRTMASDFNLPPIV AKEIVANCDKCQLKGEAMHGQVDCSPGI WQLDCTHLEGKVILVAVHVASGYIEAEVIPAETGQETAYFLLKLAGRWPVKVIHTDNGS NFTSAAVKAACWWANVRQEFGIPYNPQSQGVVESMNKELKKIIGQVREQAEHLKTAVQ MAVFIHNFKRKGGIGGYSAGERIIDIIATDIQTKELQKQITKIQNFRVYYRDSRDPIWKGPA KLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQMAGDDCVAGRQDED (SEQ ID NO: 37). In another embodiment, the Rev sequence is a homologue of SEQ ID NO: 37. In another embodiment, the sequence is a variant of SEQ ID NO: 37. In another embodiment, the sequence is a fragment of SEQ ID NO: 37. In another embodiment, the sequence is a homologue of a fragment of SEQ ID NO: 37. "Homologue" may refer to any degree of homology disclosed herein. In another embodiment, the sequence is a variant of a fragment of SEQ ID NO: 37. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the Rev protein has a sequence set forth in one of the following GenBank Accession Numbers: AF407418; AJ302646; AJ302647; AY169802-AY169813, inclusive; AY169815-AY169816; AY489739; AY618998; AY623602; L20571; L20587. In another embodiment, the Rev sequence is a homologue of one of the above sequences. In another embodiment, the sequence is a variant of one of the above sequences. In another embodiment, the sequence is a fragment of one of the above sequences. In another embodiment, the sequence is a homologue of a fragment of one of the above sequences. "Homologue" may refer to any degree of homology disclosed herein. In another embodiment, the sequence is a variant of a fragment of one of the above sequences. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the Rev protein is encoded by the sequence:

ttttttagggaaaatttggccttccaacaaggggaggccagggaattttcttcagagcagaccagagccaacagccccaccagcagagagc ttcgggttcggggaggagataaccccctctccgaagcaggagcagaaagacaaggaactgtatcccttagcttccctcaaatcactctttgg caacgaccccttgtcacagtaaaaatagggggacagctaaaggaagctctattagatacaggagcagatgatacagtattagaagaaataa atttgccaggaaaatggaaaccaaaaatgatagggggaattggaggttttatcaaagtaagacagtatgatcaaatacttatagaaatttgtgg aaaaaaggctataggtacagtattagtaggacctacacctgtcaacataattggaagaaatatgttgactcagattggttgtactttaaattrtcca attagtcctattgaaactgtaccagtaaaattaaagccaggaatggatggcccaaaggttaaacaatggccattgacagaagaaaaaataaaa gcattaacagaaatttgtacagaaatggaaaaggaaggaaaaatttcaaaaattgggcctgaaaatccatacaatactccaatatttgccataa agaaaaaagacagtactaaatggagaaaattagtagatttcagagaactcaataaaagaactcaagacttctgggaagttcaattaggaatac cacatccagcagggttaaaaaagaaaaaatcagtaacagtactggatgtgggggatgcatatttttcagttcccttagatgaagacttcaggaa gtatactgcattcaccatacctagtataaacaatgagacaccaggaattagatatcagtacaatgtgcttccacagggatggaaaggatcacc agcaatattccaaagtagcatgacaaaaatcttagagccctttagaacacaaaatccagaaatagttatctatcaatacatggatgatttgtatgt aggatctgacttagaaatagggcagcatagaacaaaaatagaggagttaagagaacatctattgaggtggggatttaccacaccagacaaa aaacatcagaaagaacctccatttctttggatgggatatgaactccatcctgacaaatggacagtacagcctatacagctgccagaaaaagac agctggactgtcaatgatatacagaagttagtgggaaaactaaattgggcaagtcagatttatccagggattaaagtaaagcaattatgtaaac tccttaggggagccaaagcactaacagacatagtaccactaactgaagaagcagaattagaattggcagagaacagggaaattctaaaaga accagtacatggagtatattatgacccatcaaaagacttaatagcagaaatacagaaacaagggcaagaccaatggacatatcaaatttatca agagccatttaaaaatctgaaaacaggaaaatatgcaaaaatgaggtctgcccacactaatgatgtaaaacaattaacagaagcagtgcaaa aaatagccacagaaagcatagtaatatggggaaagactcctaaatttagactacccatacaaaaagaaacatgggaaacatggtggacaga gtattggcaagccacctggattcctgagtgggagtttgtcaatacccctcctctagtaaaattatggtaccagttagaaaaagaacccatagta ggagcagaaactttctatgtagatggggcagctaatagggagactaaactaggaaaagcaggatatgttactgacagaggaagacaaaaa gttgtttccctaactgagacaacaaatcagaagactgaattacaagcaattcatctagctttgcaggattcaggatcagaagtaaacatagtaac agactcacagtatgcattaggaatcattcaagcacaaccagataagagtgaatcagagttagtcaatcaaataatagagcagttaataaaaaa ggaaaaggtctacctgtcatgggtaccagcacacaaaggaattggaggaaatgaacaagtagataaattagtcagtactggaatcaggaaa gtactatttttagatgggatagataaggctcaagaagaacatgaaaaatatcacagcaattggagagcaatggctagtgattttaatctgccac ctatagtagcaaaagaaatagtagccagctgtgataaatgtcagctaaaaggggaagccatgcatggacaagtagactgtagtccaggaat atggcaattagattgtacacatttagaaggaaaaattatcctggtagcagtccatgtagccagtggctatatagaagcagaagttatcccagca gaaacaggacaggaaacagcatactttatactaaaattagcaggaagatggccagtaaaagtaatacatacagacaatggcagcaatttcac cagtgctgcagttaaggcagcctgttggtgggcaggtatccaacaggaatttggaattccctacaatccccaaagtcaaggagtagtagaat ctatgaataaagaattaaagaaaatcatagggcaggtaagagatcaagctgaacaccttaagacagcagtacaaatggcagtattcattcaca attttaaaagaaaaggggggattggggggtacagtgcaggggaaagaataatagacataatagcaacagacatacaaactaaagaattaca aaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcagagacccaatttggaaaggaccagcaaaactactctggaaagg tgaaggggcagtagtaatacaagacaatagtgaaataaaggtagtaccaagaagaaaagcaaagatcattagggattatggaaaacagatg gcaggtgatgattgtgtggcaggtagacaggatgaggattag (SEQ ID NO: 19). In another embodiment, the Rev sequence is encoded by a homologue of SEQ ID NO: 19. In another embodiment, the sequence is encoded by a variant of SEQ ID NO: 19. In another embodiment, the sequence is encoded by a fragment of SEQ ID NO: 19. In another embodiment, the sequence is encoded by a homologue of a fragment of SEQ ID NO: 19. "Homologue" may refer to any degree of homology disclosed herein. In another embodiment, the sequence is encoded by a variant of a fragment of SEQ ID NO: 19. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the Rev sequence is encoded by a sequence set forth in one of the following GenBank Accession Numbers: AF407418; AJ302646; AJ302647; AY169802- AY169813, inclusive; AY169815-AY169816; AY489739; AY618998; AY623602; L20571; L20587. In another embodiment, the Rev sequence is encoded by a homologue of one of the above sequences. In another embodiment, the sequence is encoded by a variant of one of the above sequences. In another embodiment, the sequence is encoded by a fragment of one of the above sequences. In another embodiment, the sequence is encoded by a homologue of a fragment of one of the above sequences. "Homologue" may refer to any degree of homology disclosed herein. In another embodiment, the sequence is a variant of a fragment of one of the above sequences. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the Rev sequence of methods and compositions of the present invention is at least 60% homologous to a Rev or Gag-Pol sequence disclosed herein. In another embodiment, the HIV-I REV sequence is at least 65% homologous to a sequence disclosed herein. In another embodiment, the sequence is at least 70% homologous to a sequence disclosed herein. In another embodiment, the sequence is at least 72% homologous to a sequence disclosed herein. In another embodiment, the sequence is at least 74% homologous to a sequence disclosed herein. In another embodiment, the sequence is at least 76% homologous to a sequence disclosed herein. In another embodiment, the sequence is at least 78% homologous to a sequence disclosed herein. In another embodiment, the sequence is at least 80% homologous to a sequence disclosed herein. In another embodiment, the sequence is at least 82% homologous to a sequence disclosed herein. In another embodiment, the sequence is at least 84% homologous to a sequence disclosed herein. In another embodiment, the sequence is at least 86% homologous to a sequence disclosed herein. In another embodiment, the sequence is at least 88% homologous to a sequence disclosed herein. In another embodiment, the sequence is at least 90% homologous to a sequence disclosed herein. In another embodiment, the sequence is at least 92% homologous to a sequence disclosed herein. In another embodiment, the sequence is at least 94% homologous to a sequence disclosed herein. In another embodiment, the sequence is at least 95% homologous to a sequence disclosed herein. In another embodiment, the sequence is at least 96% homologous to a sequence disclosed herein. In another embodiment, the sequence is at least 97% homologous to a sequence disclosed herein. In another embodiment, the sequence is at least 98% homologous to a sequence disclosed herein. In another embodiment, the sequence is at least 99% homologous to a sequence disclosed herein. In another embodiment, the sequence is over 99% homologous to a sequence disclosed herein. Each possibility represents a separate embodiment of the present invention.

As provided herein, peptides of the present invention possess a superior ability to inhibit HIV-I replication and HIV-I viral integrase 3 '-end processing activity, both in vitro, in cells, and in vivo. Further, as provided herein, peptides of the present invention sharply inhibited integration of viral DNA and HIV-I replication in cell culture (Example 6). Thus, peptides of the present invention possess a number of superior properties relative to previously known methods of combating HIV-I infection.

In another embodiment, a peptide of the present invention further comprises an additional (i.e. non- HIV-I Rev) peptide sequence, attached to an end of the HIV-I Rev-derived peptide. In another embodiment, the additional peptide sequence is attached to the N-terminal end of the HIV-I Rev- derived peptide. In another embodiment, the additional peptide sequence is attached to the C- terminal end of the HIV-I Rev-derived peptide, hi another embodiment, the additional peptide sequences are attached to the N-terminal and C-terminal ends of the HIV-I Rev-derived peptide. Each possibility represents a separate embodiment of the present invention.

In another embodiment, a peptide of the present invention further comprises an organic, non- peptidic moiety, hi another embodiment, the peptide of the present invention comprises a hydrophobic moiety attached to the end of the HIV-I Rev-derived peptide. In another embodiment, the hydrophobic moiety is a linear hydrocarbon. In another embodiment, the hydrophobic moiety is a branched hydrocarbon. In another embodiment, the hydrophobic moiety is a linear hydrocarbon. In another embodiment, the hydrophobic moiety is a cyclic hydrocarbon. In another embodiment, the hydrophobic moiety is a polycyclic hydrocarbon. In another embodiment, the hydrophobic moiety is a heterocyclic hydrocarbon. In another embodiment, the hydrophobic moiety is a hydrocarbon derivative. In another embodiment, the hydrophobic moiety is a protecting group. In another embodiment, the protecting group serves to decrease degradation (e.g. of a linear compound).

hi another embodiment, the non-peptidic moiety is attached to the N-terminal end of the HIV-I Rev- derived peptide, hi another embodiment, the non-peptidic moiety is attached to the C-terminal end of the HIV-I Rev-derived peptide. In another embodiment, the non-peptidic moieties are attached to the N-terminal and C-terminal ends of the HIV-I Rev-derived peptide. Each possibility represents a separate embodiment of the present invention.

In another embodiment, an additional peptide sequence is attached to the N-terminal end of the HIV- 1 Rev-derived peptide and a non-peptidic moiety is attached to the C-terminal end. In another embodiment, an additional peptide sequence is attached to the C-terminal end of the HIV-I Rev- derived peptide and a non-peptidic moiety is attached to the N-terminal end. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the additional sequence(s) or moiety(ies) improves a pharmacological property of the peptide. In another embodiment, the additional sequence(s) or moiety(ies) improves a physiological property of the peptide. In another embodiment, the property is penetration into cells (e.g. moieties which enhance penetration through membranes or barriers, generally termed "leader sequences"). In another embodiment, the modified peptides exhibit slower degradation in vivo. In another embodiment, the modified peptides exhibit slower clearance in vivo. In another embodiment, the modified peptides exhibit decreased repulsion by various cellular pumps. In another embodiment, the modified peptides exhibit decreased immunogenicity. In another embodiment, the modified peptides exhibit improved administration to a subject in need. In another embodiment, the modified peptides exhibit improved penetration through an in vivo barriers (e.g. the gut). In another embodiment, the modified peptides exhibit increased specificity for HIV-I integrase. In another embodiment, the modified peptides exhibit increased affinity for HIV-I integrase. In another embodiment, the modified peptides exhibit decreased toxicity. In another embodiment, the modified peptides exhibit improvement in another pharmacological or physiological property. In another embodiment, the modified peptides exhibit improvement in ability to be imaged using an existing technology. Each possibility represents a separate embodiment of the present invention.

The association between the amino acid sequence component of the compound and other components of the compound may be by covalent linking, by non-covalent complexion, for example, by complexion to a hydrophobic polymer, which can be degraded or cleaved producing a compound capable of sustained release; by entrapping the amino acid part of the compound in liposomes or micelles to produce the final compound of the invention. The association may be by the entrapment of the amino acid sequence within the other component (liposome, micelle) or the impregnation of the amino acid sequence within a polymer to produce the final compound of the invention.

Preferably, the HIV-I Rev-derived amino acid sequence is in association with (in the meaning described above) a moiety for transport across cellular membranes.

The term "moiety for transport across cellular membranes" refers to a chemical entity, or a composition of matter (comprising several entities) that causes the transport of members associated (e.g. a HIV-I Rev-derived amino acid sequence) with it through phospholipidic membranes. Examples of such moieties are hydrophobic moieties such as linear, branched, cyclic, polycyclic or hetrocyclic substituted or non-substituted hydrocarbons. Another example of such a moiety are short peptides that cause transport of molecules attached to them into the cell by, gradient derived, active, or facilitated transport. Other examples of other non-peptidic moieties known to be transported through membranes are glycosylated steroid derivatives, which are well known in the art. The moiety of the compound may be a polymer, liposome or micelle containing, entrapping or incorporating the amino acid sequence therein. In the above examples, the compound of the invention is the polymer, liposome micelle etc. impregnated with the amino acid sequence.

Suitable functional groups for increasing transport across cellular membranes are described in Green and Wuts, "Greene 's Protective Groups in Organic Synthesis " John Wiley and Sons, 2007, the teachings of which are incorporated herein by reference. Preferred protecting groups are those that facilitate transport of the compound attached thereto into a cell, for example, by reducing the hydrophilicity and increasing the lipophilicity of the compounds.

These moieties can be cleaved in vivo, either by hydrolysis or enzymatically, inside the cell. (Ditter et al, J. Pharm. Sci. 57:783 (1968); Ditter et al., J. Pharm. Sci. 57:828 (1968); Ditter et al., J. Pharm.

Sci. 58:557 (1969); King et al, Biochemistry 26:2294 (1987); Lindberg et al, Drug Metabolism and

Disposition 17:311 (1989); and Tunek et al, Biochem. Pharm. 37:3867 (1988), Anderson et al,

Arch. Biochem. Biophys. 239:538 (1985) and Singhal et al, FASEB J. 1:220 (1987)). Hydroxyl protecting groups include esters, carbonates and carbamate protecting groups. Amine protecting groups include alkoxy and aryloxy carbonyl groups, as described above for N-terminal protecting groups. Carboxylic acid protecting groups include aliphatic, benzylic and aryl esters, as described above for C-terminal protecting groups. In one embodiment, the carboxylic acid group in the side chain of one or more glutamic acid or aspartic acid residue in a compound of the present invention is protected, preferably with a methyl, ethyl, benzyl or substituted benzyl ester, more preferably as a benzyl ester.

Examples of N-terminal protecting groups include acyl groups (-CO-R1) and alkoxy carbonyl or aryloxy carbonyl groups (-CO-O-R1), wherein Rl is an aliphatic, substituted aliphatic, benzyl, substituted benzyl, aromatic or a substituted aromatic group. Specific examples of acyl groups include acetyl, (ethyl)-CO-, n-propyl-CO-, iso-propyl-CO-, n-butyl-CO-, sec-butyl-CO-, t-butyl-CO-, hexyl, lauroyl, palmitoyl, myristoyl, stearyl, oleoyl phenyl-CO-, substituted phenyl-CO-, benzyl-CO- and (substituted benzyl)-CO~. Examples of alkoxy carbonyl and aryloxy carbonyl groups include CH3-O-CO-, (ethyl)-O-CO-, n-propyl-O-CO-, iso-propyl-O-CO-, n-butyl-O-CO-, sec-butyl-O-CO, t-butyl-O-CO, phenyl-O- CO-, substituted phenyl-O-CO- and benzyl-O-CO-, (substituted benzyl)- O-CO-. Adamantan, naphtalen, myristoleyl, tuluen, biphenyl, cinnamoyl, nitrobenzoy, toluoyl, furoyl, benzoyl, cyclohexane, norbornane, Z-caproic. In order to facilitate the N-acylation, one to four glycine residues can be present in the N-terminus of the molecule.

The carboxyl group at the C-terminus of the compound can be protected, for example, by an amide (i.e., the hydroxyl group at the C-terminus is replaced with -NH 2, -NHR2 and -NR2R3) or ester (i.e. the hydroxyl group at the C-terminus is replaced with -OR2). R2 and R3 are independently an aliphatic, substituted aliphatic, benzyl, substituted benzyl, aryl or a substituted aryl group. In addition, taken together with the nitrogen atom, R2 and R3 can form a C4 to C8 heterocyclic ring with from about 0-2 additional heteroatoms such as nitrogen, oxygen or sulfur. Examples of suitable heterocyclic rings include piperidinyl, pyrrolidinyl, morpholino, thiomorpholino or piperazinyl. Examples of C-terminal protecting groups include -NH2, -NHCH3, -N(CH3)2, -NH(ethyl),

-N(ethyl)₂, -N(methyl) (ethyl), -NH(benzyl), -N(C₁-C₄ alkyl)(benzyl), -NH(phenyl), -N(Ci-C₄ alkyl) (phenyl), -OCH3, -O-(ethyl), -O-(n-propyl), -O-(n-butyl), -O-(iso-propyl), -O-(sec- butyl), -O-(t-butyl), -O-benzyl and -O-phenyl.

Derivatives

There are several types of derivation that can be applied to HIV-I Rev-derived amino acid sequences of the present invention. The derivative may include several types of derivation (replacements and deletions, chemical modification, change in peptidic backbone etc.)

Replacements

Typically no more than 40% of the amino acids are replaced by a naturally or non-naturally occurring amino acid or with a peptidomimetic organic moiety. Preferably no more than 35%, 30%, 25%, 20%, 15%, 10%, or 5%. The replacement may be by naturally occurring amino acids (both conservative and non-conservative substitutions), by non-naturally occurring amino acids (both conservative and non-conservative substitutions), or with organic moieties which serve either as true peptidomimetics (i.e. having the same steric and electrochemical properties as the replaced amino acid), or merely serve as spacers in lieu of an amino acid, so as to keep the spatial relations between the amino acid spanning this replaced amino acid. Guidelines for the determination of the replacements and substitutions are provided below. Preferably no more than, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the amino acids are replaced.

The term "naturally occurring amino acid" refers to a moiety found within a peptide and is represented by -NH-CHR-CO-, wherein R is the side chain of a naturally occurring amino acid.

The term "non-naturally occurring amino acid" (amino acid analog) is either a peptidomimetic, or is a D or L residue having the following formula: -NH-CHR-CO-, wherein R is an aliphatic group, a substituted aliphatic group, a benzyl group, a substituted benzyl group, an aromatic group or a substituted aromatic group and wherein R does not correspond to the side chain of a naturally-occurring amino acid. This term also refers to the D-amino acid counterpart of naturally occurring amino acids. Amino acid analogs are well-known in the art; a large number of these analogs are commercially available. Many times the use of non-naturally occurring amino acids in the peptide has the advantage that the peptide is more resistant to degradation by enzymes which fail to recognize them.

The term "conservative substitution" in the context of the present invention refers to the replacement of an amino acid present in the native sequence in the specific peptide with a naturally or non- naturally occurring amino or a peptidomimetic having similar steric properties. Where the side-chain of the native amino acid to be replaced is either polar or hydrophobic, the conservative substitution should be with a naturally occurring amino acid, a non-naturally occurring amino acid or with a peptidomimetic moiety which is also polar or hydrophobic (in addition to having the same steric properties as the side-chain of the replaced amino acid).

As the naturally occurring amino acids are grouped according to their properties, conservative substitutions by naturally occurring amino acids can be easily determined, bearing in mind the fact that, in accordance with the invention, replacement of charged amino acids by sterically similar non- charged amino acids are considered as conservative substitutions.

For producing conservative substitutions by non-naturally occurring amino acids, it is also possible to use amino acid analogs (synthetic amino acids) that are known in the art. When affecting conservative substitutions, the substituting amino acid should have the same or a similar functional group in the side chain as the original amino acid.

The following are some non-limiting examples of groups of naturally occurring amino acids or of amino acid analogs are listed bellow. Replacement of one member in the group by another member of the group will be considered herein as conservative substitutions:

Group I includes leucine, isoleucine, valine, methionine, phenylalanine, cysteine, and modified amino acids having the following side chains: ethyl, n-butyl, -CH₂CH₂OH, -CH₂CH₂CH₂OH, - CH₂CHOHCH₃ and -CH₂SCH₃. Preferably Group I includes leucine, isoleucine, valine and methionine.

Group π includes glycine, alanine, valine, cysteine, and a modified amino acid having an ethyl side chain. Preferably Group II includes glycine and alanine.

Group III includes phenylalanine, phenylglycine, tyrosine, tryptophan, cyclohexylmethyl, and modified amino residues having substituted benzyl or phenyl side chains. Preferred substituents include one or more of the following: halogen, methyl, ethyl, nitro, methoxy, ethoxy and -CN. Preferably, Group III includes phenylalanine, tyrosine and tryptophan.

Group IV includes glutamic acid, aspartic acid, a substituted or unsubstituted aliphatic, aromatic or benzylic ester of glutamic or aspartic acid (e.g., methyl, ethyl, n-propyl iso-propyl, cyclohexyl, benzyl or substituted benzyl), glutamine, asparagine, CO-NH-alkylated glutamine or asparagine (e.g., methyl, ethyl, n-propyl and iso-propyl) and modified amino acids having the side chain -(CH₂) ₃_COOH, an ester thereof (substituted or unsubstituted aliphatic, aromatic or benzylic ester), an amide thereof and a substituted or unsubstituted N-alkylated amide thereof. Preferably, Group IV includes glutamic acid, aspartic acid, glutamine, asparagine, methyl aspartate, ethyl aspartate, benzyl aspartate and methyl glutamate, ethyl glutamate and benzyl glutamate.

Group V includes histidine, lysine, arginine, N-nitroarginine, β-cycloarginine, μ-hydroxyarginine, N-amidinocitruline and 2-amino-4- guanidinobutanoic acid, homologs of lysine, homologs of arginine and ornithine. Preferably, Group V includes histidine, lysine, arginine, and ornithine. A homolog of an amino acid includes from 1 to about 3 additional methylene units in the side chain. Group VI includes serine, threonine, cysteine and modified amino acids having C1-C5 straight or branched alkyl side chains substituted with -OH or -SH. Preferably, Group VI includes serine, cysteine, and threonine.

The term "non-conservative substitutions" concerns replacement of the amino acid as present in the native peptide by another naturally or non-naturally occurring amino acid, having different electrochemical and/or steric properties, for example as determined by the fact the replacing amino acid is not in the same group as the replaced amino acid of the native peptide sequence. Those non- conservative substitutions which fall under the scope of the present invention are those which still constitute a compound having integrase inhibiting activities.

In another embodiment, a "non-conservative substitution" is a substitution in which the substituting amino acid (naturally occurring or modified) has significantly different size, configuration and/or electronic properties compared with the amino acid being substituted. Thus, the side chain of the substituting amino acid can be significantly larger (or smaller) than the side chain of the native amino acid being substituted and/or can have functional groups with significantly different electronic properties than the amino acid being substituted. Examples of non-conservative substitutions of this type include the substitution of phenylalanine or cycohexylmethyl glycine for alanine, isoleucine for glycine, or -MH-CH[(-CH2)5_COOH]-CO- for aspartic acid.

In another embodiment, a functional group may be added to the side chain, deleted from the side chain or exchanged with another functional group. Examples of non-conservative substitutions of this type include adding an amine or hydroxyl, carboxylic acid to the aliphatic side chain of valine, leucine or isoleucine, exchanging the carboxylic acid in the side chain of aspartic acid or glutamic acid with an amine or deleting the amine group in the side chain of lysine or ornithine. In yet another alternative, the side chain of the substituting amino acid can have significantly different steric and electronic properties from the functional group of the amino acid being substituted. Examples of such modifications include tryptophan for glycine, lysine for aspartic acid and -(CH2)4

COOH for the side chain of serine. These examples are not meant to be limiting.

A "peptidomimetic organic moiety" can be substituted for amino acid residues in the compounds of this invention both as conservative and as non-conservative substitutions. These peptidomimetic organic moieties either replace amino acid residues of essential and non-essential amino acids or act as spacer groups within the peptides in lieu of deleted amino acids (of non-essential amino acids). The peptidomimetic organic moieties often have steric, electronic or configurational properties similar to the replaced amino acid and such peptidomimetics are used to replace amino acids in the essential positions, and are considered conservative substitutions. However, such similarities are not necessarily required. The only restriction on the use of peptidomimetics is that the peptides retain their integrase inhibiting properties or HIV-replication inhibiting properties/ or equilibrium shifting properties as defined above.

Peptidomimetics are often used to inhibit degradation of the peptides by enzymatic or other degradative processes. The peptidomimetics can be produced by organic synthetic techniques.

Examples of suitable peptidomimetics include D amino acids of the corresponding L amino acids, tetrazol (Zabrocki et ah, J. Am. Chem. Soc. 110:5875-5880 (1988)); isosteres of amide bonds (Jones et ah, Tetrahedron Lett. 29: 3853-3856 (1988));

LL-3-amino-2-propenidone-6-carboxylic acid (LL- Acp) {Kemp et ah, J. Org. Chem. 50:5834-5838 (1985)). Similar analogs are shown in Kemp et ah, Tetrahedron Lett. 29:5081-5082 (1988) as well as Kemp et ah, Tetrahedron Lett. 29:5057-5060 (1988), Kemp et ah, Tetrahedron Lett. 29:4935-4938 (1988) and Kemp et ah, J. Org. Chem. 54:109-115 (1987). Other suitable peptidomimetics are shown in Nagai and Sato, Tetrahedron Lett. 26:647-650 (1985); Di Maio et ah, J. Chem. Soc. Perkin Trans., 1687 (1985); Kahn et ah, Tetrahedron Lett. 30:2317 (1989); Olson et ah, J. Am. Chem. Soc. 112:323-333 (1990); Garvey et ah, J. Org. Chem. 56:436 (1990). Further suitable peptidomimetics include hydroxy- 1,2,3,4-tetrahydroisoquinoline- 3-carboxylate (Miyake et ah, J. Takeda Res. Labs 43:53-76 (1989)); 1,2,3,4-tetrahydro- isoquinoline-3-carboxylate (Kazmierski et ah, J. Am. Chem. Soc. 133:2275-2283 (1991)); histidine isoquinolone carboxylic acid (HIC) (Zechel et ah, Int. J. Pep. Protein Res. 43 (1991)); (2S, 3S)-methyl-phenylalanine, (2S, 3R)-methyl-phenylalanine, (2R, 3S)-methyl- phenylalanine and (2R, 3R)-methyl-phenylalanine (Kazmierski and Hruby, Tetrahedron Lett. (1991)).

Chemical modifications: Typically no more than 40%, preferably 35%, 30%, 25%, 15%, 10%, or 5% of the amino acids have their side chains modified. The modification means the same type of amino acid residue, but to its side chain a functional group has been added. For example, the side chain may be phosphorylated, glycosylated, fatty acylated, acylated, iondiated or carboxyacylated.

Deletions: The deletion may be of terminal or non-terminal amino acids to result either in deletion of non terminal amino acid or in a fragment having at least 3,4,5,6,7,8,9,10,11,12 amino acids.

Combination of modifications;

Generally at least 50%, at least 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10, or 5% of the amino acids in the parent sequence of (a)-(b) are maintained so that the combination of the deletions, chemical modifications, and replacements are no more than 50% of the total peptides as long as they retain the property of equilibrium shifting, integrase inhibition or HIV-I replication inhibition.

It should be appreciated that some of the derivatives are not active. Those derivatives that fall under the scope of the invention are those that can inhibit the HIV-I replication, preferably those that inhibit the viral integrase activity, most preferably those that can cause a shift in the oligeramization equilibrium shift in a similar manner to the parent protein of (a)-(b)).

Typically "essential amino acids" (as will be detailed below) are maintained or replaced by conservative substitutions while non-essential amino acids may be maintained, deleted or replaced by conservative or non-conservative replacements. Generally, essential amino acids are determined by various Structure-Activity-Relationship (SAR) techniques (for example amino acids when replaced by Ala cause loss of activity) are replaced by conservative substitution while non-essential amino acids can be deleted or replaced by any type of substitution. Guidelines for the determination of the deletions, replacements and substitutions are given

Other types of derivatives: D-amino acids

The term "corresponding D-amino acid" refers to the replacement of the naturally occurring L- configuration of the natural amino acid residue by the D-configuration of the same residue. Peptidic backbone modifications (peptidomimetics)

The term "at least one peptidic backbone has been altered to a non-naturally occurring peptidic backbone" means that the bond between the N- of one amino acid residue to the C- of the next has been altered to non-naturally occurring bonds by reduction (to -CH₂-NH-), alkylation (methylation) on the nitrogen atom, or the bonds have been replaced by, urea bonds, or sulfonamide bond, etheric bond (-CH₂-O-), thioetheric bond (-CH₂-S-), or to -CS-NH-. The side chain of the residue may be shifted to the backbone nitrogen to obtain N-alkylated-Gly (a peptoid) as well as aza peptides

Reverse order: The term "in reverse order" refers to the fact that the sequence of (a) to (c) may have the order of the amino acids as it appears in the native protein, or may have the reversed order (as read in the C-to N- direction). It has been found that many times sequences having such a reverse order can have the same properties, in small peptides, as the "correct" order, probably due to the fact that the side chains, and not the peptidic backbones are those responsible for interaction with other cellular components. Particularly preferred are "retro inverso" peptides - i.e. peptides that have both a reverse order as explained above, and in addition each and every single one of the amino acids, has been replaced by the non-naturally occurring D- amino acid counterpart, so that the net end result, as regards the positioning of the side chains, (the combination of reverse order and the change from L to D) is zero change. Such retro-inverso peptides, while having similar binding properties to the native peptide, were found to be resistant to degradation

Preparation of peptides of the present invention

Peptide sequences for producing any of the sequence of the compounds of the invention can be synthesized by solid phase peptide synthesis (e.g., t-BOC or F-MOC) method, by solution phase synthesis, or by other suitable techniques including combinations of the foregoing methods. The t-BOC and F-MOC methods, which are established and widely used, are described in Merrifield, J Am. Chem. Soc, 88:2149 (1963); Meienhofer, Hormonal Proteins and Peptides, CH. Li, Ed., Academic Press, 1983, pp. 48-267; and Barany and Aarifield, in The Peptides, E. Gross and J. Meienhofer, Eds., Academic Press, New York, 1980, pp. 3-285. Methods of solid phase peptide synthesis are described in Aarifield, R.B., Science, 232:341 (1986); Carpino, L.A. and Han, G. Y., J. Org. Chem., 37:3404 (1972); and Gauspohl, H. et al, Synthesis, 5:315 (1992)). The teachings of these references are incorporated herein by reference.

As demonstrated herein, peptides of the present invention inhibit HIV-I IN activity. Without wishing to be limited by theory, is believed that the observed inhibition of IN is due to steric hindrance of its active site, which is accessible only to the short Rev-derived peptides but not to the full-length Rev protein or oligopeptides. HIV-I Rev is a karyophilic protein, which is required at the late phase of the viral life cycle for promoting nuclear export of partially-spliced or un-spliced viral RNA.

Further, the present invention demonstrates that peptides of the present invention inhibit HIV-I replication in cultured cells at low micromolar concentrations, as shown by three different, unrelated assay systems. Without wishing to be limited by theory, is believed that these peptides inhibit viral IN in vivo, as indicated by the correlation between their inhibition of IN enzymatic activity in vitro and their ability to reduce HIV-I replication.

Without wishing to be limited by theory, it is believed that the shift in IN oligomerization equilibrium, caused by the Rev derived peptides, affects the DNA binding of IN. When the peptide is added to free IN, it shifts its oligomerization equilibrium towards a tetramer with significantly reduced affinity for DNA. When DNA was added before the peptides, most IN is DNA-bound when the peptide is added. The peptide can then shift the equilibrium only of the free IN fraction. Indeed, inhibition at this order of addition (peptide added to a preformed IN- DNA complex) became more pronounced when the peptide: IN ratio increased (Figure 12C-D). Around 60% inhibition was observed at 5:1 peptide: IN ratio, compared to only around 20% inhibition at 1:1 ratio. This is likely because the peptide more efficiently shifted the equilibrium of the free IN fraction towards the inactive tetramer, causing in turn dissociation of the IN: DNA complex.

The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention. EXPERIMENTAL DETAILS SECTION

MATERIALS AND EXPERIMENTAL METHODS

Cells - Monolayer adherent HeLa and HEK293T cells were grown in Dulbecco's Modified Eagle's Medium (DMEM). The T-lymphocyte cell lines Sup Tl and H9, provided by the NIH Reagent Program (Division of AIDS, NIAID, NIH, USA), was grown in RPMI 1640 medium. HeLa MAGI cells (TZM-bl) were obtained through the NIH Reagent Program and were grown in DMEM. Cells were incubated at 37⁰C in a 5% CO₂ atmosphere. All media were supplemented with 10% (v/v) fetal calf serum (FCS), 0.3 g/L L-glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin (Biological Industries, Beit Haemek, Israel).

Viruses - Wild-type HIV-I was generated by transfection of HEK293T cells with pSVC21 plasmid containing the full-length HIV-1_HXB2 viral DNA. Wild type and Δenv/VSV-G viruses were harvested from HEK293T cells 48 and 72 h post-transfection with pSVC21 Δenv. The viruses were stored at -75⁰C.

Infection of cultured cells- Cultured lymphocytes (1 x 10⁵) were centrifuged for 5 min at 2000 rpm, the supernatant was aspirated, and the cells were resuspended in 0.2 to 0.5 ml medium containing virus at a multiplicity of infection (m.o.i.) 0.1 and 2. Following absorption for 1 h at 37⁰C, cells were washed to discard unbound virus and incubated for an additional 1 to 10 days.

HIV-I titration multinuclear activation of a galactosidase indicator (MAGI) assay - Titration of HIV-I was carried out by the MAGI assay, as follows: TZM-bl cells were transferred to 96-well plates at 10 x 10³ cells per well. On the following day, the cells were infected with 50 μl of serially diluted HIV-I Δenv/VSV-G virus in the presence of 20 μg/ml DEAE-dextran (Pharmacia). Following a 2-hour incubation, 150 μl of DMEM media was added. Two days postinfection, cultured cells were fixed with 1% (v/v) formaldehyde and 0.2% (v/v) glutaraldehyde in PBS. Following an intensive wash with PBS, cells were stained with a solution of 4 mM potassium ferrocyanide, 4 mM potassium ferricyanide, 2 mM MgCl₂ and 0.4 mg/ml X-GaI (Ornat, Israel). Blue cells were counted under a light microscope at 200 X magnification. P24 assay - H9 lymphoid cells were incubated with the indicated peptides for 2 h, infected with wild type HIV-I at a m.o.i of 0.1, and incubated for 10 days. P24 content was determined using the capture assay kit (SAIC, AIDS Vaccine Program, Frederick, MD)₅ according to the manufacturer's instructions.

Plasmid construction- AU plasmids used in this study were constructed using PCR cloning techniques with the high-fidelity enzyme Platinum Pβc DNA polymerase (Invitrogen). Clones were subjected to automated DNA sequencing. For bimolecular fluorescence complementation (BiFC) experiments, yeast multicopy shuttle vectors pRS423 (with HIS3 as the selective marker) and pRS426 (with URA3 as the selective marker), both with the ADHl promoter, were used as the cloning plasmids (provided by D. Engelberg, Alexander Silberman Institute, The Hebrew University of Jerusalem). The DNA-coding region of the two yeast Green Fluorescent Protein (GFP) fragments, namely the N terminus (GN; GFP amino acids 1-154), and the C terminus (GC; GFP amino acids 155-239), were cloned into pRS423 and pRS426, respectively. A linker consisting of (GGS)₅ was used to separate the inserted genes. The final vectors were termed "GN-linker" and "linker-GC," respectively. The coding sequences of full-length HIV-I IN, Rev and Tat were amplified by PCR and inserted in-frame into the corresponding sites of the GN- linker in the C-terminal fragments of the GN, resulting in GN-IN, GN-Rev and GN-Tat.

For co-immunoprecipitation experiments, mammalian IN and Rev expression vectors were constructed by PCR amplification of HIV-I Rev and IN proteins and ligation into the pcDNA3.1 (Invitrogen) expression vector.

BiFC assay to study protein-protein interactions - The plasmids described above were transformed into the yeast strain EGY48 (Clontech), and cells were grown on Yeast Nitrogen Base (YNB) medium lacking histidine and uracil. After 48 h at 3O⁰C, plates were transferred to a 23⁰C incubator for 2 to 3 days. A sample of colonies was transferred to a PBS solution, and appearance of fluorescence was visualized by confocal microscope (MRC 1024 confocal imaging system, Bio-Rad).

Study of in vivo protein-protein interactions by co-immunoprecipitation- HEK293T cells were transfected with 5 μg of pcDNA3.1 bearing the Rev and IN genes using the calcium phosphate method. After 48 h, cells were harvested and washed three times with PBS and then lysed by the addition of PBS containing 1% (v/v) NP-40. Following centrifugation, a monoclonal Anti-Rev antibody (mabRev) (NIH AIDS Research & Reference Reagent Program, catalog number 7376) was added to the supernatant for 1 h at 4⁰C. Following 3 h incubation with protein-G-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4⁰C, the samples were washed three times with PBS containing 1% NP-40. SDS buffer was added to the samples and after boiling and Western blotting, membranes were incubated with antiserum raised against IN amino acids 276- 288 (NIH AIDS Research & Reference Reagent Program catalog number 758). Finally, membranes were stained with secondary anti-rabbit-HRP-conjugated antibody (Santa Cruz Biotechnology).

ELISA-based binding assays — Maxisorp plates (Nunc) were incubated at room temperature for 2 h with 200 μl of solution from a stock solution of 25 μg/ml of Rev-GFP in carbonate buffer. After incubation, solution was removed, plates were washed three times with PBS, and 200 μl of 5% BSA (Sigma) in PBS (w/v) was added for 2 h at room temperature. After rewashing with PBS, biotin-labeled BSA-IN was dissolved in PBS containing 5% BSA and further incubated for 1 h at room temperature. Following three washes with PBS, streptavidin- horseradish peroxidase (HRP) conjugate (Sigma) was added and the concentration of bound biotinylated molecules was estimated. Enzymatic activity of HRP was estimated by monitoring the optical density (OD) at 490 nm of the product using an ELISA plate reader (Tecan Sunrise). Results given are averages of at least three ELISA determinations wherein standard deviations never exceeded ±20%. Each measurement was performed in duplicate.

Protein expression and purification - The GST-IN expression vector was provided by Dr. A. Cereseto. The histidine-tagged IN expression vector was provided by Dr. A. Engelman

GST pull-down - 15 μg GST-IN or GST were incubated for 30 min at room temperature with 10 μl glutathione beads (Sigma) in 200 μl of buffer A (100 mM NaCl, 5% glycerol, 1 mM DTT and 50 mM Tri-HCl pH 7.5) containing 0.25% NP-40. After washing with buffer A, beads were re- suspended in 200 μl of buffer A supplemented with 0.25% NP-40, 0.1% (v/v) Na-deoxycholate, and 2 μg histidine-tagged GFP or Rev-GFP, for 30 min at room temperature. Following three washes with buffer A, SDS buffer was added, and samples were boiled and analyzed by western blotting using anti-His-tag antibody (Santa Cruz Biotechnology).

Cell-penetration experiments - Fluorescein-labeled peptides at a final concentration of 10 μM in PBS were incubated with HeLa cells for 2 h at 37⁰C. After three washes in PBS, non-fixed cells were visualized by fluorescence microscopy.

In-vitro 3 '-end processing and strand transfer assays (Examples 1-7)

The following gel-purified oligonucleotides were used in the enzymatic assays of HIV-I IN: A (21-mer), 5'-GTGTGGAAAATCTCTAGCAGT-S'- SEQ ID NO: 20; B (21-mer), 5'-

ACTGCTAGAGATTTTCCACAC-3'- SEQ ID NO: 21; C (19-mer), 5'-

GTGTGGAAAATCTCTAGCA-S'- SEQ ID NO: 22; D (38-mer), 5'-

TGCTAGTTCTAGCAGGCCCTTGGGCCGGCGCTTGCGCC-3'- SEQ ID NO: 23.

Oligonucleotides A-C correspond to the U5 end of the HIV-I long terminal repeat. Underlined letters indicate the highly conserved CA/TG dinucleotide pair. Oligonucleotide C is identical to

A, after the removal of the GT dinucleotides from its 3 '-end and thus after annealing to oligonucleotide B, creating a dinucleotide overhang at the 5 '-end of oligonucleotide B.

Oligonucleotide D, termed "dumbbell," folds to form a structure mimicking the integration intermediate. In order to test the 3'-end processing and the resulting strand transfer activity of IN, the 5'-end-labeled oligonucleotide A, annealed to its complementary strand, oligonucleotide B

(both 21 nucleotides long), was used. The duplex of oligonucleotides C and B were used for assaying the 3 '-end processing activity.

5 '-End Labeling and Substrate Preparations — Fifty pmol of oligonucleotides A, C, or D were 5'- end-labeled using 1 unit of T4 polynucleotide kinase and 50 μCi Of³²P-ATP, in a final volume of 50 μl of the appropriate buffer (supplied by the manufacturer) for 30 min at 37 ⁰C. Samples were then heat-inactivated. 5'-end-labeled oligonucleotides A or C were annealed each to an equimolar amount of oligonucleotide B in 55 niM Tris-HCl (pH 7.5) and 0.27 M NaCl.

Assays of the 3 '-End Processing and Strand Transfer (or DNA Joining). In the strand transfer assays described, the labeled 5 '-end substrate employed served as both the target and donor DNA leading to an increase in the molecular size of the substrate, whereas in the 3 '-end processing assays, unique cleavage of the labeled substrates was followed. All reactions were performed in 10-μl reaction mixtures with 0.33 pmol of the labeled duplex DNA substrate and the reaction buffer, containing 90 rnM NaCl₃ 7.5 mM MnCl₂, 10 mM DTT, 0.1 mg/ml BSA, 25 mM MOPS (pH 7.2) and 5% glycerol. 500 ng of HIV-I IN (which equals 8 pmol, assuming IN dimers of the 32-kDa subunits) was assayed. The HIV-I IN was pre-incubated on ice for 5 min the absence or the presence of increasing concentrations of the tested peptides. Reactions were initiated after adding the labeled DNA substrate in the reaction buffer, incubated for 30 min at 37 ⁰C, and then stopped by adding 10 μl of foπnamide loading buffer (90% formamide, 10 mM EDTA, 1 mg/ml bromphenol blue, 1 mg/ml xylene cyanole). Samples were heat-denatured, cooled on ice, and loaded onto 6 M urea, 14% polyacrylamide denaturing gels, followed by electrophoresis (urea- PAGE). The gels were dried and subjected to autoradiography at -80 ⁰C or at room temperature to obtain essentially linear exposures.

Quantitative estimation ofintegrase catalytic activity in vitro (Examples 8-10) An oligodinucleotide IN substrate was used, in which one oligo (5'- ACTGCTAGAGATTTTCCACACTGACTAAAAGGGTC; SEQ ID NO: 24) was labeled with biotin on the 3' end and the other oligo (5'-

GACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGT; SEQ ID NO: 25) was labeled with digoxigenin at the 5' end. The final reaction mixture contained 390 nM IN, lμM double strand oligonucleotide DNA, 20 mM Hepes (pH 7.5), 10 mM MgCl₂, 10 mM dithiothreitol, 10%Me₂SO, 5% PEG-8000, 0.1 mg/ml BSA at 40 μl. When peptide inhibition was tested the IN was preincubated with the peptide for 10 min prior to addition of the DNA substrate. Following a 1-h incubation at 37 °C, 60 μl of a buffer containing 20 mM Tris-HCl (pH 8), 400 mM NaCl, 10 mM EDTA, and salmon sperm DNA was added. In other experiments, IN was first incubated with the DNA substrate, and then peptides were added at the indicated molar ratios. The integrase reaction was followed by an immunosorbent assay on avidin-coated plates, as described above.

Peptide synthesis, labeling and purification - Peptides were synthesized on an Applied Biosystems (ABI) 433A peptide synthesizer using standard Fmoc chemistry. Amino acids were purchased from NOVAbiochem and Bio-Lab (Jerusalem, Israel). Peptides were labeled at the N terminus with 5' (and 6') carboxyfluorescein succinimidyl ester (Molecular Probes) using a fourfold excess of fluorescein and of hydroxybenzotriazole (HoBt). Peptides were purified on a Gilson HPLC using a reverse-phase C8 semi-preparative column (ACE, C-8 RP) with a gradient from 5 to 60% buffer B in buffer A [buffer A, 0.001% (v/v) trifluoroacetic acid (TFA) in water and buffer B 0.001% (v/v) TFA in acetonitrile]. Identity of the peptides was confirmed using an ABI voyager MALDI TOF mass spectrometer. Sequences of the peptides are summarized in Table 1.

Fluorescence anisotropy - Binding studies were performed at 1O⁰C using a PerkinElmer LS-50b luminescence spectrofluorometer equipped with a Hamilton Microlab M dispenser. Fluorescein- labeled peptides were dissolved in 20 mM Tris buffer pH 7.4 at the desired ionic strength to a final concentration of 0.05 to 0.1 μM. A 1 ml aliquot of the peptide solution was placed in a cuvette, and 200 μl of 100 μM IN protein (IN molarity was calculated assuming IN monomer) was titrated into the peptide solution in 20 steps of 10 μl each at 15 min intervals. Fluorescence anisotropy was measured after each addition, using excitation and emission wavelengths of 480 and 530 nm, respectively. Bandwidths were changed depending on the concentration of the labeled molecule used. Data were fitted to the Hill equation:

wherein R is the measured fluorescence anisotropy value, ΔR is the amplitude of the fluorescence change from the initial value (peptide only) to the final value (peptide in complex), [IN] is the protein concentration added, R₀ is the starting fluorescence anisotropy value, corresponding to the free peptide, and K_a is the association constant, which is equal to 1/Kd (the dissociation constant). Quantization of Integrated HIV-I DNA in the cellular genome - Following incubation of SupTl cells with the indicated peptides for 2 h, cells were infected with a HIV-I Δenv/VSV-G virus at a m.o.i of 2 for 24 h. During first-round PCR, integrated HIV-I sequences were amplified with the HIV-I LTR-specific primer (LTR-TAG-F 5'- ATGCCACGTAAGCGAAACTCTGGCTAACTAGGGAACCCACTG-S'; SEQ ID NO: 26) and Alu-targeting primers (first-Alu-F 5'-AGCCTCCCGAGTAGCTGGGA-S'; SEQ ID NO: 27) and first-Alu-R 5'-TTACAGGCATGAGCCACCG-S'; SEQ ID NO: 28) that annealed to conserved regions of the AIu repeat element. AIu-LTR fragments were amplified from 1/10 of the total cell DNA in a 25-μl reaction mixture containing IX PCR buffer, 3.5 mM MgCl₂, 200 μM dNTPs, 300 nM primers, and 0.025 U/μl Taq polymerase. First-round PCR cycle conditions were as follows: a DNA denaturation and polymerase activation step of 10 min at 95⁰C and then 12 cycles of amplification (95⁰C for 15 s, 6O⁰C for 30 s, 72⁰C for 5 min).

During second-round PCR, the first-round PCR product could be specifically amplified by using the tag-specific primer (tag-F 5'-ATGCCACGTAAGCGAAACTC-S'; SEQ ID NO: 29) and the LTR primer (LTR-R 5'-AGGCAAGCTTTATTGAGGCTTAAG-S'; SEQ ID NO: 30) designed by PrimerExpress™ (Applied Biosystems) using default settings. Second-round PCR was performed on 1/25 of the first-round PCR product in a mixture containing 300 nM of each primer, 12.5 μl of 2X SYBR green master mix (Applied Biosystems) at a final volume of 25 μl, run on an ABI PRIZM 7700 (Applied Biosystems). Second-round PCR cycles began with a DNA-denaturation and polymerase-activation step (95⁰C for 10 min), followed by 50 cycles of amplification (95⁰C for 15 s, 6O⁰C for 60 s). SVC21 plasmid containing full-length HIV-1_HXB2 viral DNA was used to generate a standard linear curve in a range of 5 ng to 0.25 fg (R = 0.99). DNA samples were assayed with quadruplets of each sample.

Effect of peptides on cell viability using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay - Following incubation with the indicated peptides, medium was removed, and cells were incubated in Earl's solution containing 0.3 mg/ml MTT for 1 h. Subsequently, solution was removed, and cells were dissolved in 100 μl DMSO for 10 min at room temperature. DMSO-solubilized cells were transferred to a 96-well ELISA plate, and OD values were monitored at a wavelength of 570 ran. Yeast two-hybrid screening - Peptide aptamer screening was performed as described (17). Yeast strain KFl (MATa trpl-901,leu2-3, 112 his3-200 gal4Δgal80ΔLYS2::GALl-HIS3 GAL2-ADE2 met2::GAL7- lacZ SPAL10-URA3) was used for the screening. The HIV-I integrase was fused in frame with the GAL4 DNA-binding domain in vector pPC97, used as a bait. The yeast prey vector pADTRX encodes the Escherichia coli thioredoxin A (trxA) gene fused to the Gal4 activation domain. The 20 amino acid randomized peptide library was inserted into the RsrII site of trxA, which corresponds to a constrained loop and was estimated to have a complexity of 2 x 10⁸. Yeast strain KFl₅ containing HIV-I integrase, was transformed with the peptide aptamer expression library by the LiAC method. First, growth was selected for growth on media lacking adenine. Subsequently, KFl positive transformants were transferred into replicas plated on media lacking histidine and uracil. The plasmid, encoding the interacting peptide aptamer, was isolated from the yeast strain. Recovered plasmids were transferred into E. coli DH5α for isolation and the DNA sequence of the insert determined. Finally, recovered plasmids were retransformed into the yeast bait strains to confirm a specific interaction.

EXAMPLE 1: HIV-I IN and Rev proteins interact when expressed in yeast and mammalian cultured cells

HIV-I Rev and IN proteins were shown to interact with each other in yeast cells using the BiFC assay. In this assay, two proteins of interest are fused to non-fluorescent N- or C terminal halves of the GFP molecule ("GN" and "GC"). Intracellular restoration of the GFP's fluorescence indicates interaction between the two fused proteins (4,7). Interaction between the Rev and IN proteins was shown by the appearance of fluorescence within yeast cells expressing fusions of these two proteins to GFP fragments (Figure IE-F). Fluorescence was seen within the cells' nuclei, suggesting that the Rev-IN interaction either did not disturb the karyophilic properties of these two proteins or occurred following their nuclear import (Figure IF). No fluorescence was observed when one of the expressing vectors lacked the interacting protein, either Rev (Figure IG-F) or IN (Figure H-J), and contained only the GFP fragment, demonstrating the specificity of the assay. As an additional control for specificity, no interaction was observed when the HIV-I Tat protein was allowed to interact with IN (Figure IK-L), indicating the specificity of the interaction. When expressed in yeast cells, the IN and Rev proteins retained their ability to form homo-oligomers (Figure IA-D), indicating preservation of their three-dimensional structures. Rev-IN interaction had no effect on the biological function of the IN protein, as shown by the finding that HIV-I replication was unaffected by overexpression of Rev protein (Figure IM).

Co-immunoprecipitation in mammalian cells was utilized to confirm the interaction between Rev and IN. Un-tagged Rev and IN interacted with each other in mammalian cells (Figure 2).

EXAMPLE 2: Interaction of IN and Rev in cell-free systems

In vitro binding assay systems, with recombinant purified proteins, were used to further characterize the interaction between Rev and IN. Since the recombinant Rev protein is highly insoluble, a Rev-GFP conjugate that is soluble and functional (6) was used. Purified IN molecules are also highly insoluble and therefore were linked covalently to BSA (BSA-IN) (8). ELISA demonstrated that biotin-labeled BSA-IN (Bb-IN) binds to Rev-GFP with an apparent Kd of 13 nM (Figure 3A). No binding was observed between Bb alone and Rev-GFP, or between Bb-IN and GFP alone (Figure 3A), demonstrating the specificity of the assay. As further indication of the specificity of the Rev-IN interaction, the interaction was inhibited with an excess of Rev-GFP but not by an unrelated protein such as carbonic anhydrase (Figure 3B).

An in vitro pull-down assay system further confirmed the specific interaction between HIV-I Rev and IN. Rev-GFP bound to GST-IN conjugates (Figure 3C). Rev-GFP did not bind to GST alone, and GST-IN did not interact with GFP, again verifying the specificity of the interaction.

EXAMPLE 3: Elucidation of the Rev regions that mediate interaction with IN

Peptide mapping of the HIV-I Rev subtype B consensus sequence was performed to identify the regions that mediate binding to IN. Five fluorescein-labeled oligopeptides (Table 1) covering the full length of Rev were synthesized, and their interaction with IN was studied using fluorescence anisotropy. Revl-30 and Rev49-74 bound to IN with K_d values at the low micromolar range, and a Hill coefficient of around 4, indicating binding of IN tetramer to the peptides. Peptides Rev31- 48, 74-93, and Rev94-116 did not exhibit binding to IN (Figure 4A and Table 1). Table 1: Binding to HIV-I IN of Rev-derived long and short peptides

EXAMPLE 4; Inhibition of IN catalytic activity by Rev-derived peptides

No significant inhibition of IN strand transfer activity or 3 '-end processing activity by full-length Rev protein was observed, nor was it observed by Revl-30 or Rev49-74 (compare Figure 5D and E to Figure 5B.). To determine whether Rev-derived short peptides inhibit IN catalytic activity, a peptide library comprising the full-length HIV-I Rev subtype B consensus sequence was screened for inhibition of IN enzymatic activities. This library, obtained from the NIH AIDS Research & Reference Reagent Program, contains small samples of 27 peptides, each 15 amino acids in length, with an 11-amino-acid overlap between sequential peptides (Table 2). Six peptides corresponding to two regions of the Rev protein inhibited IN 3 '-end processing activity (Figure 6 A and Table T). Four of these six inhibitory peptides blocked IN strand-transfer activity (Figure 6B and Table 2). Table 2: Summary of in vitro inhibition of HIV-I IN by Rev-derived peptides. The remaining peptides from the library did not inhibit IN catalytic activity.

* Peptide numbers are as given in the NIH AIDS Research & Reference Reagent Program. **Peptide sequences are from the HIV-I Rev consensus B sequence.

EXAMPLE 5: Binding to IN and penetration into cultured cells of the Rev-derived inhibitory short peptides

Of the six Rev-derived inhibitory peptides, the sequences of peptides 5993 and 6004 (Table 2) were selected for further study, including binding to IN, penetration into cultured cells and inhibition of HIV-I replication. For these studies, two peptides were synthesized (Table 1): one corresponding to 5993 but lacking its first four amino acids (DEEL) (designated "Revl3-23"), in order to obtain a cell-permeable short peptide lacking the negatively charged amino acids, and another bearing the complete sequence of peptide 6004 (designated "Rev53-67"). Similar to peptides 5993 and 6004 from the NIH peptide library, peptides Revl3-23 and Rev53-67 inhibited IN strand transfer and 3 '-end processing activities (compare Figure 5F-G with Figure 5H-I). Fluorescence anisotropy studies (Figure 4B) revealed that peptides Revl3-23 and Rev53-67 interacted with IN, showing binding affinities similar to those obtained with the longer Rev peptides (Figure 4B and Table 1).

Incubation of the fluorescently labeled Rev 13 -23 and Rev53-67 with HeLa cultured cells resulted in the appearance of intracellular fluorescence, indicating their translocation via the cells' plasma membrane (Figure 7).

EXAMPLE 6: Reyl3-23 and Rev53-67 inhibit HIV-I replication in cultured cells at nontoxic concentrations

Rev 13 -23 and Rev53-67 on HIV-I propagation was studied using TZM-bl (MAGI) cells, which express the β-galactosidase gene under trans-activation response (TAR) element regulation, with percentage of blue cells following infection with HIV-I indicating infectious virus titer. Both peptides inhibited HIV-I replication in a concentration-dependent manner (Figure 8A). Almost complete inhibition of HIV-I replication was observed with peptide Rev 13-23 at a concentration as low as 2.5 μM, while about 70% inhibition was observed with the same concentration of peptide Rev53-67. No synergetic effect was observed when a mixture of the two peptides was added, suggesting that both peptides interact with the same IN domain. A sharp decrease in the amount of viral P24 was also obtained in infected lymphoid cells incubated with peptides Rev 13- 23 and Rev53-67 (Figure 8B). The degree of inhibition observed with both peptides was similar to that obtained with the anti-HIV-1 RT inhibitor AZT.

The Rev-derived peptides blocked HIV-I replication by inhibiting the viral DNA integration step, as estimated by real-time PCR in infected lymphoid cells (Figure 8C). At the concentrations used, the Rev-derived peptides were not toxic, as demonstrated by MTT assay (Figure 8D).

EXAMPLE 7: Identification of an additional IN-binding peptide

IN-binding peptides were also selected using the yeast two-hybrid screening system with a random peptide library (9,10). By this assay, an IN-binding peptide was identified and designated IN5 (Table 1). IN5 interacts with the IN protein as was determined by fluorescence anisotropy (Figure 9A). However, IN5 was unable to block IN enzymatic activity (Figure 5C) or HIV-I replication (Figure 8A-C), despite its ability to penetrate cultured cells (Figure 9B). Thus, those IN-binding peptides that do not inhibit IN activity in vifro cannot block HIV replication in vivo. EXAMPLE 8: The Rev-derived peptides bind the ENf tetramer: gel filtration studies

The effect of the Rev-derived peptides on the IN oligomeric state was tested using analytical gel filtration. Unbound IN eluted as a high order oligomer. IN was tetrameric in presence of the Rev derived peptides, but dimeric in the presence of LTR DNA, indicating a shift in the oligomerization equilibrium in presence of the peptides (Figure 10; also see Hill coefficients values in Table 3).

Table 3: Effect of Rev-derived peptides on binding of HIV-I IN to the viral LTR DNA.

* K_ά values for peptide binding are taken from ref 16.

EXAMPLE 9; The Re- derived peptides inhibit IN binding to the viral LTR DNA

Fluorescence anisotropy was used to study whether the Rev derived peptides affect the DNA binding by IN. IN bound to a fluorescein-labeled 36-bp double stranded viral LTR DNA with K_& of 37 nM and a Hill coefficient of 2, showing that IN binds the LTR DNA tightly and as a dimer. The Rev-derived peptides significantly inhibited the binding of IN to the LTR DNA. The affinity of IN for the DNA was reduced 10-fold from 34 nM without the Rev peptides to 320 nM and 300 nM in presence of 1 μM Rev 13-23 and Rev 53-67, respectively (Figure 11 and Table 3). Thus, the Rev peptides inhibit DNA binding by IN. EXAMPLE 10: The extent of IN inhibition by the Rev peptides in vitro depends on the peptide: DNA relative order of addition and peptide; IN molar ratio

To further characterize inhibition of IN catalytic activity by the Rev-derived peptides, a quantitative in vitro integration assay was performed with different orders of addition of the peptide and the DNA as well as different IN: peptide molar ratios. When viral LTR DNA was added to a preformed IN-peptide complex, IN catalytic activity was inhibited by 70% (Figure

12A-B). On the other hand, when the peptides were added to a pre-formed IN-DNA complex at the same molar ratio, IN catalytic activity was only slightly inhibited (by 20%). Inhibition became more pronounced when the peptide: IN ratio increased (Figure 12C-D). Around 60% inhibition was observed at 5:1 peptide: IN ratio.

EXAMPLE 11: Identifying essential and non-essential amino acids in the subsequence chosen

Deletion and replacement by non-conservative amino acids generally is performed in "nonessential" amino acids, while essential amino acids should be maintained or replaced by conservative substitutions. To identify essential vs. non-essential amino acids, the following techniques are utilized:

(1) Shortening of sequence:

To determine the minimum sequence of the lead peptide required for IN binding; integrase inhibition and/or HIV-I replication inhibition, a series of truncated partly overlapping peptides derived from Rev 13-23 and Rev 53-67 is prepared. Peptides are shortened by one residue at a time from the N terminus and from the C terminus. The short peptides are labeled with fluorescein for the fluorescence anisotropy and cellular uptake studies.

(2) Systematic Amino Acid Replacement: To improve the binding affinity of the lead peptides to IN, a new series of peptides are prepared wherein the residues that do not contribute to IN binding are replaced by other natural and non-natural amino acids. Design of the mutations is based on a bioinformatics search. Rev homologous sequences are identified, and amino acids in the lead peptide are mutated according to naturally occurring residues in these positions. In parallel, a combinatorial approach is used to discover which type of amino acid will best fit the mutation positions. Fluorescein-labeled peptides are synthesized, wherein amino acids not important for IN binding are replaced by mixtures of 4-5 natural and non-natural amino acids that have the same character (e.g. polar, hydrophobic etc.). Non-natural amino acids will be primarily utilized, since their incorporation increases peptide stability. Binding of the peptides to IN is tested using fluorescent anisotropy, mixtures that bind IN are separated by HPLC, and binding of individual peptides to IN is tested. To identify synergy between the different mutations, peptides are synthesized with multiple amino acid replacements, wherein mutations resulting in tightest IN binding are combined.

(3) Increasing peptide stability: To stabilize the lead peptides against proteolysis, non-natural amino acids such as N-methyl amino acids and D-amino acids are introduced into the peptide sequence. A D-amino acid scan of the lead peptides is performed, and a series of peptides is synthesized wherein each amino acid is systematically replaced by its D enantiomer. In other experiments, an N-methyl amino acids scan of the lead peptides is performed (similarly to the D- amino acid scan). N-methylation is known to stabilize peptide to enzymatic degradation and increase their oral bioavailability due to the lack of the amide protons, which reduces their polarity.

(4) Conversion into peptidomimetics and Aza-scan: The lead peptides at this stage bear an optimized side chain composition with only the required pharmacophores present, have a shorter sequence and are stable against proteolysis. Next, backbone modifications are introduced. The lead peptides are subject to AZA scan, to improve the peptide stability and its binding affinity and specificity. Aza peptides are peptide analogs in which the α-carbon of one or more of the amino acid residues is replaced with a nitrogen atom. This reduces the flexibility of the parent linear peptide due to replacement of the rotatable Cα-CO bond by a more rigid urea N-CO structure, and leads to improved pharmacological properties such as increased duration of action, potency, and/or selectivity, as was shown for aza-analogues of angiotensin II and somatostatin and of serine and cysteine protease inhibitors. Each amino acid in the lead peptides is systematically replaced by the corresponding Aza amino acid, followed by assays for binding IN and inhibiting IN activity and HIV-I replication as described above. In other experiments, the backbone amide bonds are each systematically converted into a peptoid bond, wherein the side chain of the peptide is moved from the α-carbon to the α-nitrogen.

(6) Conversion into small molecules: Next, further modifications are introduced into the most potent peptidomimetic and in its backbone and side chains to further convert it into a small molecule. The modifications are in further modification of the peptide bonds (e.g. reduction of the carbonyl to methylene), and replacement of the side chains by organic groups with similar properties, to fine-tune the interaction with IN.

Omission scan In other experiments, identification of essential vs. non-essential amino acids in the peptide is achieved by preparing several peptides in which each amino acid is sequentially omitted ("omission-scan"). Amino acids whose loss results in reduction in physiological activity can be defined as "essential," while those whose loss does not cause significant change of activity can be defined as "non-essential" (Morrison et al, Chemical Biology 5:302-307, 2001).

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

References

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Claims

CLAIMS:

1. An isolated fragment of an HIV-I Rev protein, wherein said fragment of an HIV-I Rev protein is 6-25 amino acids in length, and the sequence of said isolated fragment of an HIV-I Rev protein comprises either: a. the sequence DEELLKTVRLIKFLY (SEQ ID NO: 11);

b. a portion of the sequence DEELLKTVRLIKFLY (SEQ ID NO: 11), wherein said portion is 6-14 amino acids in length; or c. a mutant version of the sequence DEELLKTVRLIKFLY (SEQ ID NO: 11), wherein said mutant comprises 1-3 amino acid modifications relative to SEQ ID NO: 11, wherein each of said amino acid modifications is independently selected from the group consisting of a substitution, an insertion, and a deletion.

2. The isolated fragment of an HIV-I Rev protein of claim 1, wherein the sequence of said isolated fragment of an HIV-I Rev protein is set forth in SEQ ID NO: 11 or is a portion thereof.

3. The isolated fragment of an HIV-I Rev protein of claim 2, wherein the sequence of said portion of SEQ ID NO: 11 comprises LKTVRLIKFLY (SEQ ID NO: 6).

4. The isolated fragment of an HIV-I Rev protein of claim 1, wherein said isolated fragment of an HIV-I Rev protein is an isolated mutated fragment of an HIV-I Rev protein that comprises 1-3 amino acid modifications relative to SEQ ID NO: 11, wherein each of said amino acid modifications is independently selected from the group consisting of a substitution, an insertion, and a deletion.

5. An isolated fragment of an HIV-I Rev protein, wherein said fragment of an HIV-I Rev protein is 6-25 amino acids in length, and the sequence of said isolated fragment of an HIV-I Rev protein comprises either:

a. the sequence RSISGWILSTYLGRP (SEQ ID NO: 7); or

b. a portion of the sequence RSISGWILSTYLGRP (SEQ ID NO: 7), wherein said portion is 6-14 amino acids in length.

6. A mutated fragment of an HIV-I Rev protein, wherein (a) said mutated fragment of an HIV-I Rev protein is 13-25 amino acids in length; (b) said mutated fragment of an HIV-I Rev protein comprises a mutated version of the sequence RSISGWILSTYLGRP (SEQ ID NO: 7); and (c) said mutated version of SEQ ID NO: 7 comprises 1-3 amino acid modifications relative to SEQ ID NO: 7, wherein each of said amino acid modifications is independently selected from the group consisting of a substitution, an insertion, and a deletion.

7. An isolated fragment of an HIV-I Rev protein, wherein said fragment of an HIV-I Rev protein is 6-25 amino acids in length, and the sequence of said isolated fragment of an

HIV-I Rev protein comprises either:

a. the sequence LKTVRLIKFLYQSNP (SEQ ID NO : 12);

b. a portion of the sequence LKTVRLIKFLYQSNP (SEQ ID NO: 12), wherein said portion is 6-14 amino acids in length; c. the sequence QRQIRSISGWILSTY (SEQ ID NO: 15); or

d. a portion of the sequence QRQIRSISGWILSTY (SEQ ID NO: 15), wherein said portion is 6-14 amino acids in length.

8. A mutated fragment of an HIV-I Rev protein, wherein (a) said mutated fragment of an HIV-I Rev protein is 13-25 amino acids in length; (b) said mutated fragment of an HIV-I Rev protein comprises a mutated version of a sequence selected from the group consisting of LKTVRLIKFLYQSNP (SEQ ID NO: 12) and QRQIRSISGWILSTY (SEQ ID NO: 15); and (c) said mutated version of SEQ ID NO: 12 or SEQ ID NO: 15 comprises 1-3 amino acid modifications relative to SEQ ID NO: 12 or SEQ ID NO: 15, wherein each of said amino acid modifications is independently selected from the group consisting of a substitution, an insertion, and a deletion.

9. The isolated fragment of an HIV-I Rev protein of any of claims 1, 4, 6, or 8, wherein one or both of said amino acid modifications eliminates a negatively charged amino acid from the native HIV-I Rev sequence corresponding to said isolated fragment of an HIV-I Rev protein.

10. The isolated fragment of an HIV- 1 Rev protein of any of claims 1 -9, wherein said isolated peptide is capable of inhibiting 3'-end processing activity of an HIV-I integrase protein.

11. The isolated fragment of an HIV-I Rev protein of any of claims 1-10, wherein said isolated peptide is capable of inhibiting HIV-I replication in a target cell.

12. The isolated fragment of an HIV-I Rev protein of any of claims 1-11, wherein said isolated peptide is capable of inhibiting binding of an HIV-I integrase protein to an HIV- 1 long terminal repeat DNA terminus.

13. An isolated peptide comprising the isolated fragment of an HIV-I Rev protein of any of claims 1-12, wherein said isolated peptide is 12-100 amino acids in length.

14. A pharmaceutical composition, comprising the isolated fragment of an HIV-I Rev protein of any of claims 1-13 and a pharmaceutically acceptable carrier, diluent, or additive.

15. A pharmaceutical composition, comprising: (a) the isolated fragment of an HIV-I Rev protein of any of claims 1-4 or a peptide comprising said isolated fragment of an HIV-I Rev protein; (b) the isolated fragment of an HIV-I Rev protein of any of claims 5-6 or a peptide comprising said isolated fragment of an HIV-I Rev protein; and (c) a pharmaceutically acceptable carrier, diluent, or additive.

16. A pharmaceutical composition, comprising: (a) the isolated fragment of an HIV-I Rev protein of any of claims 1-4 or a peptide comprising said isolated fragment of an HIV-I Rev protein; (b) the isolated fragment of an HIV-I Rev protein of any of claims 7-8 or a peptide comprising said isolated fragment of an HIV-I Rev protein; and (c) a pharmaceutically acceptable carrier, diluent, or additive.

17. A pharmaceutical composition, comprising: (a) the isolated fragment of an HIV-I Rev protein of any of claims 5-6 or a peptide comprising said isolated fragment of an HIV-I Rev protein; (b) the isolated fragment of an HIV-I Rev protein of any of claims 7-8 or a peptide comprising said isolated fragment of an HIV-I Rev protein; and (c) a pharmaceutically acceptable carrier, diluent, or additive.

18. A method of inhibiting replication of an HIV-I in a target cell, comprising administering to said target cell the isolated fragment of an HIV-I Rev protein of any of claims 1-12, thereby inhibiting replication of an HIV- 1 in a target cell.

19. A method for treating HIV-I infection in a subject in need thereof, comprising administering to said subject the isolated fragment of an HIV-I Rev protein of any of claims 1-12, thereby treating HIV-I infection in a subject in need thereof.

20. A method for inhibiting 3'-end processing of an HIV-I integrase protein, comprising contacting said HIV-I integrase protein with the isolated fragment of an HIV-I Rev protein of any of claims 1-12, thereby inhibiting 3'-end processing of an HIV-I integrase protein.

21. A method for inhibiting binding of an HIV-I integrase protein to an HIV-I long terminal repeat DNA terminus, comprising contacting said HIV-I integrase protein with the isolated fragment of an HIV-I Rev protein of any of claims 1-12, thereby inhibiting binding of an HIV-I integrase protein to an HIV-I long terminal repeat DNA terminus.

22. The pharmaceutical composition of any of claims 14-17 for inhibiting replication of an HIV-I in a target cell.

23. The pharmaceutical composition of any of claims 14-17 for treating HIV-I infection in a subj ect in need thereof.

24. The pharmaceutical composition of any of claims 14-17 for inhibiting 3'-end processing of an HIV-I integrase protein.

25. The pharmaceutical composition of any of claims 14-17 for inhibiting binding of an HIV- 1 integrase protein to an HIV-I long terminal repeat DNA terminus.

26. Use of the isolated fragment of an HIV-I Rev protein of any of claims 1-12 in the preparation in a pharmaceutical composition for inhibiting binding of an HIV-I integrase protein to an HIV-I long terminal repeat DNA terminus, inhibiting replication of an HIV- 1 in a target cell, treating HIV-I infection in a subject in need thereof, or inhibiting 3 '-end processing of an HIV-I integrase protein.