Exploration of the effect of sequence variations located inside the binding pocket of HIV-1 and HIV-2 proteases.

HIV-2 protease (PR2) is naturally resistant to most FDA (Food and Drug Administration)-approved HIV-1 protease inhibitors (PIs), a major antiretroviral class. In this study, we compared the PR1 and PR2 binding pockets extracted from structures complexed with 12 ligands. The comparison of PR1 and PR2 pocket properties showed that bound PR2 pockets were more hydrophobic with more oxygen atoms and fewer nitrogen atoms than PR1 pockets. The structural comparison of PR1 and PR2 pockets highlighted structural changes induced by their sequence variations and that were consistent with these property changes. Specifically, substitutions at residues 31, 46, and 82 induced structural changes in their main-chain atoms that could affect PI binding in PR2. In addition, the modelling of PR1 mutant structures containing V32I and L76M substitutions revealed a cooperative mechanism leading to structural deformation of flap-residue 45 that could modify PR2 flexibility. Our results suggest that substitutions in the PR1 and PR2 pockets can modify PI binding and flap flexibility, which could underlie PR2 resistance against PIs. These results provide new insights concerning the structural changes induced by PR1 and PR2 pocket variation changes, improving the understanding of the atomic mechanism of PR2 resistance to PIs.

Amino acid residues in HIV-2 reverse transcriptase that restrict the development of nucleoside analogue resistance through the excision pathway.

Nucleoside reverse transcriptase (RT) inhibitors (NRTIs) are the backbone of current antiretroviral treatments. However, the emergence of viral resistance against NRTIs is a major threat to their therapeutic effectiveness. In HIV-1, NRTI resistance-associated mutations either reduce RT-mediated incorporation of NRTI triphosphates (discrimination mechanism) or confer an ATP-mediated nucleotide excision activity that removes the inhibitor from the 3' terminus of DNA primers, enabling further primer elongation (excision mechanism). In HIV-2, resistance to zidovudine (3'-azido-3'-deoxythymidine (AZT)) and other NRTIs is conferred by mutations affecting nucleotide discrimination. Mutations of the excision pathway such as M41L, D67N, K70R, or S215Y (known as thymidine-analogue resistance mutations (TAMs)) are rare in the virus from HIV-2-infected individuals. Here, we demonstrate that mutant M41L/D67N/K70R/S215Y HIV-2 RT lacks ATP-dependent excision activity, and recombinant virus containing this RT remains susceptible to AZT inhibition. Mutant HIV-2 RTs were tested for their ability to unblock and extend DNA primers terminated with AZT and other NRTIs, when complexed with RNA or DNA templates. Our results show that Met73 and, to a lesser extent, Ile75 suppress excision activity when TAMs are present in the HIV-2 RT. Interestingly, recombinant HIV-2 carrying a mutant D67N/K70R/M73K RT showed 10-fold decreased AZT susceptibility and increased rescue efficiency on AZT- or tenofovir-terminated primers, as compared with the double-mutant D67N/K70R. Molecular dynamics simulations reveal that Met73influences ß3-ß4 hairpin loop conformation, whereas its substitution affects hydrogen bond interactions at position 70, required for NRTI excision. Our work highlights critical HIV-2 RT residues impeding the development of excision-mediated NRTI resistance.

High-Frequency Illegitimate Strand Transfers of Nascent DNA Fragments During Reverse Transcription Result in Defective Retrovirus Genomes.

BACKGROUND: Two strand transfers of nascent DNA fragments during reverse transcription are required for retrovirus replication. However, whether strand transfers occur at illegitimate sites and how this may affect retrovirus replication are not well understood. METHODS: The reverse transcription was carried out with reverse transcriptases (RTs) from HIV-1, HIV-2, and murine leukemia virus. The nascent complementary DNA fragments were directly cloned without polymerase chain reaction amplification. The sequences were compared with the template sequence to determine if new sequences contained mismatched sequences caused by illegitimate strand transfers. RESULTS: Among 1067 nascent reverse transcript sequences, most of them (72%) matched to the template sequences, although they randomly stopped across the RNA templates. The other 28% of them contained mismatched 3'-end sequences because of illegitimate strand transfers. Most of the illegitimate strand transfers (81%) were disassociated from RNA templates and realigned onto opposite complementary DNA strands. Up to 3 strand transfers were detected in a single sequence, whereas most of them (93%) contained 1 strand transfer. Because most of the illegitimate strand-transfer fragments were generated from templates at 2 opposite orientations, they resulted in defective viral genomes and could not be detected by previous methods. Further analysis showed that mutations at pause/disassociation sites resulted in significantly higher strand-transfer rates. Moreover, illegitimate strand-transfer rates were significantly higher for HIV-2 RT (38.2%) and murine leukemia virus RT (44.6%) than for HIV-1 RT (5.1%). CONCLUSIONS: Illegitimate strand transfers frequently occur during reverse transcription and can result in a large portion of defective retrovirus genomes.

dUTPase: the frequently overlooked enzyme encoded by many retroviruses.

Retroviruses are among the best studied viruses in last decades due to their pivotal involvement in cellular processes and, most importantly, in causing human diseases, most notably-acquired immunodeficiency syndrome (AIDS) that is triggered by human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2, respectively). Numerous studied were conducted to understand the involvement of the three cardinal retroviral enzymes, reverse transcriptase, integrase and protease, in the life cycle of the viruses. These studies have led to the development of many inhibitors of these enzymes as anti-retroviral specific drugs that are used for routine treatments of HIV/AIDS patients. Interestingly, a fourth virus-encoded enzyme, the deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is also found in several major retroviral groups. The presence and the importance of this enzyme to the life cycle of retroviruses were usually overlooked by most retrovirologists, although the occurrence of dUTPases, particularly in beta-retroviruses and in non-primate retroviruses, is known for more than 20 years. Only more recently, retroviral dUTPases were brought into the limelight and were shown in several cases to be essential for viral replication. Therefore, it is likely that future studies on this enzyme will advance our knowledge to a level that will allow designing novel, specific and potent anti-dUTPase drugs that are effective in combating retroviral diseases. The aim of this review is to give concise background information on dUTPases in general and to summarize the most relevant data on retroviral dUTPases and their involvement in the replication processes and pathogenicity of the viruses, as well as in possibly-associated human diseases.

A modular system to evaluate the efficacy of protease inhibitors against HIV-2.

The human immunodeficiency virus (HIV) protease is a homodimeric aspartyl protease that is crucial for the viral life-cycle, cleaving proviral polyproteins, hence creating mature protein components that are required for the formation of an infectious virus. With diagnostic measures and clinically used protease inhibitors focusing on HIV-1, due to its higher virulence and prevalence, studies of the efficacy of those inhibitors on HIV-2 protease remain widely lacking. Utilizing a wild-type HIV-2 vector backbone and cloning techniques we have developed a cassette system where the efficacy of clinically used protease inhibitors can be studied for various serotypes of HIV-2 protease both in enzymatic and cell culture assays. In our experiments, optimization of the expression protocol led to a relatively stable enzyme, for cell culture assays, the efficiency of transfection and transduction capability of the modified vector was tested and was not found to differ from that of the wild-type, moreover, a 2nd generation protease inhibitor was used to demonstrate the usefulness of the system. The combination of assays performed with our cassette system is expected to provide an accurate measure of the efficacy of currently used; as well as experimental protease inhibitors on HIV-2.

SAMHD1 specifically affects the antiviral potency of thymidine analog HIV reverse transcriptase inhibitors.

Sterile alpha motif and histidine-aspartic domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase recently recognized as an antiviral factor that acts by depleting dNTP availability for viral reverse transcriptase (RT). SAMHD1 restriction is counteracted by the human immunodeficiency virus type 2 (HIV-2) accessory protein Vpx, which targets SAMHD1 for proteosomal degradation, resulting in an increased availability of dNTPs and consequently enhanced viral replication. Nucleoside reverse transcriptase inhibitors (NRTI), one of the most common agents used in antiretroviral therapy, compete with intracellular dNTPs as the substrate for viral RT. Consequently, SAMHD1 activity may be influencing NRTI efficacy in inhibiting viral replication. Here, a panel of different RT inhibitors was analyzed for their different antiviral efficacy depending on SAMHD1. Antiviral potency was measured for all the inhibitors in transformed cell lines and primary monocyte-derived macrophages and CD4(+) T cells infected with HIV-1 with or without Vpx. No changes in sensitivity to non-NRTI or the integrase inhibitor raltegravir were observed, but for NRTI, sensitivity significantly changed only in the case of the thymidine analogs (AZT and d4T). The addition of exogenous thymidine mimicked the change in viral sensitivity observed after Vpx-mediated SAMHD1 degradation, pointing toward a differential effect of SAMHD1 activity on thymidine. Accordingly, sensitivity to AZT was also reduced in CD4(+) T cells infected with HIV-2 compared to infection with the HIV-2ΔVpx strain. In conclusion, reduction of SAMHD1 levels significantly decreases HIV sensitivity to thymidine but not other nucleotide RT analog inhibitors in both macrophages and lymphocytes.

Synthesis, biological evaluation and molecular modeling of 4,6-diarylpyrimidines and diarylbenzenes as novel non-nucleosides HIV-1 reverse transcriptase inhibitors.

A series of novel 4,6-diarylpyrimidines (4,6-DAPY) and diarylbenzenes (DABE) compounds were synthesized and evaluated as inhibitors of human immunodeficiency virus type-1 (HIV-1). Among them, the most potent HIV-1 inhibitors were 8b, 8d, 14b and 18 (EC(50) = 0.049, 0.381, 0.599 and 0.398 µM, respectively), with HIV-1 inhibitory activity improved or similar to nevirapine (NVP, EC(50) = 0.097 µM) and delavirdine (DEV, EC(50) = 0.55 µM). The other compounds displayed moderate activity (8c, EC(50) = 5.25 µM) or were inactive (8a and 14a) against HIV-1 replication. Molecular modeling studies were performed with the synthesized compounds in complex with the wild-type reverse transcriptase (RT). A correlation was found between the anti-HIV activity and the electrostatic energy of interaction with Lys101 residue. These findings enrich the SAR of these Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) families.

The early years of retroviral protease crystal structures.

Soon after its discovery, the attempts to develop anti-AIDS therapeutics focused on the retroviral protease (PR)-an enzyme used by lentiviruses to process the precursor polypeptide into mature viral proteins. An urgent need for the three-dimensional structure of PR to guide rational drug design prompted efforts to produce milligram quantities of this enzyme. However, only minute amounts of PR were present in the HIV-1 and HIV-2 viruses, and initial attempts to express this protein in bacteria were not successful. This review describes X-ray crystallographic studies of the retroviral proteases carried out at NCI-Frederick in the late 1980s and early 1990s and puts into perspective the crucial role that the total protein chemical synthesis played in unraveling the structure, mechanism of action, and inhibition of HIV-1 PR. Notably, the first fully correct structure of HIV-1 PR and the first cocrystal structure of its complex with an inhibitor (a substrate-derived, reduced isostere hexapeptide MVT-101) were determined using chemically synthesized protein. Most importantly, these sets of coordinates were made freely available to the research community and were used worldwide to solve X-ray structures of HIV-1 PR complexes with an array of inhibitors and set in motion a variety of theoretical studies. Publication of the structure of chemically synthesized HIV-1 PR complexed with MVT-101 preceded only by six years the approval of the first PR inhibitor as an anti-AIDS drug.

Novel N-3 substituted TSAO-T derivatives: synthesis and anti-HIV-evaluation.

Novel derivatives of the anti-HIV-1 agent, TSAO-T, bearing at the N-3 position alkylating groups or photoaffinity labels were prepared and evaluated for their anti-HIV activity. All of these compounds demonstrated pronounced anti-HIV-1 activity and inhibited HIV-1 RT; however, we were unable to detect stable covalent linkages between inhibitor and enzyme. In addition, compounds with an alcohol functional group connected to the N-3 position through a cis or trans double bond have been prepared. These compounds have been useful to study how the conformational restriction of the linker affects in the interaction between the N-3 substituent and the HIV-1 RT enzyme.

Nitroimidazoles. V. Synthesis and anti-HIV evaluation of new 5-substituted piperazinyl-4-nitroimidazole derivatives.

A series of 2-alkylthio-1-[4-(1-benzyl-2-ethyl-4-nitro-1H- -imidazol-5-yl)-piperazin-1-yl]ethanones (3-9) and alkyl-[4-(1-benzyl-2-ethyl-4-nitro-1H-imidazol-5-yl)-piperazin-1-yl)ketones (11-20) as well as the indole analogue 22 were synthesized from 4-nitro-5-piperazinyl imidazole derivative 1, with the aim to develop newly non-nucleoside reverse transcriptase inhibitors (NNRTIs). The newly synthesized compounds were assayed against HIV-1 and HIV-2 in MT-4 cells. Compound 4 showed inhibition of HIV-1 (EC50 0.45 microg mL-1) and HIV-2 (0.50 microg mL-1), while 11 showed inhibition of HIV-1 (EC50 2.48 microg mL-1, SI = 4).

Amino acid preferences for a critical substrate binding subsite of retroviral proteases in type 1 cleavage sites.

The specificities of the proteases of 11 retroviruses representing each of the seven genera of the family Retroviridae were studied using a series of oligopeptides with amino acid substitutions in the P2 position of a naturally occurring type 1 cleavage site (Val-Ser-Gln-Asn-Tyr Pro-Ile-Val-Gln; the arrow indicates the site of cleavage) in human immunodeficiency virus type 1 (HIV-1). This position was previously found to be one of the most critical in determining the substrate specificity differences of retroviral proteases. Specificities at this position were compared for HIV-1, HIV-2, equine infectious anemia virus, avian myeloblastosis virus, Mason-Pfizer monkey virus, mouse mammary tumor virus, Moloney murine leukemia virus, human T-cell leukemia virus type 1, bovine leukemia virus, human foamy virus, and walleye dermal sarcoma virus proteases. Three types of P2 preferences were observed: a subgroup of proteases preferred small hydrophobic side chains (Ala and Cys), and another subgroup preferred large hydrophobic residues (Ile and Leu), while the protease of HIV-1 preferred an Asn residue. The specificity distinctions among the proteases correlated well with the phylogenetic tree of retroviruses prepared solely based on the protease sequences. Molecular models for all of the proteases studied were built, and they were used to interpret the results. While size complementarities appear to be the main specificity-determining features of the S2 subsite of retroviral proteases, electrostatic contributions may play a role only in the case of HIV proteases. In most cases the P2 residues of naturally occurring type 1 cleavage site sequences of the studied proteases agreed well with the observed P2 preferences.

Apelin peptides block the entry of human immunodeficiency virus (HIV).

The orphan G protein-coupled receptor APJ has been shown to be a coreceptor for human and simian immunodeficiency virus (HIV and SIV) strains. We have determined that some HIV and SIV strains use APJ as a coreceptor to infect the brain-derived NP-2/CD4 cells. Because apelin is an endogenous ligand for the APJ receptor, we examined the inhibitory effects of apelin peptides on HIV infection, and found that the apelin peptides inhibit the entry of some HIV-1 and HIV-2 into the NP-2/CD4 cells expressing APJ. The inhibitory efficiency has been found to be in the order of apelin-36>apelin-17>apelin-13>apelin-12.

Effect of substrate residues on the P2' preference of retroviral proteinases.

The substrate sequence requirements for preference toward P2' Glu residue by human immunodeficiency virus type 1 (HIV-1) proteinase were studied in both the matrix protein/ capsid protein (MA/CA) and CA/p2 cleavage site sequence contexts. These sequences represent typical type 1 (-aromatic*Pro-) and type 2 (-hydrophobic* hydrophobic-) cleavage site sequences, respectively. While in the type 1 sequence context, the preference for P2' Glu over Ile or Gln was found to be strongly dependent on the ionic strength and the residues being outside the P2-P2' region of the substrate, it remained preferable in the type 2 substrates when typical type 1 substrate sequence residues were substituted into the outside regions. The pH profile of the specificity constants suggested a lower pH optimum for substrates having P2' Glu in contrast to those having uncharged residues, in both sequence contexts. The very low frequency of P2' Glu in naturally occurring retroviral cleavage sites of various retroviruses including equine infectious anemia virus (EIAV) and murine leukemia virus (MuLV) suggests that such a residue may not have a general regulatory role in the retroviral life cycle. In fact, unlike HIV-1 and HIV-2, EIAV and MuLV proteinases do not favor P2' Glu in either the MA/CA or CA/p2 sequence contexts.

Depsides and depsidones as inhibitors of HIV-1 integrase: discovery of novel inhibitors through 3D database searching.

Seventeen lichen acids comprising despides, depsidones, and their synthetic derivatives have been examined for their inhibitory activity against HIV-1 integrase, and two pharmacophores associated with inhibition of this enzyme have been identified. A search of the NCI 3D database of approximately 200,000 structures yielded some 800 compounds which contain one or the other pharmacophore. Forty-two of these compounds were assayed for HIV-1 integrase inhibition, and of these, 27 had inhibitory IC50 values of less than 100 microM; 15 were below 50 microM. Several of these compounds were also examined for their activity against HIV-2 integrase and mammalian topoisomerase I.

Bile pigments as HIV-1 protease inhibitors and their effects on HIV-1 viral maturation and infectivity in vitro.

Using recently developed molecular-shape description algorithms, we searched the Available Chemical Directory for known compounds similar in shape to the potent HIV-1 protease inhibitor Merck L-700,417; 15 compounds most similar in shape to the inhibitor were selected for testing in vitro. Four of these inhibited the protease at 100 microM or less and the most active of the four were the naturally occurring pigments biliverdin and bilirubin. Biliverdin and bilirubin inhibited recombinant HIV-1 protease in vitro at pH 7.8 with K1 values of approx. 1 microM, and also inhibited HIV-2 and simian immunodeficiency virus proteases. The related pyrrolic pigments stercobilin, urobilin, biliverdin dimethyl ester and xanthobilirubic acid showed similar inhibitory activity at low micromolar concentrations. Biliverdin, bilirubin and xanthobilirubic acid did not inhibit viral polyprotein processing in cultured cells, but they reduced viral infectivity significantly. At 100 microM, xanthobilirubic acid affected viral assembly, resulting in a 50% decrease in the generation of infectious particles. In contrast, at the same concentrations biliverdin and bilirubin exerted little or no effect on viral assembly but blocked infection of HeLaT4 cells by 50%. These results suggest that bile pigments might be a new class of potential lead compounds for developing protease inhibitors and they raise the question of whether hyperbilirubinaemia can influence the course of HIV infection.

Expression, characterisation and mutagenesis of the aspartic proteinase from equine infectious anaemia virus.

The gene encoding the proteinase from equine infectious anaemia virus (EIAV) was cloned and expressed in Escherichia coli. The recombinant EIAV proteinase was purified to homogeneity and shown to have the ability to process polyprotein and synthetic peptide substrates of human immunodeficiency virus (HIV) origin with an efficiency that can approach that exhibited by HIV proteinase. EIAV proteinase, however, was not susceptible to inhibition by a wide variety of inhibitors of HIV-1 proteinase, including those which have been licenced as anti-AIDS drugs. In this respect, EIAV proteinase behaves like an extreme case of a drug-resistant mutant of HIV-1 proteinase that has arisen under selective drug pressure. Only one potent inhibitor (HBY-793) of HIV-1 proteinase showed comparable efficiency against the EIAV enzyme; the compounds A-77003 and A-76889, which differ only in their stereochemistry and which are otherwise structurally identical to HBY-793 from residues P2 to P2' [nomenclature of Schechter, I. & Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162], were not effective inhibitors of EIAV proteinase. Mutant forms of EIAV proteinase (Thr30-->Asp and Ile54-->Gly) were generated and their ability to interact with substrates and inhibitors was characterised. HBY-793 inhibited [Gly54]proteinase as effectively as the wild-type proteinase but was tenfold less potent against [Asp30]proteinase. Data interpretations are presented, based on the structure solved for the complex between HBY-793 and EIAV [Gly54]proteinase [Gustchina A., Kervinen, J., Powell, D. J., Zdanov, A., Kay, J. & Wlodawer, A. (1996) Protein Sci. 5, 1453-1465].

Inhibition of human and simian immunodeficiency virus protease function by targeting Vpx-protease-mutant fusion protein into viral particles.

The human immunodeficiency virus type I (HIV-1) Vpr and HIV-2 Vpx proteins package into virions through interactions with their cognate Gag polyprotein precursor. The targeting properties of Vpr and Vpx have been exploited to incorporate foreign proteins into virions by expression as heterologous fusion molecules (X. Wu, H.-M. Liu, H. Xiao, J. Kim, P. Seshaiah, G. Natsoulis, J. D. Boeke, B. H. Hahn, and J. C. Kappes, J. Virol. 69:3389-3398, 1995). To explore the possibility of utilizing Vpx and Vpr to target dominant negative mutants of the HIV Pol proteins into virions, we fused HIV-2 Vpx with an enzymatically defective protease (PR) mutant. Using a vector system to facilitate transient coexpression with HIV provirus, Vpx-PR-mutant (VpxPR(M)) fusion protein was expressed and packaged efficiently into HIV-2 and simian immunodeficiency virus virions. Immunoblot analysis of purified virions demonstrated that the packaging of VpxPR(M) interfered with the processing of the Gag and Gag/Pol precursor proteins, similar to that of a well-characterized active-site PR inhibitor. The incomplete processing of Gag and Gag/Pol was consistent with a 25-fold reduction in virion infectivity. The coexpression of a packaging defective VpxPR(M) fusion protein with HIV-2 provirus produced virions with fully processed Gag protein, similar to wild-type virions. Importantly, virions trans complemented with a Vpx-chloramphenicol acetyltransferase fusion protein were normal with respect to the processing of Gag protein and the ability to infect and replicate in vitro. These results indicate that VpxPR(M) specifically inhibited the function of the viral protease and provide for the first time proof of principle that the incorporation of foreign proteins into virions via fusion with Vpx can inhibit HIV replication. The use of accessory proteins as vehicles to deliver deleterious proteins to virions, including dominant negative mutants of Pol proteins, may provide new opportunities for application of gene therapy-based antiretroviral strategies. The ability to package PR by expression in trans, independent of the Gag/Pol precursor, also represents a novel approach that may be exploited to study the function of the Pol proteins.

Synthesis and anti-HIV activity of 10,11-dihydropyrrolo [1,2-b][1,2,5]benzothiadiazepine-11-acetic acid 5,5-dioxide derivatives and related compounds.

The synthesis and the in vitro anti-HIV-1 activity of novel pyrrolo annulated benzothiadiazepine acetic acids and some related derivatives are reported. The new compounds share chemical features with pyrrolo[1,2-d][1,4]benzodiazepin-6-one 1 and Ro 5-3335 pyrrylbenzodiazepinone 4, two inhibitors of HIV-replication at the level of reverse transcriptase (RT) and transcriptional transactivation by Tat, respectively. Two derivatives, namely methyl 10,11-dihydropyrrolo[1,2-b][1,2,5]benzothiadiazepine-11-acetic-5,5 -dioxide (5a) and 1,12b-dihydro-2H-azeto[2,1-d]pyrrolo[1,2-b][1,2,5]benzoth iadiazepin-2-one 8,8-dioxide (7a), were found to exhibit a significant, although not very potent, activity against human immunodeficiency virus Type 1 (HIV-1).

Proteolytic activity of human immunodeficiency virus Vpr- and Vpx-protease fusion proteins.

In addition to Gag, Pol, and Env, primate lentiviruses encode other virion-associated proteins, including Vpr, Vpx, and Vif. Vpr- and Vpx-staphylococcal nuclease and chloramphenicol acetyltransferase fusion proteins incorporate into human immunodeficiency virus (HIV) virions and retain enzyme activity when expressed in trans with HIV proviruses (Wu et al., J. Virol. 69, 3389, 1995). To explore whether the viral protease (PR) could be expressed as a proteolytically active fusion protein, the HIV PR coding region was fused in-frame with the HIV-2 vpx and HIV-1 vpr genes. Using a vaccinia virus-T7 expression system, the Vpx-PR fusion protein was expressed and formed homodimers. Coexpression with Pr55Gag demonstrated that Vpx-PR possessed Gag-specific proteolytic activity and inhibited the production of Gag virus-like particles. Trans-expression of a PR-Vpr fusion protein with HIV-1 provirus caused a profound reduction in viral protein expression and virion production. Importantly, the PR-Vpr fusion protein caused a similar level of inhibition and intracellular cleavage of Pr55Gag precursor protein when coexpressed with protease defective HIV-1 provirus. The inhibitory effect of PR-Vpr expression on virion production was markedly greater than that of PR alone. These results indicate that Vpr arguments the intracellular proteolytic activity of PR when expressed as a fusion protein and thus may be relevant for the expression of PR in intracellular immunization strategies against HIV infection. Moreover, the ability to express and package enzymatically active PR-Vpr fusion protein, independent of Gag/Pol, may provide a novel means to study enzyme function.

Prediction of human immunodeficiency virus protease cleavage sites in proteins.

Knowledge of the polyprotein cleavage sites by HIV protease will refine our understanding of its specificity and the information thus acquired is useful for designing specific and efficient HIV protease inhibitors. The pace in searching for the proper inhibitors of HIV protease will be greatly expedited if one can find an accurate and rapid method for predicting the cleavage sites in proteins by HIV protease. Various prediction models or algorithms have been developed during the past 5 years. This Review is devoted to addressing the following problems: (1) Why is it important to predict the cleavability of a peptide by HIV protease? (2) What progresses have been made in developing the prediction methods, and what merits and weakness does each of these methods carry? The attention is focused on the state-of-the-art, which is featured by a discriminant function algorithm developed very recently as well as an improved database (the program and database are available upon request) established according to new experimental results.