Genetically Intact but Functionally Impaired HIV-1 Env Glycoproteins in the T-Cell Reservoir.

HIV-infected subjects under antiretroviral treatment (ART) harbor a persistent viral reservoir in resting CD4+ T cells, which accounts for the resurgence of HIV replication after ART interruption. A large majority of HIV reservoir genomes are genetically defective, but even among intact proviruses few seem able to generate infectious virus. To understand this phenomenon, we examined the function and expression of HIV envelope glycoproteins reactivated from the reservoir of four HIV-infected subjects under suppressive ART. We studied full-length genetically intact env sequences from both replicative viruses and cell-associated mRNAs. We found that these Env proteins varied extensively in fusogenicity and infectivity, with strongest functional defects found in Envs from cell-associated mRNAs. Env functional impairments were essentially explained by defects in Env protein expression. Our results support the idea that defects in HIV Env expression, preventing cytopathic or immune HIV clearance, contribute to the persistence of the HIV T-cell reservoir in vivoIMPORTANCE In most individuals, evolution of HIV infection is efficiently controlled on the long-term by combination antiviral therapies. These treatments, however, fail to eradicate HIV from the infected subjects, a failure that results both in resurgence of virus replication and in resumption of HIV pathogenicity when the treatment is stopped. HIV resurgence, in these instances, is widely assumed to emerge from a reservoir of silent virus integrated in the genomes of a small number of T lymphocytes. The silent HIV reservoir is mostly composed of heavily deleted or mutated HIV DNA. Moreover, among the seemingly intact remaining HIV, only very few are actually able to efficiently propagate in tissue culture. In this study, we find that intact HIV in the reservoir often carry strong defects in their capacity to promote fusion to neighboring cells and infection of target cells, a defect related to the function and expression of the HIV envelope glycoprotein. Impaired envelope glycoprotein expression and function could explain why cells harboring these viruses tend to remain undetected and unharmed in the reservoir.

Single-dose pharmacokinetics and pharmacodynamics of oral tenofovir and emtricitabine in blood, saliva and rectal tissue: a sub-study of the ANRS IPERGAY trial.

OBJECTIVES: In the ANRS IPERGAY pre-exposure prophylaxis (PrEP) trial, a single dose of tenofovir disoproxil fumarate and emtricitabine was taken orally 2-24 h before sexual intercourse. A sub-study was conducted to assess the pharmacokinetics of tenofovir and emtricitabine in blood, saliva and rectal tissue following this initial oral intake. METHODS: Plasma, PBMC, saliva and rectal tissue sampling was performed over 24 h in 12 seronegative men before enrolment in the ANRS IPERGAY trial, following a single dose of 600 mg tenofovir disoproxil fumarate/400 mg emtricitabine. Ex vivo HIV infectibility of rectal biopsies was also assessed. RESULTS: The median plasma Tmax of tenofovir (median Cmax: 401 µg/L) and emtricitabine (median Cmax: 2868 µg/L) was obtained 1 h (range: 0.5-4) and 2 h (range: 1-4) after dosing, respectively. The median C24 of tenofovir and emtricitabine was 40 and 63 µg/L, respectively. The median PBMC tenofovir diphosphate and emtricitabine triphosphate levels were 12.2 and 16.7 fmol/106 cells and 2800 and 2000 fmol/106 cells at 2 and 24 h after dosing, respectively. Saliva/plasma AUC0-24 ratios were 2% and 17% for tenofovir and emtricitabine, respectively. Emtricitabine was detected in rectal tissue 30 min after dosing, whereas tenofovir was only detectable at 24 h. Ex vivo HIV infectibility assays of rectal biopsies showed partial protection after dosing (P < 0.07). DISCUSSION: A single high dose of oral tenofovir disoproxil fumarate/emtricitabine provides rapid and high blood levels of tenofovir and emtricitabine, with rapid diffusion of emtricitabine in saliva and rectal tissue.

Microtubule-associated proteins 1 (MAP1) promote human immunodeficiency virus type I (HIV-1) intracytoplasmic routing to the nucleus.

After cell entry, HIV undergoes rapid transport toward the nucleus using microtubules and microfilaments. Neither the cellular cytoplasmic components nor the viral proteins that interact to mediate transport have yet been identified. Using a yeast two-hybrid screen, we identified four cytoskeletal components as putative interaction partners for HIV-1 p24 capsid protein: MAP1A, MAP1S, CKAP1, and WIRE. Depletion of MAP1A/MAP1S in indicator cell lines and primary human macrophages led to a profound reduction in HIV-1 infectivity as a result of impaired retrograde trafficking, demonstrated by a characteristic accumulation of capsids away from the nuclear membrane, and an overall defect in nuclear import. MAP1A/MAP1S did not impact microtubule network integrity or cell morphology but contributed to microtubule stabilization, which was shown previously to facilitate infection. In addition, we found that MAP1 proteins interact with HIV-1 cores both in vitro and in infected cells and that interaction involves MAP1 light chain LC2. Depletion of MAP1 proteins reduced the association of HIV-1 capsids with both dynamic and stable microtubules, suggesting that MAP1 proteins help tether incoming viral capsids to the microtubular network, thus promoting cytoplasmic trafficking. This work shows for the first time that following entry into target cells, HIV-1 interacts with the cytoskeleton via its p24 capsid protein. Moreover, our results support a role for MAP1 proteins in promoting efficient retrograde trafficking of HIV-1 by stimulating the formation of stable microtubules and mediating the association of HIV-1 cores with microtubules.

Impact of the HIV integrase genetic context on the phenotypic expression and in vivo emergence of raltegravir resistance mutations.

OBJECTIVES: HIV resistance to the integrase inhibitor raltegravir in treated patients is characterized by distinct resistance pathways. We hypothesize that differences in the in vivo dynamics of HIV resistance to raltegravir are due to the genetic context of the integrase present at baseline. PATIENTS AND METHODS: We studied four patients whose viruses evolved towards different resistance pathways. The integrase baseline sequences were inserted into a reference clone. Primary resistance mutations were then introduced and their impact on viral replication capacity (RC) and resistance was measured. RESULTS: Patients A and B experienced emergence and persistence of mutation N155H under raltegravir therapy. In the integrase sequence from Patient A, N155H conferred potent resistance coupled with a lower impact on RC than Q148H. In Patient B, instead, selection of N155H could be explained by the dramatic loss of RC induced by the alternative Q148H mutation. In Patient C, N155H initially emerged and was later replaced by Q148H. In this integrase context, N155H resulted in higher RC but lower resistance than Q148H. In Patient D, Q148H rapidly emerged without appearance of N155H. This was the only patient for whom Q148H conferred higher RC and resistance than N155H. CONCLUSIONS: The emergence of different resistance mutations in patients was in full agreement with the impact of mutations in different baseline integrase contexts. Evolution towards different resistance genotypes is thus largely determined by the capacity of different integrase sequences present at baseline to minimize the effect of mutations on virus RC while allowing expression of resistance.

Pressure from TRIM5α contributes to control of HIV-1 replication by individuals expressing protective HLA-B alleles.

The expression of certain HLA class I alleles, including HLA-B*27 and HLA-B*57, is associated with better control of human immunodeficiency virus type 1 (HIV-1) infection, but the mechanisms responsible are not fully understood. We sought evidence that pressure from the human restriction factor TRIM5α (hTRIM5α) could contribute to viral control. The hTRIM5α sensitivity of viruses from both HLA-B*57-positive (HLA-B*57(+)) and HLA-B*27(+) patients who spontaneously controlled viral replication, but not viruses from viremic patients expressing these alleles, was significantly greater than that of viruses from patients not expressing these protective HLA-B alleles. Overall, a significant negative correlation between hTRIM5α sensitivity and viral load was observed. In HLA-B*57(+) patients, the T242N mutation in the HLA-B*57-restricted TW10 CD8(+) T lymphocyte (CTL) epitope was strongly associated with hTRIM5α sensitivity. In HLA-B*27(+) controllers, hTRIM5α sensitivity was associated with a significant reduction in emergence of key CTL mutations. In several patients, viral evolution to avoid hTRIM5α sensitivity was observed but could be associated with reduced viral replicative capacity. Thus, in individuals expressing protective HLA-B alleles, the combined pressures exerted by CTL, hTRIM5α, and capsid structural constraints can prevent viral escape both by impeding the selection of necessary resistance/compensatory mutations and forcing the selection of escape mutations that increase hTRIM5α sensitivity or impair viral replicative capacity.

High level of susceptibility to human TRIM5α conferred by HIV-2 capsid sequences.

BACKGROUND: HIV-2, which was transmitted to humans from a distant primate species (sooty mangabey), differs remarkably from HIV-1 in its infectivity, transmissibility and pathogenicity. We have tested the possibility that a greater susceptibility of HIV-2 capsid (CA) to the human restriction factor TRIM5α (hTRIM5α) could contribute to these differences. RESULTS: We constructed recombinant clones expressing CA from a variety of HIV-2 viruses in the context of HIV-1 NL4-3-luciferase. CA sequences were amplified from the plasma of HIV-2 infected patients, including 8 subtype A and 7 subtype B viruses. CA from 6 non-epidemic HIV-2 subtypes, 3 HIV-2 CRF01_AB recombinants and 4 SIVsmm viruses were also tested. Susceptibility to hTRIM5α was measured by comparing single-cycle infectivity in human target cells expressing hTRIM5α to that measured in cells in which hTRIM5α activity was inhibited by overexpression of hTRIM5γ.The insertion of HIV-2 CA sequences in the context of HIV-1 did not affect expression and maturation of the HIV-2 CA protein. The level of susceptibility hTRIM5α expressed by viruses carrying HIV-2 CA sequences was up to 9-fold higher than that of HIV-1 NL4-3 and markedly higher than a panel of primary HIV-1 CA sequences. This phenotype was found both for viruses carrying CA from primary HIV-2 sequences and viruses carrying CA from laboratory-adapted HIV-2 clones. High hTRIM5α susceptibility was found in all HIV-2 subtypes. In this series of viruses, susceptibility to hTRIM5α was not significantly affected by the presence of a proline at position 119 or by the number of prolines at positions 119, 159 or 178 in HIV-2 CA. No significant correlation was found between HIV-2 viremia and sensitivity to hTRIM5α. CONCLUSIONS: HIV-2 capsid sequences expressed high levels of susceptibility to hTRIM5α. This property, common to all HIV-2 sequences tested, may contribute in part to the lower replication and pathogenicity of this virus in humans.

Host cell factors in HIV replication: partners and opponents.

Cycles of virus replication within the cell are often presented in a simplistic manner, viruses being considered as intracellular parasites simply diverting the cellular machinery for their own benefit. Accumulated knowledge on the relationship between the human immunodeficiency virus (HIV) and host cells illustrate the complexity of these relationships. Some cellular factors are partners of the virus and are essential for the proper performance of the multiplication cycle, whereas other factors are adversaries with antiviral activity, called restriction factors. This review aims to give an overview of these partners and opponents.

DNA ultra-sensitive quantification, a technology for studying HIV unintegrated linear DNA.

Unintegrated HIV DNA represents between 20% and 35% of the total viral DNA in infected patients. Only the linear forms (unintegrated linear DNAs [ULDs]) can be substrates for integration and for the completion of a full viral cycle. In quiescent cells, these ULDs may be responsible for pre-integrative latency. However, their detection remains difficult due to the lack of specificity and sensitivity of existing techniques. We developed an ultra-sensitive, specific, and high-throughput technology for ULD quantification called DUSQ (DNA ultra-sensitive quantification) combining linker-mediated PCR and next-generation sequencing (NGS) using molecular barcodes. Studying cells with different activity levels, we determined that the ULD half-life goes up to 11 days in resting CD4+ T cells. Finally, we were able to quantify ULDs in samples from patients infected with HIV-1, providing a proof of concept for the use of DUSQ in vivo to track pre-integrative latency. DUSQ can be adapted to the detection of other rare DNA molecules.

Gag cytotoxic T lymphocyte escape mutations can increase sensitivity of HIV-1 to human TRIM5alpha, linking intrinsic and acquired immunity.

Although laboratory-adapted HIV-1 strains are largely resistant to the human restriction factor TRIM5α (hTRIM5α), we have recently shown that some viruses carrying capsid (CA) sequences from clinical isolates can be more sensitive to this restriction factor. In this study we evaluated the contribution to this phenotype of CA mutations known to be associated with escape from cytotoxic T lymphocyte (CTL) responses. Recombinant viruses carrying HIV-1 CA sequences from NL4-3 and three different clinical isolates were prepared, along with variants in which mutations associated with CTL resistance were modified by site-directed mutagenesis, and the infectivities of these viruses in target cells expressing hTRIM5α and cells in which TRIM5α activity had been inhibited by overexpression of TRIM5γ were compared. For both hTRIM5α-sensitive viruses studied, CTL-associated mutations were found to be responsible for this phenotype. Both CTL resistance mutations occurring within HLA-restricted CA epitopes and compensatory mutations occurring outside CTL epitopes influenced hTRIM5α sensitivity, and mutations associated with CTL resistance selected in prior hosts can contribute to this effect. The impact of CTL resistance mutations on hTRIM5α sensitivity was context dependent, because mutations shown to be responsible for the TRIM5α-sensitive phenotype in viruses from one patient could have little or no impact on this parameter when introduced into another virus. No fixed relationship between changes in hTRIM5α sensitivity and infectivity was discernible in our studies. Taken together, these findings suggest that CTL mutations may influence HIV-1 replication by modifying both viral infectivity and sensitivity to TRIM5α.

Modulation of TRIM5alpha activity in human cells by alternatively spliced TRIM5 isoforms.

TRIM5α is a restriction factor that can block an early step in the retroviral life cycle by recognizing and causing the disassembly of incoming viral capsids, thereby preventing the completion of reverse transcription. Numerous other isoforms of human TRIM5 exist, and isoforms lacking a C-terminal SPRY domain can inhibit the activity of TRIM5α. Thus, TRIM5α activity in a given cell type could be dependent on the relative proportions of TRIM5 isoforms expressed, but little information concerning the relative expression of TRIM5 isoforms in human cells is available. In this study, we demonstrate that mRNAs coding for TRIM5α represent only 50% of total TRIM5 transcripts in human cell lines, CD4(+) T cells, and macrophages. Transcripts coding for, in order of abundance, TRIM5ι (TRIM5-iota), a previously uncharacterized isoform, TRIM5γ, TRIM5δ, and TRIM5κ are also present. Like TRIM5γ and TRIM5δ, TRIM5ι and TRIM5κ do not inhibit HIV-1 replication, but both have dominant-negative activity against TRIM5α. Specific knockdown of TRIM5ι increases TRIM5α activity in human U373-X4 cells, indicating that physiological levels of expression of truncated TRIM5 isoforms in human cells can reduce the activity of TRIM5α.

Genotypic and Phenotypic Diversity of the Replication-Competent HIV Reservoir in Treated Patients.

In HIV infection, viral rebound after treatment discontinuation is considered to originate predominantly from viral genomes integrated in resting CD4+ T lymphocytes. Replication-competent proviral genomes represent a minority of the total HIV DNA. While the quantification of the HIV reservoir has been extensively studied, the diversity of genomes that compose the reservoir was less explored. Here, we measured the genotypic and phenotypic diversity in eight patients with different treatment histories. Between 4 and 14 (mean, 8) individual viral isolates per patient were obtained using a virus outgrowth assay, and their near-full-length genomes were sequenced. The mean pairwise distance (MPD) observed in different patients correlated with the time before undetectable viremia was achieved (r = 0.864, P = 0.0194), suggesting that the complexity of the replication-competent reservoir mirrors that present at treatment initiation. No correlation was instead observed between MPD and the duration of successful treatment (mean, 8 years; range, 2 to 21 years). For 5 of the 8 patients, genotypically identical viral isolates were observed in independent wells, suggesting clonal expansion of infected cells. Identical viruses represented between 25 and 60% of the isolates (mean, 48%). The proportion of identical viral isolates correlated with the duration of treatment (r = 0.822, P = 0.0190), suggesting progressive clonal expansion of infected cells during ART. A broader range of infectivity was also observed among isolates from patients with delayed viremia control (r = 0.79, P = 0.025). This work unveiled differences in the genotypic and phenotypic features of the replication-competent reservoir from treated patients and suggests that delaying treatment results in increased diversity of the reservoir. IMPORTANCE In HIV-infected and effectively treated individuals, integrated proviral genomes may persist for decades. The vast majority of the genomes, however, are defective, and only the replication-competent fraction represents a threat of viral reemergence. The quantification of the reservoir has been thoroughly explored, while the diversity of the genomes has been insufficiently studied. Its characterization, however, is relevant for the design of strategies aiming the reduction of the reservoir. Here, we explored the replication-competent near-full-length HIV genomes of eight patients who experienced differences in the delay before viremia control and in treatment duration. We found that delayed effective treatment was associated with increased genetic diversity of the reservoir. The duration of treatment did not impact the diversity but was associated with higher frequency of clonally expanded sequences. Thus, early treatment initiation has the double advantage of reducing both the size and the diversity of the reservoir.

Strain-specific differences in the impact of human TRIM5alpha, different TRIM5alpha alleles, and the inhibition of capsid-cyclophilin A interactions on the infectivity of HIV-1.

HIV-1 infectivity is strongly restricted by TRIM5α from certain primate species but has been described as being only marginally susceptible to human TRIM5α. In this study, we evaluated the effects of the modulation of human TRIM5α activity (pretreatment of target cells with alpha interferon, expression of a pre-miRNA targeting TRIM5α, and/or overexpression of TRIM5γ), the inhibition of cyclophilin A (CypA)-CA interactions, and the expression of different allelic variants of human TRIM5α on the infectivity of a series of recombinant viruses carrying different patient-derived Gag-protease sequences. We show that HIV-1 displays virus-specific differences in its sensitivity to human TRIM5α and in its sensitivity to different TRIM5α alleles. The effect of inhibiting CypA-CA interactions is also strain specific, and blocking these interactions can either inhibit or improve viral infectivity, depending on the isolate studied. The inhibition of CypA-CA interactions also modulates viral sensitivity to human TRIM5α. In the absence of CypA-CA interactions, most viruses displayed increased sensitivity to the inhibitory effects of TRIM5α on viral replication, but one isolate showed a paradoxical decrease in sensitivity to TRIM5α. Taken together, these findings support a model in which three interlinked factors--capsid sequence, CypA levels, and TRIM5α--interact to determine capsid stability and therefore viral infectivity.

Gag mutations strongly contribute to HIV-1 resistance to protease inhibitors in highly drug-experienced patients besides compensating for fitness loss.

Human immunodeficiency virus type 1 (HIV-1) resistance to protease inhibitors (PI) results from mutations in the viral protease (PR) that reduce PI binding but also decrease viral replicative capacity (RC). Additional mutations compensating for the RC loss subsequently accumulate within PR and in Gag substrate cleavage sites. We examined the respective contribution of mutations in PR and Gag to PI resistance and RC and their interdependence using a panel of HIV-1 molecular clones carrying different sequences from six patients who had failed multiple lines of treatment. Mutations in Gag strongly and directly contributed to PI resistance besides compensating for fitness loss. This effect was essentially carried by the C-terminal region of Gag (containing NC-SP2-p6) with little or no contribution from MA, CA, and SP1. The effect of Gag on resistance depended on the presence of cleavage site mutations A431V or I437V in NC-SP2-p6 and correlated with processing of the NC/SP2 cleavage site. By contrast, reverting the A431V or I437V mutation in these highly evolved sequences had little effect on RC. Mutations in the NC-SP2-p6 region of Gag can be dually selected as compensatory and as direct PI resistance mutations, with cleavage at the NC-SP2 site behaving as a rate-limiting step in PI resistance. Further compensatory mutations render viral RC independent of the A431V or I437V mutations while their effect on resistance persists.

HIV-1 Gag processing intermediates trans-dominantly interfere with HIV-1 infectivity.

Protease inhibitors (PI) act by blocking human immunodeficiency virus (HIV) polyprotein processing, but there is no direct quantitative correlation between the degree of impairment of Gag processing and virion infectivity at low PI concentrations. To analyze the consequences of partial processing, virus particles were produced in the presence of limiting PI concentrations or by co-transfection of wild-type proviral plasmids with constructs carrying mutations in one or more cleavage sites. Low PI concentrations caused subtle changes in polyprotein processing associated with a pronounced reduction of particle infectivity. Dissection of individual stages of viral entry indicated a block in accumulation of reverse transcriptase products, whereas virus entry, enzymatic reverse transcriptase activity, and replication steps following reverse transcription were not affected. Co-expression of low amounts of partially processed forms of Gag together with wild-type HIV generally exerted a trans-dominant effect, which was most prominent for a construct carrying mutations at both cleavage sites flanking the CA domain. Interestingly, co-expression of low amounts of Gag mutated at the CA-SP1 cleavage site also affected processing activity at this site in the wild-type virus. The results indicate that low amounts (<5%) of Gag processing intermediates can display a trans-dominant effect on HIV particle maturation, with the maturation cleavage between CA and SP1 being of particular importance. These effects are likely to be important for the strong activity of PI at concentrations achieved in vivo and also bear relevance for the mechanism of action of the antiviral drug bevirimat.

Selective-advantage profile of human immunodeficiency virus type 1 integrase mutants explains in vivo evolution of raltegravir resistance genotypes.

The emergence of human immunodeficiency virus type 1 resistance to raltegravir, an integrase strand transfer inhibitor, follows distinct and independent genetic pathways, among which the N155H and Q148HKR pathways are the most frequently encountered in treated patients. After prolonged viral escape, mutants of the N155H pathway are replaced by mutants of the Q148HKR pathway. We have examined the mechanisms driving this evolutionary pattern using an approach that assesses the selective advantage of site-directed mutant viruses as a function of drug concentration. These selective-advantage curves revealed that among single mutants, N155H had the highest and the widest (1 to 500 nM) selective-advantage profile. Despite the higher 50% inhibitory concentration, Q148H displayed a lower and narrower (10 to 100 nM) selective-advantage profile. Among double mutants, the highest and widest selective-advantage profile was seen with G140S+Q148H. This finding likely explains why N155H can be selected early in the course of RAL resistance evolution in vivo but is later replaced by genotypes that include Q148HKR.

Modulation of HIV-1 infectivity and cyclophilin A-dependence by Gag sequence and target cell type.

BACKGROUND: HIV-1 Gag proteins are essential for virion assembly and viral replication in newly infected cells. Gag proteins are also strong determinants of viral infectivity; immune escape mutations in the Gag capsid (CA) protein can markedly reduce viral fitness, and interactions of CA with host proteins such as cyclophilin A (CypA) and TRIM5alpha can have important effects on viral infectivity. Little information, however, is available concerning the extent that different primary Gag proteins affect HIV-1 replication in different cell types, or the impact on viral replication of differences in the expression by target cells of proteins that interact with CA. To address these questions, we compared the infectivity of recombinant HIV-1 viruses expressing Gag-protease sequences from primary isolates in different target cells in the presence or absence of agents that disrupt cyclophilin A - CA interactions and correlated these results with the viral genotype and the expression of cyclophilin A and TRIM5alpha by the target cells. RESULTS: Viral infectivity was governed by the nature of the Gag proteins in a target cell-specific fashion. The treatment of target cells with agents that disrupt CypA-CA interactions often produced biphasic dose-response curves in which viral infectivity first increased and subsequently decreased as a function of the dose used. The extent that treatment of target cells with high-dose CypA inhibitors impaired viral infectivity was dependent on several factors, including the viral genotype, the nature of the target cell, and the extent that treatment with low-dose CypA inhibitors increased viral infectivity. Neither the presence of polymorphisms in the CA CypA-binding loop, the level of expression of CypA, or the level of TRIM5alpha expression could, alone, explain the differences in the shape of the dose-response curves observed or the extent that high-dose CypA inhibitors reduced viral infectivity. CONCLUSION: Multiple interactions between host-cell factors and Gag can strongly affect HIV-1 infectivity, and these vary according to target cell type and the origin of the Gag sequence. Two of the cellular activities involved appear to be modulated in opposite directions by CypA-CA interactions, and Gag sequences determine the intrinsic sensitivity of a given virus to each of these cellular activities.

In vivo selection by enfuvirtide of HIV type-1 env quasispecies with optimal potential for phenotypic expression of HR1 mutations.

BACKGROUND: HIV type-1 (HIV-1) resistance to enfuvirtide (ENF) is mediated by mutations in the HR1 domain of gp41. We have previously shown that some of these mutations are selected in the context of env backgrounds that are not dominant before exposure to ENF, suggesting that particular env environments could facilitate phenotypic expression of HR1-mediated ENF resistance. METHODS: Envelope clones, representing the viral quasi-species present in the longitudinal follow-up of a patient who failed ENF-based salvage therapy, were tested for ENF susceptibility and Env-related replicative capacity. ENF resistance mutations in HR1 were introduced or back-mutated in representative clones to evaluate their phenotypic effect in different genetic contexts. RESULTS: The ENF resistance levels produced by the introduction of mutation V38A in pretherapeutic env sequences were significantly lower than those of env clones harvested after viral escape, and in which V38A was naturally selected. Back-mutation of V38A from these clones resulted in a strong loss in ENF resistance, but these clones retained significant residual resistance, again strongly suggesting the role of determinants outside of HR1 in HIV-1 resistance to ENF. By contrast with changes in resistance, addition or removal of HR1 mutations in env clones had little effect on viral replicative capacity. CONCLUSIONS: The development of ENF resistance in vivo is a concerted coevolutionary process whereby HR1 mutations are selected within env variants that permit their optimal phenotypic expression.

HIV resistance to raltegravir.

Similar to all antiretroviral drugs, failure of raltegravir-based treatment regimens to fully supress HIV replication almost invariably results in emergence of HIV resistance to this new drug. HIV resistance to raltegravir is the consequence of mutations located close to the integrase active site, which can be divided into three main evolutionary pathways: the N155H, the Q148R/H/K and the Y143R/C pathways. Each of these primary mutations can be accompanied by a variety of secondary mutations that both increase resistance and compensate for the variable loss of viral replicative capacity that is often associated with primary resistance mutations. One unique property of HIV resistance to raltegravir is that each of these different resistance pathways are mutually exclusive and appear to evolve separately on distinct viral genomes. Resistance is frequently initiated by viruses carrying mutations of the N155H pathway, followed by emergence and further dominance of viral genomes carrying mutations of the Q148R/H/K or of the Y143R/C pathways, which express higher levels of resistance. Even if some natural integrase polymorphisms can be part of this evolution process, these polymorphisms do not affect HIV susceptibility in the absence of primary mutations. Therefore, all HIV-1 subtypes and groups, together with HIV-2, are naturally susceptible to raltegravir. Finally, because interaction of integrase strand transfer inhibitors with the HIV integrase active site is comparable from one compound to another, raltegravir-resistant viruses express significant cross resistance to most other compounds of this new class of antiretroviral drugs.

Peptide P5 (residues 628-683), comprising the entire membrane proximal region of HIV-1 gp41 and its calcium-binding site, is a potent inhibitor of HIV-1 infection.

The membrane proximal region (MPR) of the transmembrane subunit, gp41, of the HIV envelope glycoprotein plays a critical role in HIV-1 infection of CD4+ target cells and CD4-independent mucosal entry. It contains continuous epitopes recognized by neutralizing IgG antibodies 2F5, 4E10 and Z13, and is therefore considered to be a promising target for vaccine design. Moreover, some MPR-derived peptides, such as T20 (enfuvirtide), are in clinical use as HIV-1 inhibitors. We have shown that an extended MPR peptide, P5, harbouring the lectin-like domain of gp41 and a calcium-binding site, is implicated in the interaction of HIV with its mucosal receptor. We now investigate the potential antiviral activities of P5 and other such long MPR-derived peptides. Structural studies of gp41 MPR-derived peptides using circular dichroism showed that the peptides P5 (a.a.628-683), P1 (a.a.648-683), P5L (a.a.613-683) and P7 (a.a.613-746) displayed a well-defined alpha-helical structure. Peptides P5 inhibited HIV-1 envelope mediated cell-cell fusion and infection of peripheral blood mononuclear cells by both X4- and R5-tropic HIV-1 strains, whereas peptides P5 mutated in the calcium binding site or P1 lacked antiviral activity, when P5L blocked cell fusion in contrast to P7. Strikingly, P5 inhibited CD4-dependent infection by T20-resistant R5-tropic HIV-1 variants. Cell-cell fusion studies indicated that the anti-HIV-1 activity of P5, unlike T20, could not be abrogated in the presence of the N-terminal leucine zipper domain (LZ). These results suggested that P5 could serve as a potent fusion inhibitor.

Functional diversity of HIV-1 envelope proteins expressed by contemporaneous plasma viruses.

BACKGROUND: Numerous studies have shown that viral quasi-species with genetically diverse envelope proteins (Env) replicate simultaneously in patients infected with the human immunodeficiency virus type 1 (HIV-1). Less information is available concerning the extent that envelope sequence diversity translates into a diversity of phenotypic properties, including infectivity and resistance to entry inhibitors. METHODS: To study these questions, we isolated genetically distinct contemporaneous clonal viral populations from the plasma of 5 HIV-1 infected individuals (n = 70), and evaluated the infectivity of recombinant viruses expressing Env proteins from the clonal viruses in several target cells. The sensitivity to entry inhibitors (enfuvirtide, TAK-799), soluble CD4 and monoclonal antibodies (2G12, 48d, 2F5) was also evaluated for a subset of the recombinant viruses (n = 20). RESULTS: Even when comparisons were restricted to viruses with similar tropism, the infectivity for a given target cell of viruses carrying different Env proteins from the same patient varied over an approximately 10-fold range, and differences in their relative ability to infect different target cells were also observed. Variable region haplotypes associated with high and low infectivity could be identified for one patient. In addition, clones carrying unique mutations in V3 often displayed low infectivity. No correlation was observed between viral infectivity and sensitivity to inhibition by any of the six entry inhibitors evaluated, indicating that these properties can be dissociated. Significant inter-patient differences, independent of infectivity, were observed for the sensitivity of Env proteins to several entry inhibitors and their ability to infect different target cells. CONCLUSION: These findings demonstrate the marked functional heterogeneity of HIV-1 Env proteins expressed by contemporaneous circulating viruses, and underscore the advantage of clonal analyses in characterizing the spectrum of functional properties of the genetically diverse viral populations present in a given patient.