Role of the envelope genetic context in the development of enfuvirtide resistance in human immunodeficiency virus type 1-infected patients

Acquired human immunodeficiency virus type 1(HIV-1) resistance to the fusion inhibitor enfuvirtide (ENF) is primarily associated with mutations within the highly conserved first heptad repeat (HR1) region of gp41. Viral env sequences, however, are remarkably variable, and the envelope genetic background could have an important impact on optimal expression of HR1 mutations. We have examined the genetic evolution of env sequences, ENF susceptibility, and Env replicative capacity in patients failing ENF treatment. Sequential plasma-derived virus populations, obtained from six patients initiating ENF treatment as part of a salvage therapy, were studied using a recombinant phenotypic assay evaluating the entire gp120 and the gp41 ectodomains. Regardless of major differences in the baseline ENF susceptibilities, viral populations with similar phenotypic ENF resistance (50% inhibitory concentration, >3,000 ng/ml) were selected under treatment in four of six patients. As expected, in all patients ENF-resistant viruses harbored one or more HR1 mutations (positions 36, 38, and 43). Interestingly, in five patients the emergence of resistance mutations was not associated with reduced Env replicative capacity. Phylogenetic analysis of env sequences in sequential samples from two patients showed that the HR1 mutations had emerged in the context of env quasi-species that were different from those prevalent at baseline. Thus, the envelope genetic context appears to play a critical role in the selection of HR1 mutations and the expression of ENF resistance, thereby conditioning the evolution of HIV-1 under fusion inhibitor selective pressure.     

Relative replicative fitness of human immunodeficiency virus type 1 mutants resistant to enfuvirtide (T-20)

Resistance to enfuvirtide (ENF; T-20), a fusion inhibitor of human immunodeficiency virus type 1 (HIV-1), is conferred by mutations in the first heptad repeat of the gp41 ectodomain. The replicative fitness of recombinant viruses carrying ENF resistance mutations was studied in growth competition assays. ENF resistance mutations, selected in vitro or in vivo, were introduced into the env gene of HIV-1(NL4-3) by site-directed mutagenesis and expressed in HIV-1 recombinants carrying sequence tags in nef. The doubling time of ENF-resistant viruses was highly correlated with decreasing ENF susceptibility (R(2) = 0.859; P < 0.001). Initial fitness experiments focused on mutants identified by in vitro selection in the presence of ENF (L. T. Rimsky, D. C. Shugars, and T. J. Matthews, J. Virol. 72:986-993, 1998). In the absence of drug, these mutants displayed reduced fitness compared to wild-type virus with a relative order of fitness of wild type > I37T > V38 M > D36S/V38 M; this order was reversed in the presence of ENF. Likewise, recombinant viruses carrying ENF resistance mutations selected in vivo displayed reduced fitness in the absence of ENF with a relative order of wild type > N42T > V38A > N42T/N43K approximately N42T/N43S > V38A/N42D approximately V38A/N42T. Fitness and ENF susceptibility were inversely correlated (r = -0.988; P < 0.001). Similar results were obtained with recombinants expressing molecularly cloned full-length env genes obtained from patient-derived HIV-1 isolates before and after ENF treatment. Further studies are needed to determine whether the reduced fitness of ENF-resistant viruses alters their pathogenicity in vivo.     

Vicriviroc resistance decay and relative replicative fitness in HIV-1 clinical isolates under sequential drug selection pressures

We previously described an HIV-1-infected individual who developed resistance to vicriviroc (VCV), an investigational CCR5 antagonist, during 28 weeks of therapy (Tsibris AM et al., J. Virol. 82:8210-8214, 2008). To investigate the decay of VCV resistance mutations, a standard clonal analysis of full-length env (gp160) was performed on plasma HIV-1 samples obtained at week 28 (the time of VCV discontinuation) and at three subsequent time points (weeks 30, 42, and 48). During 132 days, VCV-resistant HIV-1 was replaced by VCV-sensitive viruses whose V3 loop sequences differed from the dominant pretreatment forms. A deep-sequencing analysis showed that the week 48 VCV-sensitive V3 loop form emerged from a preexisting viral variant. Enfuvirtide was added to the antiretroviral regimen at week 30; by week 48, enfuvirtide treatment selected for either the G36D or N43D HR-1 mutation. Growth competition experiments demonstrated that viruses incorporating the dominant week 28 VCV-resistant env were less fit than week 0 viruses in the absence of VCV but more fit than week 48 viruses. This week 48 fitness deficit persisted when G36D was corrected by either site-directed mutagenesis or week 48 gp41 domain swapping. The correction of N43D, in contrast, restored fitness relative to that of week 28, but not week 0, viruses. Virus entry kinetics correlated with observed fitness differences; the slower entry of enfuvirtide-resistant viruses corrected to wild-type rates in the presence of enfuvirtide. These findings suggest that while VCV and enfuvirtide select for resistance mutations in only one env subunit, gp120 and gp41 coevolve to maximize viral fitness under sequential drug selection pressures.     

Clinical resistance to enfuvirtide does not affect susceptibility of human immunodeficiency virus type 1 to other classes of entry inhibitors

The clinical use of the human immunodeficiency virus (HIV) fusion inhibitor enfuvirtide (ENF) can select for drug-resistant HIV-1 strains bearing mutations in the HR1 region of the viral envelope (Env) protein. We analyzed the properties of multiple Env proteins isolated from five patients who experienced an initial decline in viral load after ENF therapy followed by subsequent rebound due to emergence of ENF-resistant HIV-1. Prior to ENF therapy, each patient harbored genetically and phenotypically diverse Env proteins that used CCR5 and/or CXCR4 to elicit membrane fusion. Coreceptor usage patterns of the Envs isolated from two patients underwent homogenization following ENF therapy, whereas in the other three patients, recombination appeared to allow the introduction of a single HR1 sequence with ENF resistance mutations into phenotypically distinct Env proteins. Analysis of individual Env clones also revealed that prior to ENF therapy, there was sometimes marked heterogeneity in the susceptibility of individual Env proteins to coreceptor inhibitors. After virologic failure, all Envs acquired resistance to ENF but exhibited no consistent change in their sensitivity to the fusion inhibitor T-1249 or to coreceptor inhibitors. In summary, using patient-derived Env proteins, we found that ENF failure was associated with emergence of high-level resistance to ENF due largely to mutations in HR1 but that susceptibility to other entry inhibitors was unaffected, that in these late-stage patients there was greater clonal variability to coreceptor than to fusion inhibitors, and that recombination events in vivo could sometimes restore Env genotypic and phenotypic heterogeneity by introducing drug-resistant gp41 sequences into heterologous gp120 backgrounds.     

Enfuvirtide resistance mutations: impact on human immunodeficiency virus envelope function, entry inhibitor sensitivity, and virus neutralization

Enfuvirtide (ENF/T-20/Fuzeon), the first human immunodeficiency virus (HIV) entry inhibitor to be licensed, targets a structural intermediate of the entry process. ENF binds the HR1 domain in gp41 after Env has bound CD4, preventing conformational changes needed for membrane fusion. Mutations in HR1 that confer ENF resistance can arise following ENF therapy. ENF resistance mutations were introduced into an R5- and X4-tropic Env to examine their impact on fusion, infection, and sensitivity to different classes of entry inhibitors and neutralizing antibodies. HR1 mutations could reduce infection and fusion efficiency and also delay fusion kinetics, likely accounting for their negative impact on viral fitness. HR1 mutations had minimal effect on virus sensitivity to other classes of entry inhibitors, including those targeting CD4 binding (BMS-806 and a CD4-specific monoclonal antibody [MAb]), coreceptor binding (CXCR4 inhibitor AMD3100 and CCR5 inhibitor TAK-779), or fusion (T-1249), indicating that ENF-resistant viruses can remain sensitive to other entry inhibitors in vivo. Some HR1 mutations conferred increased sensitivity to a subset of neutralizing MAbs that likely target fusion intermediates or with epitopes preferentially exposed following receptor interactions (17b, 48D, 2F5, 4E10, and IgGb12), as well as sera from some HIV-positive individuals. Mechanistically, enhanced neutralization correlated with reduced fusion kinetics, indicating that, in addition to steric constraints, kinetics may also limit virus neutralization by some antibodies. Therefore, escape from ENF comes at a cost to viral fitness and may confer enhanced sensitivity to humoral immunity due to prolonged exposure of epitopes that are not readily accessible in the native Env trimer. Resistance to other entry inhibitors was not observed.     

Determinants of human immunodeficiency virus type 1 baseline susceptibility to the fusion inhibitors enfuvirtide and T-649 reside outside the peptide interaction site

The peptide fusion inhibitor (PFI) enfuvirtide is the first of a new class of entry inhibitors to receive FDA approval. We previously determined the susceptibility of 55 PFI-na?ve-patient isolates to enfuvirtide and a second peptide inhibitor, T-649. Seven of the 55 viral isolates were insusceptible to enfuvirtide, T-649, or both inhibitors in the absence of prior exposure. To determine the molecular basis of the insusceptible phenotypes, we PCR amplified and cloned five PFI-insusceptible and one PFI-susceptible, full-length, biologically functional env genes and characterized viruses pseudotyped with the Env proteins in a single-round drug sensitivity assay. Overall, the mean 50% inhibitory concentrations of enfuvirtide and T-649 for the PFI-insusceptible Env pseudotypes were 1.4 to 1.7 log(10) and 1.2 to 1.8 log(10) greater, respectively, than those for a PFI-susceptible lab strain, NLHX; however, all of the PFI-insusceptible Env proteins conserved the sequence of a critical enfuvirtide interaction site (residues 36 to 38 of gp41, GIV) in HR-1. In contrast, multiple amino acid changes were observed C-terminal to HR-1, many of which were located in regions of HR-2 corresponding to the PFI. Nevertheless, peptides based on patient-derived HR-2 sequences were not more potent inhibitors than enfuvirtide or T-649, arguing that the basis of PFI susceptibility is not a higher-affinity, competitive HR-1/HR-2 interaction. These results demonstrate that regions of Env outside the enfuvirtide interaction site can significantly impact the PFI susceptibility of patient-derived Env, even prior to drug exposure. We hypothesize that both gp120 gene- and gp41 gene-encoded determinants that minimize the window of opportunity for PFI to bind provide a growth advantage and possibly a predisposition to resistance to this new class of drugs in vivo.     

Impact of mutations in the coreceptor binding site on human immunodeficiency virus type 1 fusion, infection, and entry inhibitor sensitivity

An increasingly large number of antiviral agents that prevent entry of human immunodeficiency virus (HIV) into cells are in preclinical and clinical development. The envelope (Env) protein of HIV is the major viral determinant that affects sensitivity to these compounds. To understand how changes in Env can impact entry inhibitor sensitivity, we introduced six mutations into the conserved coreceptor binding site of the R5 HIV-1 strain YU-2 and measured the effect of these changes on CD4 and coreceptor binding, membrane fusion levels and rates, virus infection, and sensitivity to the fusion inhibitors enfuvirtide (T-20) and T-1249, the CCR5 inhibitor TAK-779, and an antibody to CD4. The mutations had little effect on CD4 binding but reduced CCR5 binding to various extents. In general, reductions in coreceptor binding efficiency resulted in slower fusion kinetics and increased sensitivity to TAK-779 and enfuvirtide. In addition, low CCR5 binding usually reduced overall fusion and infection levels. However, one mutation adjacent to the bridging sheet beta21 strand, P438A, had little effect on fusion activity, fusion rate, infectivity, or sensitivity to enfuvirtide or T-1249 despite causing a marked reduction in CCR5 binding and a significant increase in TAK-779 sensitivity. Thus, our findings indicate that changes in the coreceptor binding site of Env can modulate its fusion activity, infectivity, and entry inhibitor sensitivity by multiple mechanisms and suggest that reductions in coreceptor binding do not always result in prolonged fusion kinetics and increased sensitivity to enfuvirtide.     

Loss of asparagine-linked glycosylation sites in variable region 5 of human immunodeficiency virus type 1 envelope is associated with resistance to CD4 antibody ibalizumab

Ibalizumab (formerly TNX-355) is a first-in-class, monoclonal antibody inhibitor of CD4-mediated human immunodeficiency type 1 (HIV-1) entry. Multiple clinical trials with HIV-infected patients have demonstrated the antiviral activity, safety, and tolerability of ibalizumab treatment. A 9-week phase Ib study adding ibalizumab monotherapy to failing drug regimens led to transient reductions in HIV viral loads and the evolution of HIV-1 variants with reduced susceptibility to ibalizumab. This report characterizes these variants by comparing the phenotypic susceptibilities and envelope (env) sequences of (i) paired baseline and on-treatment virus populations, (ii) individual env clones from selected paired samples, and (iii) env clones containing site-directed mutations. Viruses with reduced susceptibility to ibalizumab were found to exhibit reduced susceptibility to the anti-CD4 antibody RPA-T4. Conversely, susceptibility to soluble CD4, which targets the HIV-1 gp120 envelope protein, was enhanced. No changes in susceptibility to the fusion inhibitor enfuvirtide or the CCR5 antagonist maraviroc were observed. Functionally, viruses with reduced ibalizumab susceptibility also displayed high levels of infectivity relative to those of paired baseline viruses. Individual env clones exhibiting reduced ibalizumab susceptibility contained multiple amino acid changes in different regions relative to the paired baseline clones. In particular, clones with reduced susceptibility to ibalizumab contained fewer potential asparagine-linked glycosylation sites (PNGSs) in variable region 5 (V5) than did paired ibalizumab-susceptible clones. The reduction in ibalizumab susceptibility due to the loss of V5 PNGSs was confirmed by site-directed mutagenesis. Taken together, these findings provide important insights into resistance to this new class of antiretroviral drug.     

trans-Dominant Interference with Human Immunodeficiency Virus Type 1 Replication and Transmission in CD4+ Cells by an Envelope Double Mutant

We previously reported that a human immunodeficiency virus type 1 (HIV-1) envelope (Env) mutant with the whole cytoplasmic domain deleted, denoted mutant TC, is able to dominantly interfere with wild-type (wt) virus infectivity. In the present study, the feasibility of developing a dominant negative mutant-based genetic anti-HIV strategy targeting the gp41 cytoplasmic domain was investigated. Mutants TC and 427,TC, a TC derivative with a Trp-to-Ser substitution introduced into residue 427 in the CD4-binding site, and a series of mutants with deletions in the cytoplasmic domain, effectively trans-dominantly interfered with wt Env-mediated viral infectivity, as demonstrated by an env trans-complementation assay. The syncytium formation-defective 427, TC double mutant not only inhibited heterologous LAV and ELI Env-mediated viral infectivity but also interfered with syncytium formation and infectivity mediated by the Env proteins of the two primary isolates 92BR and 92US. Stable HeLa-CD4-LTR-beta-gal clones that harbored Tat-controlled expression cassettes encoding the control DeltaKS, which had a deletion in the env gene, wt, or mutant env gene were generated. Viral transmission mediated by laboratory-adapted T-cell-tropic HXB2 and NL4-3 viruses was greatly reduced in the TC and 427,TC transfectants compared to that observed in the control DeltaKS and wt transfectants. Viral replication caused by HXB2 and NL4-3 viruses and by macrophage-tropic ConB and ADA-GG viruses was delayed or reduced in human CD4(+) T cells transfected with the 427,TC env construct compared to that observed in cells transfected with the control DeltaKS or TC env construct. The lack of significant interference by TC mutant was due neither to the lack of TC env gene integration into host DNA nor to the lack of TC Env expression upon Tat induction. These results indicate that this 427,TC Env double mutant has a role in the development of trans-dominant mutant-based genetic anti-HIV strategies.     

Nef decreases HIV-1 sensitivity to neutralizing antibodies that target the membrane-proximal external region of TMgp41

Primate lentivirus nef is required for sustained virus replication in vivo and accelerated progression to AIDS. While exploring the mechanism by which Nef increases the infectivity of cell-free virions, we investigated a functional link between Nef and Env. Since we failed to detect an effect of Nef on the quantity of virion-associated Env, we searched for qualitative changes by examining whether Nef alters HIV-1 sensitivity to agents that target distinct features of Env. Nef conferred as much as 50-fold resistance to 2F5 and 4E10, two potent neutralizing monoclonal antibodies (nAbs) that target the membrane proximal external region (MPER) of TMgp41. In contrast, Nef had no effect on HIV-1 neutralization by MPER-specific nAb Z13e1, by the peptide inhibitor T20, nor by a panel of nAbs and other reagents targeting gp120. Resistance to neutralization by 2F5 and 4E10 was observed with Nef from a diverse range of HIV-1 and SIV isolates, as well as with HIV-1 virions bearing Env from CCR5- and CXCR4-tropic viruses, clade B and C viruses, or primary isolates. Functional analysis of a panel of Nef mutants revealed that this activity requires Nef myristoylation but that it is genetically separable from other Nef functions such as the ability to enhance virus infectivity and to downregulate CD4. Glycosylated-Gag from MoMLV substituted for Nef in conferring resistance to 2F5 and 4E10, indicating that this activity is conserved in a retrovirus that does not encode Nef. Given the reported membrane-dependence of MPER-recognition by 2F5 and 4E10, in contrast to the membrane-independence of Z13e1, the data here is consistent with a model in which Nef alters MPER recognition in the context of the virion membrane. Indeed, Nef and Glycosylated-Gag decreased the efficiency of virion capture by 2F5 and 4E10, but not by other nAbs. These studies demonstrate that Nef protects lentiviruses from one of the most broadly-acting classes of neutralizing antibodies. This newly discovered activity for Nef has important implications for anti-HIV-1 immunity and AIDS pathogenesis.     

Positive and negative modulation of virus infectivity and envelope glycoprotein incorporation into virions by amino acid substitutions at the N terminus of the simian immunodeficiency virus matrix protein

The matrix (MA) protein of the simian immunodeficiency viruses (SIVs) is encoded by the amino-terminal region of the Gag precursor and is the component of the viral capsid that lines the inner surface of the virus envelope. Previously, we identified domains in the SIV MA that are involved in the transport of Gag to the plasma membrane and in particle assembly. In this study, we characterized the role in the SIV life cycle of highly conserved residues within the SIV MA region spanning the two N-terminal alpha-helices H1 and H2. Our analyses identified two classes of MA mutants: (i) viruses encoding amino acid substitutions within alpha-helices H1 or H2 that were defective in envelope (Env) glycoprotein incorporation and exhibited impaired infectivity and (ii) viruses harboring mutations in the beta-turn connecting helices H1 and H2 that were more infectious than the wild-type virus and displayed an enhanced ability to incorporate the Env glycoprotein. Remarkably, among the latter group of MA mutants, the R22L/G24L double amino acid substitution increased virus infectivity eightfold relative to the wild-type virus in single-cycle infectivity assays, an effect that correlated with a similar increase in Env incorporation. Furthermore, the R22L/G24L MA mutation partially or fully complemented single-point MA mutations that severely impair or block Env incorporation and virus infectivity. Our finding that the incorporation of the Env glycoprotein into virions can be upregulated by specific mutations within the SIV MA amino terminus strongly supports the notion that the SIV MA domain mediates Gag-Env association during particle formation.     

Envelope is a major viral determinant of the distinct in vitro cellular transformation tropism of human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2

Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are related deltaretroviruses but are distinct in their disease-inducing capacity. These viruses can infect a variety of cell types, but only T lymphocytes become transformed, which is defined in vitro as showing indefinite interleukin-2-independent growth. Studies have indicated that HTLV-1 has a preferential tropism for CD4+ T cells in vivo and is associated with the development of leukemia and neurological disease. Conversely, the in vivo T-cell tropism of HTLV-2 is less clear, although it appears that CD8+ T cells preferentially harbor the provirus, with only a few cases of disease association. The difference in T-cell transformation tropism has been confirmed in vitro as shown by the preferential transformation of CD4+ T cells by HTLV-1 versus the transformation of CD8+ T cells by HTLV-2. Our previous studies showed that Tax and overlapping Rex do not confer the distinct T-cell transformation tropisms between HTLV-1 and HTLV-2. Therefore, for this study HTLV-1 and HTLV-2 recombinants were generated to assess the contribution of LTR and env sequences in T-cell transformation tropism. Both sets of proviral recombinants expressed p19 Gag following transfection into cells. Furthermore, recombinant viruses were replication competent and had the capacity to transform T lymphocytes. Our data showed that exchange of the env gene resulted in altered T-cell transformation tropism compared to wild-type virus, while exchange of long terminal repeat sequences had no significant effect. HTLV-2/Env1 preferentially transformed CD4+ T cells similarly to wild-type HTLV-1 (wtHTLV-1), whereas HTLV-1/Env2 had a transformation tropism similar to that of wtHTLV-2 (CD8+ T cells). These results indicate that env is a major viral determinant for HTLV T-cell transformation tropism in vitro and provides strong evidence implicating its contribution to the distinct pathogenesis resulting from HTLV-1 versus HTLV-2 infections.     

Retracing the evolutionary pathways of human immunodeficiency virus type 1 resistance to protease inhibitors: virus fitness in the absence and in the presence of drug

Human immunodeficiency virus type 1 (HIV-1) resistance to protease inhibitors (PI) is a major obstacle to the full success of combined antiretroviral therapy. High-level resistance to these compounds is the consequence of stepwise accumulation of amino acid substitutions in the HIV-1 protease (PR), following pathways that usually differ from one inhibitor to another. The selective advantage conferred by resistance mutations may depend upon several parameters: the impact of the mutation on virus infectivity in the presence or absence of drug, the nature of the drug, and its local concentration. Because drug concentrations in vivo are subject to extensive variation over time and display a markedly uneven tissue distribution, the parameters of selection for HIV-1 resistance to PI in treated patients are complex and poorly understood. In this study, we have reconstructed a large series of HIV-1 mutants that carry single or combined mutations in the PR, retracing the accumulation pathways observed in ritonavir-, indinavir-, and saquinavir-treated patients. We have then measured the phenotypic resistance and the drug-free infectivity of these mutant viruses. A deeper insight into the evolutionary value of HIV-1 PR mutants came from a novel assay system designed to measure the replicative advantage of mutant viruses as a function of drug concentration. By tracing the resultant fitness profiles, we determined the range of drug concentrations for which mutant viruses displayed a replicative advantage over the wild type and the extent of this advantage. Fitness profiles were fully consistent with the order of accumulation of resistance mutations observed in treated patients and further emphasise the key importance of local drug concentration in the patterns of selection of drug-resistant HIV-1 mutants.     

Impact of the enfuvirtide resistance mutation N43D and the associated baseline polymorphism E137K on peptide sensitivity and six-helix bundle structure

Enfuvirtide (ENF), the first human immunodeficiency virus type 1 (HIV-1) fusion inhibitor approved for clinical use, acts by binding to gp41 heptad repeat 1 (HR1) and preventing its interaction with the viral HR2 region. Treatment-emergent resistance to ENF has been mapped to residues within HR1, and these mutations decrease its susceptibility to ENF and may reduce viral fitness and pathogenesis, although the mechanism for these effects is not clear. N43D, a common ENF resistance mutation, was found in in vitro assays to cause a 5-50-fold in antiviral activity. We introduced this mutation into peptide models and determined the impact of this mutation by circular dichroism and X-ray crystallography. We find that the mutation results in a decrease in the thermal stability of the six-helix bundle and causes a significant change in the HR1-HR2 interface, including a loss of HR2 helicity. These data form a mechanistic basis for the decrease in ENF sensitivity and six-helix bundle stability. The E137K polymorphism, generally present at baseline in patients who develop N43D, partially compensates for the loss of stability, and we show that these residues likely form an ion pair. These data form a framework for understanding the impact of resistance mutations on viral fitness and pathogenesis and provide a pathway for the development of novel fusion inhibitor peptides.     

Wild-type-like viral replication potential of human immunodeficiency virus type 1 envelope mutants lacking palmitoylation signals

Palmitoylation of the cytoplasmic domain of the human immunodeficiency type virus type 1 (HIV-1) envelope (Env) transmembrane protein, gp41, has been implicated in Env targeting to detergent-resistant lipid rafts, Env incorporation into the virus, and viral infectivity. In contrast, we provide evidence here to show that HIV-1 infectivity, Env targeting to lipid rafts, and Env incorporation into the virus are independent of cytoplasmic tail palmitoylation. The T-cell (T)-tropic HXB2-based virus, which utilizes CXCR4 as the entry coreceptor, carrying a Cys-to-Ser mutation at residue 764 or 837 or at both replicated with wild-type (WT) virus replication kinetics in CD4+ T cells. The properties of Env expression, precursor processing, cell surface expression, and Env incorporation of these three mutant viruses were normal compared to those of the WT virus. These three mutant Env proteins all effectively mediated one-cycle virus infection. When the Cys residues were replaced by Ala residues, all single and double mutants still retained the phenotypes of infectivity, Env incorporation, and lipid raft localization of the WT Env. When Cys-to-Ala substitutions were introduced into the macrophage (M)-tropic ConB virus, which utilizes CCR5 as the coreceptor, these mutations did not affect the replication potential, Env phenotypes, lipid raft targeting, or Env assembly into the virus of the WT Env. These T- and M-tropic mutants also productively replicated in human primary CD4+ T cells. Moreover, mutations at both Cys residues significantly reduced the level of palmitoylation of the Env. Our results together support the notion that palmitoylation of the cytoplasmic tail of the HIV-1 Env is not essential for the HIV-1 virus life cycle.     

Amino acid 36 in the human immunodeficiency virus type 1 gp41 ectodomain controls fusogenic activity: implications for the molecular mechanism of viral escape from a fusion inhibitor

We have previously described a human immunodeficiency virus type 1 (HIV-1) proviral clone, pL2, derived from defective viral particles with higher fusogenicity than the prototypic NL4-3 virus. In this study, we attempted to determine the region that confers the enhanced fusion activity by creating envelope recombinants between pL2 and pNL4-3, as well as point mutants based on pNL4-3. The results indicate that amino acid 36 of gp41 is key for the fusogenic activity and infectivity enhancement and that glycine 36 (36G) of gp41 in pL2 is conserved in nearly all HIV-1 isolates except for pNL4-3. The mutation 36G-->D in a primary-isolate-derived Env decreased syncytium-forming activity and infectivity. The assays for cell-cell fusion and viral binding suggested that the enhanced fusion mediated by the 36D-->G mutation is not due to increased binding efficiency but is directly due to actual enhancement of viral fusion activity. Interestingly, this amino acid position is exactly equivalent to that at which the mutation of HIV-1 isolates that have escaped from a fusion inhibitor, enfuvirtide (T-20), has been frequently observed. The correlation between these previous findings and our findings was suggested by structural analysis. Our finding, therefore, has implications for a molecular basis of the viral escape from this drug.     

Mutations in the Leucine Zipper-Like Heptad Repeat Sequence of Human Immunodeficiency Virus Type 1 gp41 Dominantly Interfere with Wild-Type Virus Infectivity

It has been previously shown that a proline substitution for any of the conserved leucine or isoleucine residues located in the leucine zipper-like heptad repeat sequence of human immunodeficiency virus type 1 (HIV-1) gp41 renders viruses noninfectious and envelope (Env) protein unable to mediate membrane fusion (S. S.-L. Chen, C.-N. Lee, W.-R. Lee, K. McIntosh, and T.-M. Lee, J. Virol. 67:3615–3619, 1993; S. S.-L. Chen, J. Virol. 68:2002–2010, 1994). To understand whether these variants could act as trans-dominant inhibitory mutants, the ability of these mutants to inhibit wild-type (wt) virus infectivity was examined. Comparable amounts of cell- and virion-associated gag gene products as well as virion-associated gp41 were found in transfection with wt or mutant HIV-1 provirus. Viruses obtained from coexpression of wt provirus with mutant 566 or 580 provirus inhibited more potently the production of infectious virus than did viruses generated from cotransfection of wt provirus with other mutant proviruses. Nevertheless, all viruses produced from mixed transfection showed decreased infectivity compared with that of the wt virus when a multinuclear-activation β-galactosidase induction assay was performed. The ability of wt Env to induce cytopathic effects was inhibited by coexpression with mutant Env. Coexpression of mutants inhibited the ability of the wt protein to mediate virus-to-cell transmission, as demonstrated by an env trans-complementation assay with a defective HIV-1 proviral vector. These observations indicated that mutant Env, per se, interferes with wt Env function. Moreover, cotransfection of wt and mutant proviruses produced amounts of cell- and virion-associated gag gene products comparable to those produced by transfection of wt provirus. Similar amounts of gp41 were also found in virions generated from wt-mutant cotransfection as well as from wt transfection alone. These results indicated that the inhibitory effect conferred by mutants on the wt virus infectivity does not involve the late steps of Gag protein assembly and budding, but they suggest that the wt and mutant Env proteins form a dysfunctional hetero-oligomer which is impaired in an early step of the virus replication cycle. Our study demonstrates that mutations in the HIV-1 gp41 leucine zipper-like heptad repeat sequence dominantly inhibit infectious virus production.     

The genetic bottleneck in vertical transmission of subtype C HIV-1 is not driven by selection of especially neutralization-resistant virus from the maternal viral population

Subtype C human immunodeficiency virus type 1 (HIV-1C) continues to cause the majority of new cases of mother-to-child transmission (MTCT), and yet there are limited data on HIV-1C transmission. We amplified env from plasma RNA for 19 HIV-1C MTCT pairs, 10 transmitting in utero (IU) and 9 transmitting intrapartum (IP). There was a strong genetic bottleneck between all mother-infant pairs, with a majority of transmission events involving the transmission of a single virus. env genes of viruses transmitted to infants IP, but not IU, encoded Env proteins that were shorter and had fewer putative N-linked glycosylation sites in the V1-V5 region than matched maternal sequences. Viruses pseudotyped with env clones representative of each maternal and infant population were tested for neutralization sensitivity. The 50% inhibitory concentration of autologous serum was similar against both transmitted (infant) and nontransmitted (maternal) viruses in a paired analysis. Mother and infant Env proteins were also similar in sensitivity to soluble CD4, to a panel of monoclonal antibodies, and to heterologous HIV-1C sera. In addition, there was no difference in the breadth or potency of neutralizing antibodies between sera from 50 nontransmitting and 23 IU and 23 IP transmitting HIV-1C-infected women against four Env proteins from heterologous viruses. Thus, while a strong genetic bottleneck was detected during MCTC, with viruses of shorter and fewer glycosylation sites in env present in IP transmission, our data do not support this bottleneck being driven by selective resistance to antibodies.     

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.