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.     

Impact of the HIV-1 env genetic context outside HR1-HR2 on resistance to the fusion inhibitor enfuvirtide and viral infectivity in clinical isolates

Resistance mutations to the HIV-1 fusion inhibitor enfuvirtide emerge mainly within the drug's target region, HR1, and compensatory mutations have been described within HR2. The surrounding envelope (env) genetic context might also contribute to resistance, although to what extent and through which determinants remains elusive. To quantify the direct role of the env context in resistance to enfuvirtide and in viral infectivity, we compared enfuvirtide susceptibility and infectivity of recombinant viral pairs harboring the HR1-HR2 region or the full Env ectodomain of longitudinal env clones from 5 heavily treated patients failing enfuvirtide therapy. Prior to enfuvirtide treatment onset, no env carried known resistance mutations and full Env viruses were on average less susceptible than HR1-HR2 recombinants. All escape clones carried at least one of G36D, V38A, N42D and/or N43D/S in HR1, and accordingly, resistance increased 11- to 2800-fold relative to baseline. Resistance of full Env recombinant viruses was similar to resistance of their HR1-HR2 counterpart, indicating that HR1 and HR2 are the main contributors to resistance. Strictly X4 viruses were more resistant than strictly R5 viruses, while dual-tropic Envs featured similar resistance levels irrespective of the coreceptor expressed by the cell line used. Full Env recombinants from all patients gained infectivity under prolonged drug pressure; for HR1-HR2 viruses, infectivity remained steady for 3/5 patients, while for 2/5 patients, gains in infectivity paralleled those of the corresponding full Env recombinants, indicating that the env genetic context accounts mainly for infectivity adjustments. Phylogenetic analyses revealed that quasispecies selection is a step-wise process where selection of enfuvirtide resistance is a dominant factor early during therapy, while increased infectivity is the prominent driver under prolonged therapy.     

Impact of human immunodeficiency virus type 1 gp41 amino acid substitutions selected during enfuvirtide treatment on gp41 binding and antiviral potency of enfuvirtide in vitro

Enfuvirtide (ENF), a novel human immunodeficiency virus type 1 (HIV-1) fusion inhibitor, has potent antiviral activity against HIV-1 both in vitro and in vivo. Resistance to ENF observed after in vitro passaging was associated with changes in a three-amino-acid (aa) motif, GIV, at positions 36 to 38 of gp41. Patients with ongoing viral replication while receiving ENF during clinical trials acquired substitutions within gp41 aa 36 to 45 in the first heptad repeat (HR-1) of gp41 in both population-based plasma virus sequences and proviral DNA sequences from isolates showing reduced susceptibilities to ENF. To investigate their impact on ENF susceptibility, substitutions were introduced into a modified pNL4-3 strain by site-directed mutagenesis, and the susceptibilities of mutant viruses and patient-derived isolates to ENF were tested. In general, susceptibility decreases for single substitutions were lower than those for double substitutions, and the levels of ENF resistance seen for clinical isolates were higher than those observed for the site-directed mutant viruses. The mechanism of ENF resistance was explored for a subset of the substitutions by expressing them in the context of a maltose binding protein chimera containing a portion of the gp41 ectodomain and measuring their binding affinity to fluorescein-labeled ENF. Changes in binding affinity for the mutant gp41 fusion proteins correlated with the ENF susceptibilities of viruses containing the same substitutions. The combined results support the key role of gp41 aa 36 to 45 in the development of resistance to ENF and illustrate that additional envelope regions contribute to the ENF susceptibility of fusion inhibitor-na?ve viruses and resistance to ENF.     

Selection with a peptide fusion inhibitor corresponding to the first heptad repeat of HIV-1 gp41 identifies two genetic pathways conferring cross-resistance to peptide fusion inhibitors corresponding to the first and second heptad repeats (HR1 and HR2) of gp41

We generated four HIV-1 cultures that are resistant to a peptide fusion inhibitor corresponding to the first heptad repeat of gp41 in order to study mechanisms of resistance and gain insights into envelope glycoprotein-mediated membrane fusion. Two genetic pathways emerged that were defined by acquisition of a specific mutation in either the first or second heptad repeat region of gp41 (HR1 or the HR2, respectively). Each pathway was enriched in mutations that clustered in either HR2 and V3 or in HR1 and residues in or near CD4 contact sites. The gp41 mutations in both pathways not only accounted for resistance to the selecting HR1 peptide but also conferred cross-resistance to HR2 peptide fusion inhibitors and enhanced the stability of the six-helix bundle formed by the self-assembly of HR1 and HR2. The gp120 mutations alone enhanced fusion but did not appear to directly contribute to resistance. The implications of these findings for resistance mechanisms and regulation of envelope-mediated fusion are discussed.     

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.     

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.     

Role of the ectodomain of the gp41 transmembrane envelope protein of human immunodeficiency virus type 1 in late steps of the membrane fusion process

The membrane fusion process mediated by the gp41 transmembrane envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) was addressed by a flow cytometry assay detecting exchanges of fluorescent membrane probes (DiI and DiO) between cells expressing the HIV-1 envelope proteins (Env) and target cells. Double-fluorescent cells were detected when target cells expressed the type of chemokine receptor, CXCR4 or CCR5, matching the type of gp120 surface envelope protein, X4 or R5, respectively. Background levels of double-fluorescent cells were observed when the gp120-receptor interaction was blocked by AMD3100, a CXCR4 antagonist. The L568A mutation in the N-terminal heptad repeat (HR1) of gp41 resulted in parallel inhibition of the formation of syncytia and double-fluorescent cells, indicating that gp41 had a direct role in the exchange of fluorescent probes. In contrast, three mutations in the loop region of the gp41 ectodomain, located on either side of the Cys-(X)(5)-Cys motif (W596 M and W610A) or at the distal end of HR1 (D589L), had limited or no apparent effect on membrane lipid mixing between Env(+) and target cells, while they blocked formation of syncytia and markedly reduced the exchanges of cytoplasmic fluorescent probes. The loop region could therefore have a direct or indirect role in events occurring after the merging of membranes, such as the formation or dilation of fusion pores. Two types of inhibitors of HIV-1 entry, the gp41-derived peptide T20 and the betulinic acid derivative RPR103611, had limited effects on membrane exchanges at concentrations blocking or markedly reducing syncytium formation. This finding confirmed that T20 can inhibit the late steps of membrane fusion (post-lipid mixing) and brought forth an indirect argument for the role of the gp41 loop region in these steps, as mutations conferring resistance to RPR103611V were mapped in this region (I595S or L602H).     

Maturation-dependent human immunodeficiency virus type 1 particle fusion requires a carboxyl-terminal region of the gp41 cytoplasmic tail

Lentiviruses, including human immunodeficiency virus type 1 (HIV-1), typically encode fusion glycoproteins with long cytoplasmic tails (CTs). We previously reported that immature HIV-1 particles are inhibited for fusion with target cells by a mechanism requiring the 152-amino-acid CT of gp41. The gp41 CT was also shown to mediate the detergent-resistant association of the HIV-1 envelope glycoprotein complex with immature HIV-1 particles, indicating that the gp41 CT forms a stable complex with Gag in immature virions. In the present study, we analyzed the effects of progressive truncations and point mutations in the gp41 CT on the fusion of mature and immature HIV-1 particles with target cells. We also determined the effects of these mutations on the detergent-resistant association of gp41 with immature HIV-1 particles. Removal of the C-terminal 28 amino acids relieved the dependence of HIV-1 fusion on maturation. However, a mutant Env protein lacking this region remained associated with immature HIV-1 particles treated with nonionic detergent. Further mutational analysis of the C-terminal region of gp41 revealed two specific sequences required for maturation-dependent HIV-1 fusion. Collectively, our results demonstrate that the extreme C terminus of gp41 plays a key role in coupling HIV-1 fusion competence to virion maturation. They further indicate that the stable association of gp41 with Gag in immature virions is not sufficient for inhibition of immature HIV-1 particle fusion.     

Characterization of the functional properties of env genes from long-term survivors of human immunodeficiency virus type 1 infection.

A small number of persons infected with human immunodeficiency virus type 1 (HIV-1) remain clinically and immunologically healthy for more than a decade after infection. Recent reports suggest that these individuals may be infected with an attenuated strain of HIV-1; however, a common genetic basis for viral attenuation has not been found in all cases. In the present study, we examined the functional properties of the HIV-1 env genes from six long-term survivors. env clones were generated by PCR amplification of proviral env sequences, followed by cloning of the amplified regions into expression vectors. Eight to ten clones from each subject were screened by transient transfection for expression of the envelope precursor glycoprotein, gp160. Those clones expressing gp160 were then cotransfected with an HIV-1 luciferase reporter vector, pNL4-3Env(-)LUC(+) and evaluated for their ability to mediate infection of phytohemagglutinin-activated peripheral blood mononuclear cells in single-cycle infectivity assays. Clones expressing gp160 were identified for all six long-term survivors, indicating the presence of proviral env genes with intact open reading frames. For two subjects, D and DH, the encoded envelope glycoproteins yielded high levels of luciferase activity when pseudotyped onto HIV-1 virions and tested in single-cycle infectivity assays. In contrast, envelope glycoproteins cloned from four other long-term survivors were poorly processed and failed to mediate infection. Sequencing of the gp120/41 cleavage site and conserved gp41 cysteine residues of these clones did not reveal any obvious mutations to explain the functional defects. The functional activity of env clones from long-term survivors D and DH was comparable to that seen with several primary HIV-1 env genes cloned from individuals with disease progression and AIDS. These results suggest that the long-term survival of subjects D and DH is not associated with overt functional defects in env; however, functional abnormalities in env may contribute to maintaining a long-term asymptomatic state in the other four cases we studied.     

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.     

Impact of natural polymorphism within the gp41 cytoplasmic tail of human immunodeficiency virus type 1 on the intracellular distribution of envelope glycoproteins and viral assembly

The motifs involved in the various functions of the human immunodeficiency virus type 1 (HIV-1) gp41 cytoplasmic tail (CT), particularly those related to the intracellular trafficking and assembly of envelope glycoproteins (Env) onto core particles, have generally been assessed with a restricted panel of T-cell laboratory-adapted virus strains. Here, we investigated gp41 CT sequences derived from individuals infected with HIV-1 viruses of various subtypes. We identified four patients harboring HIV variants with a natural polymorphism in the membrane-proximal tyrosine-based signal Y(712)SPL or the Y(802)W(803) diaromatic motif, which are two major determinants of Env intracellular trafficking. Confocal microscopy showed that the intracellular distribution of Env with a mutation in the tyrosine or diaromatic motif differed from that of Env with no mutation in these motifs. Surprisingly, the gp41 CTs of the primary viruses also had differential effects on the intracellular distribution of Env, independently of mutations in the tyrosine or diaromatic motifs, suggesting the involvement of additional determinants. Furthermore, analyses of virus replication kinetics indicated that the effects of mutations in the tyrosine or diaromatic motifs on viral replication depended on the gp41 CT context. These effects were at least partly due to differences in the efficiency of Env incorporation into virions. Thus, polymorphisms in primary HIV-1 gp41 CTs at the quasispecies or subtype level can influence the intracellular distribution of Env, its incorporation into virions, and viral replication capacity.     

Variable Constraints on the Principal Immunodominant Domain of the Transmembrane Glycoprotein of Human Immunodeficiency Virus Type 1

Lentiviruses have in their transmembrane glycoprotein (TM) a highly immunogenic structure referred to as the principal immunodominant domain (PID). The PID forms a loop of 5 to 7 amino acids between two conserved cysteines. Previous studies showed that envelope (Env) glycoprotein functions of feline immunodeficiency virus (FIV) could be retained after extensive mutation of the PID loop sequence, in spite of its high conservation. In order to compare Env function in different lentiviruses, either random mutations were introduced in the PID loop sequence of human immunodeficiency virus type 1 (HIV-1) or the entire HIV-1 PID loop was replaced by the corresponding PID loop of FIV or simian immunodeficiency virus (SIV). In the macrophage-tropic HIV-1 ADA Env, mutations impaired the processing of the gp160 Env precursor, thereby abolishing viral infectivity. However, 6 of the 108 random Env mutants that were screened retained the capacity to induce cell membrane fusion. The SIV and FIV sequences and five random mutations were then introduced in the context of T-cell-line-adapted HIV-1 LAI which, although phenotypically distant from HIV-1 ADA, has an identical PID loop sequence. In contrast to the situation for HIV-1 ADA mutants, the cleavage of the Env precursor was unaffected in most HIV-1 LAI mutants. Such mutations, however, resulted in increased shedding of the gp120 surface glycoprotein (SU) from the gp41 TM. The HIV-1 LAI Env mutants showed high fusogenic efficiency. Three Env mutants retained the capacity to mediate virus entry in target cells, although less efficiently than the wild-type Env, and allowed the reconstitution of infectious molecular clones. These results indicated that in HIV-1, like FIV, the conserved PID sequence can be changed without impairing Env function. However, functional constraints on the PID of HIV-1 vary depending on the structural context of Env, presumably in relation to the role of the PID in the interaction of the SU and TM subunits and the stability of the Env complex.     

Regulation of human immunodeficiency virus type 1 Env-mediated membrane fusion by viral protease activity

We and others have presented evidence for a direct interaction between the matrix (MA) domain of the human immunodeficiency virus type 1 (HIV-1) Gag protein and the cytoplasmic tail of the transmembrane envelope (Env) glycoprotein gp41. In addition, it has been postulated that the MA domain of Gag undergoes a conformational change following Gag processing, and the cytoplasmic tail of gp41 has been shown to modulate Env-mediated membrane fusion activity. Together, these results raise the possibility that the interaction between the gp41 cytoplasmic tail and MA is regulated by protease (PR)-mediated Gag processing, perhaps affecting Env function. To examine whether Gag processing affects Env-mediated fusion, we compared the ability of wild-type (WT) HIV-1 Env and a mutant lacking the gp41 cytoplasmic tail to induce fusion in the context of an active (PR(+)) or inactive (PR(-)) viral PR. We observed that PR(-) virions bearing WT Env displayed defects in cell-cell fusion. Impaired fusion did not appear to be due to differences in the levels of virion-associated Env, in CD4-dependent binding to target cells, or in the formation of the CD4-induced gp41 six-helix bundle. Interestingly, truncation of the gp41 cytoplasmic tail reversed the fusion defect. These results suggest that interactions between unprocessed Gag and the gp41 cytoplasmic tail suppress fusion.     

Human immunodeficiency virus type 1 envelope glycoprotein oligomerization requires the gp41 amphipathic alpha-helical/leucine zipper-like sequence.

Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) oligomerization was investigated by coexpressing wild-type and truncated envelope glycoproteins to determine the minimum sequence required for mutant-wild-type hetero-oligomerization. The gp41 putative amphipathic alpha-helix, Leu-550 to Leu-582, was essential for hetero-oligomer formation. Alanine substitution of 9 of the 10 residues composing the gp41 amphipathic alpha-helix 4-3 hydrophobic repeat sequence was required to inhibit mutant-wild-type hetero-oligomerization and to render the envelope glycoprotein precursor, gp160, monomeric. This indicates that multiple hydrophobic contacts contribute to the stable envelope glycoprotein oligomeric structure. Single alanine substitutions within the hydrophobic repeat sequence did not affect gp160 oligomeric structure but abolished syncytium-forming function. Some mutations also diminished gp160 processing efficiency and the association between gp120 and gp41 in a position-dependent manner. These results indicate that the gp41 amphipathic alpha-helix 4-3 hydrophobic repeat sequence plays a central role in HIV-1 envelope glycoprotein oligomerization and fusion function.     

Disruption of Helix-Capping Residues 671 and 674 Reveals a Role in HIV-1 Entry for a Specialized Hinge Segment of the Membrane Proximal External Region of gp41

HIV-1 (human immunodeficiency virus type 1) uses its trimeric gp160 envelope (Env) protein consisting of non-covalently associated gp120 and gp41 subunits to mediate entry into human T lymphocytes. A facile virus fusion mechanism compensates for the sparse Env copy number observed on viral particles and includes a 22-amino-acid, lentivirus-specific adaptation at the gp41 base (amino acid residues 662–683), termed the membrane proximal external region (MPER). We show by NMR and EPR that the MPER consists of a structurally conserved pair of viral lipid-immersed helices separated by a hinge with tandem joints that can be locked by capping residues between helices. This design fosters efficient HIV-1 fusion via interconverting structures while, at the same time, affording immune escape. Disruption of both joints by double alanine mutations at Env positions 671 and 674 (AA) results in attenuation of Env-mediated cell–cell fusion and hemifusion, as well as viral infectivity mediated by both CD4-dependent and CD4-independent viruses. The potential mechanism of disruption was revealed by structural analysis of MPER conformational changes induced by AA mutation. A deeper acyl chain-buried MPER middle section and the elimination of cross-hinge rigid-body motion almost certainly impede requisite structural rearrangements during the fusion process, explaining the absence of MPER AA variants among all known naturally occurring HIV-1 viral sequences. Furthermore, those broadly neutralization antibodies directed against the HIV-1 MPER exploit the tandem joint architecture involving helix capping, thereby disrupting hinge function.     

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.     

Antibody neutralization escape mediated by point mutations in the intracytoplasmic tail of human immunodeficiency virus type 1 gp41

The persistence of human immunodeficiency virus type 1 (HIV-1) infection in the presence of robust host immunity has been associated in part with variation in viral envelope proteins leading to antigenic variation and escape from neutralizing antibodies. Previous studies of natural neutralization escape mutants have predominantly focused on gp120 and gp41 ectodomain sequence variations that alter antibody binding via changes in conformation or glycosylation pattern of the Env, likely due to the immune pressure exerted on the exposed ectodomain component of the glycoprotein. Here, we show for the first time a novel mechanism by which point mutations in the intracytoplasmic tail of the transmembrane component (gp41) of envelope can render the virus resistant to neutralization by monoclonal antibodies and broadly neutralizing polyclonal serum antibodies. Point mutations in a highly conserved structural motif within the intracytoplasmic tail resulted in decreased binding of neutralizing antibodies to the Env ectodomain, evidently due to allosteric changes both in the gp41 ectodomain and in gp120. While receptor binding and infectivity of the mutant virus remained unaltered, the changes in Env antigenicity were associated with an increase in neutralization resistance of the mutant virus. These studies demonstrate the structurally integrated nature of gp120 and gp41 and underscore a previously unrecognized potentially critical role for even minor sequence variation of the intracytoplasmic tail in modulating the antigenicity of the ectodomain of HIV-1 envelope glycoprotein complex.     

人类免疫缺陷病毒1型gp41结构与功能的研究进展

人类免疫缺陷病毒1型(HIV-1)通过其包膜糖蛋白(Env)介导侵入靶细胞.Env由受体特异性结合单位gp120和膜融合单位gp41组成.HIV-1的gp41分为3个功能区:膜外区、跨膜区和膜内区.膜外区是病毒感染时膜融合的主要结构基础;跨膜区通过疏水残基使Env锚定在脂质膜上;膜内区则表现多重功能,参与病毒的感染、复...     

Genetic evidence for an interaction between human immunodeficiency virus type 1 matrix and alpha-helix 2 of the gp41 cytoplasmic tail

The incorporation of envelope (Env) glycoproteins into virions is an essential step in the retroviral replication cycle. Lentiviruses, including human immunodeficiency virus type 1 (HIV-1), encode Env glycoproteins with unusually long cytoplasmic tails, the functions of which have not been fully elucidated. In this study, we examine the effects on virus replication of a number of mutations in a helical motif (alpha-helix 2) located near the center of the HIV-1 gp41 cytoplasmic tail. We find that, in T-cell lines, small deletions in this domain disrupt the incorporation of Env glycoproteins into virions and markedly impair virus infectivity. Through the analysis of viral revertants, we demonstrate that a single amino acid change (34VI) in the matrix domain of Gag reverses the Env incorporation and infectivity defect imposed by a small deletion near the C terminus of alpha-helix 2. These results provide genetic evidence, in the context of infected T cells, for an interaction between HIV-1 matrix and the gp41 cytoplasmic tail and identify domains of both proteins involved in this putative interaction.     

Coreceptor tropism can be influenced by amino acid substitutions in the gp41 transmembrane subunit of human immunodeficiency virus type 1 envelope protein

Many studies have demonstrated that the third variable region (V3) of the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) is a major determinant of coreceptor tropism. Other regions in the surface gp120 subunit of Env can modulate coreceptor tropism in a manner that is not fully understood. In this study, we evaluated the effect of env determinants outside of V3 on coreceptor usage through the analysis of (i) patient-derived env clones that differ in coreceptor tropism, (ii) chimeric env sequences, and (iii) site-directed mutants. The introduction of distinct V3 sequences from CXCR4-using clones into an R5-tropic env backbone conferred the inefficient use of CXCR4 in some but not all cases. Conversely, in many cases, X4- and dual-tropic env backbones containing the V3 sequences of R5-tropic clones retained the ability to use CXCR4, suggesting that sequences outside of the V3 regions of these CXCR4-using clones were responsible for CXCR4 use. The determinants of CXCR4 use in a set of dual-tropic env sequences with V3 sequences identical to those of R5-tropic clones mapped to the gp41 transmembrane (TM) subunit. In one case, a single-amino-acid substitution in the fusion peptide of TM was able to confer CXCR4 use; however, TM substitutions associated with CXCR4 use varied among different env sequences. These results demonstrate that sequences in TM can modulate coreceptor specificity and that env sequences other than that of V3 may facilitate efficient CXCR4-mediated entry. We hypothesize that the latter plays an important role in the transition from CCR5 to CXCR4 coreceptor use.