Human immunodeficiency virus strains differ in their ability to infect CD4+ cells expressing the rat homolog of CXCR-4 (fusin).

A clade B strain of human immunodeficiency virus type 1 (HIV-1(LAI)) could infect CD4+ cells expressing human CXCR-4 (fusin) or its rat homolog with similar efficacy. By contrast, cells expressing rat CXCR-4 were not permissive to HIV-1(NDK) (clade D), HIV-2(ROD), or HIV-1(LAI) with chimeric envelope protein gp120 bearing the V3 domain from HIV-1(NDK). The reciprocal chimeric gp120 (HIV-1(NDK) with V3 from HIV-1(LAI)) could mediate infection of cells expressing either human or rat CXCR-4. Genetically divergent HIV strains have different requirements for interaction with the CXCR-4 coreceptor, and the gp120 V3 domain seems to be involved in this interaction.     

Sensitivity to a nonpeptidic compound (RPR103611) blocking human immunodeficiency virus type 1 Env-mediated fusion depends on sequence and accessibility of the gp41 loop region

The triterpene RPR103611 is an efficient inhibitor of membrane fusion mediated by the envelope proteins (Env, gp120-gp41) of CXCR4-dependent (X4) human immunodeficiency virus type 1 (HIV-1) strains, such as HIV-1(LAI) (LAI). Other X4 strains, such as HIV-1(NDK) (NDK), and CCR5-dependent (R5) HIV-1 strains, such as HIV-1(ADA) (ADA), were totally resistant to RPR103611. Analysis of chimeric LAI-NDK Env proteins identified a fragment of the NDK gp41 ectodomain determining drug resistance. A single difference at position 91, leucine in LAI and histidine in NDK, apparently accounted for their sensitivity or resistance to RPR103611. We had previously identified a mutation of isoleucine 84 to serine in a drug escape LAI variant. Both I84 and L91 are located in the "loop region" of gp41 separating the proximal and distal helix domains. Nonpolar residues in this region therefore appear to be important for the antiviral activity of RPR103611 and are possibly part of its target. However, another mechanism had to be envisaged to explain the drug resistance of ADA, since its gp41 loop region was almost identical to that of LAI. Fusion mediated by chimeric Env consisting of LAI gp120 and ADA gp41, or the reciprocal construct, was fully blocked by RPR103611. The gp120-gp41 complex of R5 strains is stable, relative to that of X4 strains, and this stability could play a role in their drug resistance. Indeed, when the postbinding steps of ADA infection were performed under mildly acidic conditions (pH 6.5 or 6.0), a treatment expected to favor dissociation of gp120, we achieved almost complete neutralization by RPR103611. The drug resistance of NDK was partially overcome by preincubating virus with soluble CD4, a gp120 ligand inducing conformational changes in the Env complex. The antiviral efficacy of RPR103611 therefore depends on the sequence of the gp41 loop and the stability of the gp120-gp41 complex, which could limit the accessibility of this target.     

Analysis of the Critical Domain in the V3 Loop of Human Immunodeficiency Virus Type 1 gp120 Involved in CCR5 Utilization

Human immunodeficiency virus type 1 (HIV-1) infection of CD4(+) lymphocytes and macrophages involves interaction of the surface subunit of the envelope protein (gp120) with coreceptors. Isolates have been found with specific tropism for macrophages and/or T-cell lines, through the utilization of chemokine receptor CCR5 (R5) or CXCR4 (X4). The third hypervariable loop (V3 loop) of gp120 is the major determinant of tropism. Using chimeric envelopes between HXB2 (X4) and ADA (R5), we found that the C-terminal half of the V3 loop was sufficient to confer on HXB2 the ability to infect CCR5-expressing cells. A sequence motif was identified at positions 289 to 292 allowing 30% of wild-type levels of infection, whereas full activity was achieved with the conversion of Lys to Glu at position 287 in addition to the above motif. Moreover, V3 loops from either SF2 (X4R5) or SF162 (R5) also allowed infection of CCR5-expressing cells, supporting the importance of V3 loops in influencing CCR5 utilization. The effects of amino acid changes at position 287 on the level of infection via CCR5 showed that negatively charged residues (Glu and Asp) were optimal for efficient interaction whereas only bulky hydrophobic residues drastically reduced infection. In addition, sequences at the N terminus of the V3 loop independently modulated the level of infection via CCR5. This study also examined the susceptibility of chimeric envelopes to neutralization by anticoreceptor antibodies and suggested the presence of differential interaction between the chimeric envelopes and CCR5. These findings highlight the critical residues in the V3 loop that mediate HIV-1 infection.     

Evidence for Common Structural Determinants of Human Immunodeficiency Virus Type 1 Coreceptor Activity Provided through Functional Analysis of CCR5/CXCR4 Chimeric Coreceptors

Human immunodeficiency virus type 1 (HIV-1) infection in vivo is dependent upon the interaction of the viral envelope glycoprotein gp120 with CC chemokine receptor 5 (CCR5) or CXC chemokine receptor 4 (CXCR4). To study the determinants of the gp120-coreceptor association, we generated a set of chimeric HIV-1 coreceptors which express all possible combinations of the four extracellular domains of CCR5 and CXCR4. Stable U87 astroglioma cell lines expressing CD4 and individual chimeric coreceptor proteins were tested against a variety of R5, X4, and R5X4 envelope glycoproteins and virus strains for their ability to support HIV-1-mediated cell fusion and infection, respectively. Each of the cell lines promoted fusion with cells expressing an HIV envelope glycoprotein, except for U87.CD4.5455, which presents the first extracellular loop (ECL1) and flanking sequences of CXCR4 in the context of CCR5. However, all of the chimeric coreceptors allowed productive infection by one or more of the viral strains tested. Viral phenotype was a predictive factor for the observed activity of the chimeric molecules; X4 and R5X4 HIV strains utilized a majority of the chimeras, while R5 strains were limited in their ability to infect cells expressing these chimeric molecules. The expression of CCR5 ECL2 within the CXCR4 backbone supported infection by an R5 primary isolate, but no chimeras bearing the N terminus of CCR5 exhibited activity with R5 strains. Remarkably, the introduction of any CXCR4 domain into the CCR5 backbone was sufficient to allow utilization by multiple X4 strains. However, critical determinants within ECL2 and/or ECL3 of CXCR4 were apparent for all X4 viruses upon replacement of these domains in CXCR4 with CCR5 sequences. Unexpectedly, chimeric coreceptor-facilitated entry was blocked in all cases by the presence of the CXCR4-specific inhibitor AMD3100. Our data provide proof that CCR5 contains elements that support usage by X4 viral strains and demonstrate that the gp120 interaction sites of CCR5 and CXCR4 are structurally related.     

Identification of gp120 binding sites on CXCR4 by using CD4-independent human immunodeficiency virus type 2 Env proteins

Human immunodeficiency virus (HIV) and simian (SIV) immunodeficiency virus entry is mediated by binding of the viral envelope glycoprotein (Env) to CD4 and chemokine receptors, CCR5 and/or CXCR4. CD4 induces extensive conformational changes that expose and/or induce formation of a chemokine receptor binding site on gp120. CD4-independent Env's of HIV type 1 (HIV-1), HIV-2, and SIV have been identified that exhibit exposed chemokine receptor binding sites and can bind directly to CCR5 or CXCR4 in the absence of CD4. While many studies have examined determinants for gp120-CCR5 binding, analysis of gp120-CXCR4 binding has been hindered by the apparently lower affinity of this interaction for X4-tropic HIV-1 isolates. We show here that gp120 proteins from two CD4-independent HIV-2 Env's, VCP and ROD/B, bind directly to CXCR4 with an apparently high affinity. By use of CXCR4 N-terminal deletion constructs, CXCR4-CXCR2 chimeras, and human-rat CXCR4 chimeras, binding determinants were shown to reside in the amino (N) terminus, extracellular loop 2 (ECL2), and ECL3. Alanine-scanning mutagenesis of charged residues, tyrosines, and phenylalanines in extracellular CXCR4 domains implicated multiple amino acids in the N terminus (E14/E15, D20, Y21, and D22), ECL2 (D187, R188, F189, Y190, and D193), and ECL3 (D262, E268, E277, and E282) in binding, although minor differences were noted between VCP and ROD/B. However, mutations in CXCR4 that markedly reduced binding did not necessarily hinder cell-cell fusion by VCP or ROD/B, especially in the presence of CD4. These gp120 proteins will be useful in dissecting determinants for CXCR4 binding and Env triggering and in evaluating pharmacologic inhibitors of the gp120-CXCR4 interaction.     

Spontaneous Mutations in the env Gene of the Human Immunodeficiency Virus Type 1 NDK Isolate Are Associated with a CD4-Independent Entry Phenotype

Human immunodeficiency virus type 1 (HIV-1) entry into target cells is a multistep process initiated by envelope protein gp120 binding to cell surface CD4. The conformational changes induced by this interaction likely favor a second-step interaction between gp120 and a coreceptor such as CXCR4 or CCR5. Here, we report a spontaneous and stable CD4-independent entry phenotype for the HIV-1 NDK isolate. This mutant strain, which emerged from a population of chronically infected CD4-positive CEM cells, can replicate in CD4-negative human cell lines. The presence of CXCR4 alone renders cells susceptible to infection by the mutant NDK, and infection can be blocked by the CXCR4 natural ligand SDF-1. Furthermore, we have correlated the CD4-independent phenotype with seven mutations in the C2 and C3 regions and the V3 loop. We propose that the mutant gp120 spontaneously acquires a conformation allowing it to interact directly with CXCR4. This virus provides us with a powerful tool to study directly gp120-CXCR4 interactions.     

Molecular Recognition of CXCR4 by a Dual Tropic HIV-1 gp120 V3 Loop

HIV-1 cell entry is initiated by the interaction of the viral envelope glycoprotein gp120 with CD4, and chemokine coreceptors CXCR4 and CCR5. The molecular recognition of CXCR4 or CCR5 by the HIV-1 gp120 is mediated through the V3 loop, a fragment of gp120. The binding of the V3 loop to CXCR4 or CCR5 determines the cell tropism of HIV-1 and constitutes a key step before HIV-1 cell entry. Thus, elucidating the molecular recognition of CXCR4 by the V3 loop is important for understanding HIV-1 viral infectivity and tropism, and for the design of HIV-1 inhibitors. We employed a comprehensive set of computational tools, predominantly based on free energy calculations and molecular-dynamics simulations, to investigate the molecular recognition of CXCR4 by a dual tropic V3 loop. We report what is, to our knowledge, the first HIV-1 gp120 V3 loop:CXCR4 complex structure. The computationally derived structure reveals an abundance of polar and nonpolar intermolecular interactions contributing to the HIV-1 gp120:CXCR4 binding. Our results are in remarkable agreement with previous experimental findings. Therefore, this work sheds light on the functional role of HIV-1 gp120 V3 loop and CXCR4 residues associated with HIV-1 coreceptor activity.     

Molecular Recognition of CXCR4 by a Dual Tropic HIV-1 gp120 V3 Loop

HIV-1 cell entry is initiated by the interaction of the viral envelope glycoprotein gp120 with CD4, and chemokine coreceptors CXCR4 and CCR5. The molecular recognition of CXCR4 or CCR5 by the HIV-1 gp120 is mediated through the V3 loop, a fragment of gp120. The binding of the V3 loop to CXCR4 or CCR5 determines the cell tropism of HIV-1 and constitutes a key step before HIV-1 cell entry. Thus, elucidating the molecular recognition of CXCR4 by the V3 loop is important for understanding HIV-1 viral infectivity and tropism, and for the design of HIV-1 inhibitors. We employed a comprehensive set of computational tools, predominantly based on free energy calculations and molecular-dynamics simulations, to investigate the molecular recognition of CXCR4 by a dual tropic V3 loop. We report what is, to our knowledge, the first HIV-1 gp120 V3 loop:CXCR4 complex structure. The computationally derived structure reveals an abundance of polar and nonpolar intermolecular interactions contributing to the HIV-1 gp120:CXCR4 binding. Our results are in remarkable agreement with previous experimental findings. Therefore, this work sheds light on the functional role of HIV-1 gp120 V3 loop and CXCR4 residues associated with HIV-1 coreceptor activity.     

Effect of mutations in the second extracellular loop of CXCR4 on its utilization by human and feline immunodeficiency viruses

CCR5 and CXCR4 are the principal CD4-associated coreceptors used by human immunodeficiency virus type 1 (HIV-1). CXCR4 is also a receptor for the feline immunodeficiency virus (FIV). The rat CXCR4 cannot mediate infection by HIV-1NDK or by FIVPET (both cell line-adapted strains) because of sequence differences with human CXCR4 in the second extracellular loop (ECL2). Here we made similar observations for HIV-189.6 (a strain also using CCR5) and for a primary HIV-1 isolate. It showed the role of ECL2 in the coreceptor activity of CXCR4 for different types of HIV-1 strains. By exchanging ECL2 residues between human and rat CXCR4, we found that several amino acid differences contributed to the inactivity of the rat CXCR4 toward HIV-189.6. In contrast, its inactivity toward HIV-1NDK seemed principally due to a serine at position 193 instead of to an aspartic acid (Asp193) in human CXCR4. Likewise, a mutation of Asp187 prevented usage of CXCR4 by FIVPET. Different mutations of Asp193, including its replacement by a glutamic acid, markedly reduced or suppressed the activity of CXCR4 for HIV-1NDK infection, indicating that the negative charge was not the only requirement. Mutations of Asp193 and of arginine residues (Arg183 and Arg188) of CXCR4 reduced the efficiency of HIV-1 infection for all HIV-1 strains tested. Other ECL2 mutations tested had strain-specific effects or no apparent effect on HIV-1 infection. The ECL2 mutants allowed us to identify residues contributing to the epitope of the 12G5 monoclonal antibody. Overall, residues with different charges and interspersed in ECL2 seem to participate in the coreceptor activity of CXCR4. This suggests that a conformational rather than linear epitope of ECL2 contributes to the HIV-1 binding site. However, certain HIV-1 and FIV strains seem to require the presence of a particular ECL2 residue.     

Importance of V3 Loop Flexibility and Net Charge in the Context of Co-Receptor Recognition. A Molecular Dynamics Study on HIV gp120

The entry of HIV-1 into a host cell requires the interaction of envelope glycoprotein gp120 with CD4 receptor as well as a co-receptor, which can be either CCR5 or CXCR4. The third variable loop (V3) of HIV-1 gp120 plays an important role in co-receptor selection (CCR5 or CXCR4) and also acts as an epitope for neutralizing antibodies against gp120. Here we have performed long time molecular dynamics simulations of two gp120 structures that are representatives of a R5 and X4 strains in the CD4-free and CD4-bound states. The results indicate some conserved features in both systems, such as the rigidity of the gp120 core, the conservation of the CD4 Phe43-gp120 binding cavity contacts, a high flexibility of the V3 loop particularly in the CD4 bound form. Analysis of the distribution of V3 loop's net charge shows it to be more positive for the gp120 sequences selecting CXCR4 co-receptor, letting us to propose that V3 loop net charge and flexibility are the two main elements in the co-receptor selection.     

Molecular Recognition of CCR5 by an HIV-1 gp120 V3 Loop

The binding of protein HIV-1 gp120 to coreceptors CCR5 or CXCR4 is a key step of the HIV-1 entry to the host cell, and is predominantly mediated through the V3 loop fragment of HIV-1 gp120. In the present work, we delineate the molecular recognition of chemokine receptor CCR5 by a dual tropic HIV-1 gp120 V3 loop, using a comprehensive set of computational tools predominantly based on molecular dynamics simulations and free energy calculations. We report, what is to our knowledge, the first complete HIV-1 gp120 V3 loop : CCR5 complex structure, which includes the whole V3 loop and the N-terminus of CCR5, and exhibits exceptional agreement with previous experimental findings. The computationally derived structure sheds light into the functional role of HIV-1 gp120 V3 loop and CCR5 residues associated with the HIV-1 coreceptor activity, and provides insights into the HIV-1 coreceptor selectivity and the blocking mechanism of HIV-1 gp120 by maraviroc. By comparing the binding of the specific dual tropic HIV-1 gp120 V3 loop with CCR5 and CXCR4, we observe that the HIV-1 gp120 V3 loop residues 13–21, which include the tip, share nearly identical structural and energetic properties in complex with both coreceptors. This result paves the way for the design of dual CCR5/CXCR4 targeted peptides as novel potential anti-AIDS therapeutics.     

Identification of residues of CXCR4 critical for human immunodeficiency virus coreceptor and chemokine receptor activities

CXCR4 is a G-coupled receptor for the stromal cell-derived factor (SDF-1) chemokine, and a CD4-associated human immunodeficiency virus type 1 (HIV-1) coreceptor. These functions were studied in a panel of CXCR4 mutants bearing deletions in the NH(2)-terminal extracellular domain (NT) or substitutions in the NT, the extracellular loops (ECL), or the transmembrane domains (TMs). The coreceptor activity of CXCR4 was markedly impaired by mutations of two Tyr residues in NT (Y7A/Y12A) or at a single Asp residue in ECL2 (D193A), ECL3 (D262A), or TMII (D97N). These acidic residues could engage electrostatical interactions with basic residues of the HIV-1 envelope protein gp120, known to contribute to the selectivity for CXCR4. The ability of CXCR4 mutants to bind SDF-1 and mediate cell signal was consistent with the two-site model of chemokine-receptor interaction. Site I involved in SDF-1 binding but not signaling was located in NT with particular importance of Glu(14) and/or Glu(15) and Tyr(21). Residues required for both SDF-1 binding and signaling, and thus probably part of site II, were identified in ECL2 (Asp(187)), TMII (Asp(97)), and TMVII (Glu(288)). The first residues () of NT also seem required for SDF-1 binding and signaling. A deletion in the third intracellular loop abolished signaling, probably by disrupting the coupling with G proteins. The identification of CXCR4 residues involved in the interaction with both SDF-1 and HIV-1 may account for the signaling activity of gp120 and has implications for the development of antiviral compounds.     

Structural and functional characterization of human CXCR4 as a chemokine receptor and HIV-1 co-receptor by mutagenesis and molecular modeling studies

The human CXC chemokine receptor 4 (CXCR4) is a receptor for the chemokine stromal cell-derived factor (SDF-1alpha) and a co-receptor for the entry of specific strains of human immunodeficiency virus type I (HIV-1). CXCR4 is also recognized by an antagonistic chemokine, the viral macrophage inflammatory protein II (vMIP-II) encoded by human herpesvirus type VIII. SDF-1alpha or vMIP-II binding to CXCR4 can inhibit HIV-1 entry via this co-receptor. An approach combining protein structural modeling and site-directed mutagenesis was used to probe the structure-function relationship of CXCR4, and interactions with its ligands SDF-1alpha and vMIP-II and HIV-1 envelope protein gp120. Hypothetical three-dimensional structures were proposed by molecular modeling studies of the CXCR4.SDF-1alpha complex, which rationalize extensive biological information on the role of CXCR4 in its interactions with HIV-1 envelope protein gp120. With site-directed mutagenesis, we have identified that the amino acid residues Asp (D20A) and Tyr (Y21A) in the N-terminal domain and the residue Glu (E268A) in extracellular loop 3 (ECL3) are involved in ligand binding, whereas the mutation Y190A in extracellular loop 2 (ECL2) impairs the signaling mediated by SDF-1alpha. As an HIV-1 co-receptor, we found that the N-terminal domain, ECL2, and ECL3 of CXCR4 are involved in HIV-1 entry. These structural and mutational studies provide valuable information regarding the structural basis for CXCR4 activity in chemokine binding and HIV-1 viral entry, and could guide the design of novel targeted inhibitors.     

Ligand binding characteristics of CXCR4 incorporated into paramagnetic proteoliposomes

The G protein-coupled receptor CXCR4 is a coreceptor, along with CD4, for the human immunodeficiency virus type 1 (HIV-1) and has been implicated in breast cancer metastasis. We studied the binding of the HIV-1 gp120 envelope glycoprotein (gp) to CXCR4 but found that the gp120s from CXCR4-using HIV-1 strains bound nonspecifically to several cell lines lacking human CXCR4 expression. Therefore, we constructed paramagnetic proteoliposomes (CXCR4-PMPLs) containing pure, native CXCR4. CXCR4-PMPLs specifically bound the natural ligand, SDF-1alpha, and the gp120s from CXCR4-using HIV-1 strains. Conformation-dependent anti-CXCR4 antibodies and the CXCR4 antagonist AMD3100 blocked HIV-1 gp120 binding to CXCR4-PMPLs. The gp120-CXCR4 interaction was blocked by anti-gp120 antibodies directed against the third variable (V3) loop and CD4-induced epitopes, structures that have also been implicated in the binding of gp120 to the other HIV-1 coreceptor, CCR5. Compared with the binding of R5 HIV-1 gp120s to CCR5, the gp120-CXCR4 interaction exhibited a lower affinity (K(d) = 200 nm) and was dependent upon prior CD4 binding, even at low temperature. Thus, although similar regions of X4 and R5 HIV-1 gp120s appear to be involved in binding CXCR4 and CCR5, respectively, differences exist in nonspecific binding to cell surfaces, affinity for the chemokine receptor, and CD4 dependence at low temperature.     

The V1/V2 loop of HIV-1 gp120 is necessary for Tat binding and consequent modulation of virus entry

Preventing cell entry of human immunodeficiency virus 1 (HIV-1) is of interest for the development of innovative therapies. We previously reported a specific interaction between HIV-1 envelope glycoprotein 120 (gp120) and Tat at the cell surface, which enhances virus attachment and entry. We also identified a gp120-mimicking peptide, CT319, that competes with gp120 for Tat binding, thus inhibiting HIV-1 infection. Here we report a molecular dissection of gp120 regions involved in this mechanism. Our findings identify the V1/V2 loop of gp120 as involved in Tat binding, and define this interaction as functionally relevant for HIV-1 entry into host cells.     

Selective interactions of polyanions with basic surfaces on human immunodeficiency virus type 1 gp120

It is well established that the gp120 V3 loop of T-cell-line-adapted human immunodeficiency virus type 1 (HIV-1) binds both cell-associated and soluble polyanions. Virus infectivity is increased by interactions between HIV-1 and heparan sulfate proteoglycans on some cell types, and soluble polyanions such as heparin and dextran sulfate neutralize HIV-1 in vitro. However, the analysis of gp120-polyanion interactions has been limited to T-cell-line-adapted, CXCR4-using virus and virus-derived gp120, and the polyanion binding ability of gp120 regions other than the V3 loop has not been addressed. Here we demonstrate by monoclonal-antibody inhibition, labeled heparin binding, and surface plasmon resonance studies that a second site, most probably corresponding to the newly defined, highly conserved coreceptor binding region on gp120, forms part of the polyanion binding surface. Consistent with the binding of polyanions to the coreceptor binding surface, dextran sulfate interfered with the gp120-CXCR4 association while having no detectable effect on the gp120-CD4 interaction. The interaction between polyanions and X4 or R5X4 gp120 was readily detectable, whereas weak or undetectable binding was observed with R5 gp120. Analysis of mutated forms of X4 gp120 demonstrated that the V3 loop is the major determinant for polyanion binding whereas other regions, including the V1/V2 loop structure and the NH(2) and COOH termini, exert a more subtle influence. A molecular model of the electrostatic potential of the conserved coreceptor binding region confirmed that it is basic but that the overall charge on this surface is dominated by the V3 loop. These results demonstrate a selective interaction of gp120 with polyanions and suggest that the conserved coreceptor binding surface may present a novel and conserved target for therapeutic intervention.     

Accurate and efficient gp120 V3 loop structure based models for the determination of HIV-1 co-receptor usage

Background  

HIV-1 targets human cells expressing both the CD4 receptor, which binds the viral envelope glycoprotein gp120, as well as either the CCR5 (R5) or CXCR4 (X4) co-receptors, which interact primarily with the third hypervariable loop (V3 loop) of gp120. Determination of HIV-1 affinity for either the R5 or X4 co-receptor on host cells facilitates the inclusion of co-receptor antagonists as a part of patient treatment strategies. A dataset of 1193 distinct gp120 V3 loop peptide sequences (989 R5-utilizing, 204 X4-capable) is utilized to train predictive classifiers based on implementations of random forest, support vector machine, boosted decision tree, and neural network machine learning algorithms. An in silico mutagenesis procedure employing multibody statistical potentials, computational geometry, and threading of variant V3 sequences onto an experimental structure, is used to generate a feature vector representation for each variant whose components measure environmental perturbations at corresponding structural positions.     

Role of Naturally Occurring Basic Amino Acid Substitutions in the Human Immunodeficiency Virus Type 1 Subtype E Envelope V3 Loop on Viral Coreceptor Usage and Cell Tropism

To assess the role of naturally occurring basic amino acid substitutions in the V3 loop of human immunodeficiency virus type 1 (HIV-1) subtype E on viral coreceptor usage and cell tropism, we have constructed a panel of chimeric viruses with mutant V3 loops of HIV-1 subtype E in the genetic background of HIV-1LAI. The arginine substitutions naturally occurring at positions 8, 11, and 18 of the V3 loop in an HIV-1 subtype E X4 strain were systematically introduced into that of an R5 strain to generate a series of V3 loop mutant chimera. These chimeric viruses were employed in virus infectivity assays using HOS-CD4 cells expressing either CCR5 or CXCR4, peripheral blood mononuclear cells, T-cell lines, or macrophages. The arginine substitution at position 11 of the V3 loop uniformly caused the loss of infectivity in HOS-CD4-CCR5 cells, indicating that position 11 is critical for utilization of CCR5. CXCR4 usage was conferred by a minimum of two arginine substitutions, regardless of combination, whereas arginine substitutions at position 8 and 11 were required for T-cell line tropism. Nonetheless, macrophage tropism was not conferred by the V3 loop of subtype E R5 strain per se. We found that the specific combinations of amino acid changes in HIV-1 subtype E env V3 loop are critical for determining viral coreceptor usage and cell tropism. However, the ability to infect HOS-CD4 cells through either CXCR4 or CCR5 is not necessarily correlated with T-cell or macrophage tropism, suggesting that cellular tropism is not dictated solely by viral coreceptor utilization.     

Resistance to a drug blocking human immunodeficiency virus type 1 entry (RPR103611) is conferred by mutations in gp41.

A triterpene derived from betulinic acid (RPR103611) blocks human immunodeficiency virus type 1 (HIV-1) infection and fusion of CD4+ cells with cells expressing HIV-1 envelope proteins (gp120 and gp41), suggesting an effect on virus entry. This compound did not block infection by a subtype D HIV-1 strain (NDK) or cell-cell fusion mediated by the NDK envelope proteins. The genetic basis of drug resistance was therefore addressed by testing envelope chimeras derived from NDK and a drug-sensitive HIV-1 strain (LAI, subtype B). A drug-resistant phenotype was observed for all chimeras bearing the ectodomain of NDK gp41, while the origins of gp120 and of the membrane anchor and cytoplasmic domains of gp41 had no apparent role. The envelope gene of a LAI variant, fully resistant to the antiviral effect of RPR103611, was cloned and sequenced. Its product differed from the parental sequence at two positions in gp41, with changes of arginine 22 to alanine (R22A) and isoleucine 84 to serine (I84S), the gp120 being identical. In the context of LAI gp41, the I84S substitution was sufficient for drug resistance. Therefore, in two different systems, differences in gp41 were associated with sensitivity or resistance to RPR103611. Modifications of gp41 can affect the quaternary structure of gp120 and gp41 and the accessibility of gp120 to antiviral agents such as neutralizing antibodies. However, a direct effect of RPR103611 on a gp41 target must also be envisioned, in agreement with the blocking of apparently late steps of HIV-1 entry. This compound could be a valuable tool for structure-function studies of gp41.     

V3 loop truncations in HIV-1 envelope impart resistance to coreceptor inhibitors and enhanced sensitivity to neutralizing antibodies

The V1/V2 region and the V3 loop of the human immunodeficiency virus type I (HIV-1) envelope (Env) protein are targets for neutralizing antibodies and also play an important functional role, with the V3 loop largely determining whether a virus uses CCR5 (R5), CXCR4 (X4), or either coreceptor (R5X4) to infect cells. While the sequence of V3 is variable, its length is highly conserved. Structural studies indicate that V3 length may be important for interactions with the extracellular loops of the coreceptor. Consistent with this view, genetic truncation of the V3 loop is typically associated with loss of Env function. We removed approximately one-half of the V3 loop from three different HIV-1 strains, and found that only the Env protein from the R5X4 strain R3A retained some fusion activity. Loss of V1/V2 (DeltaV1/V2) was well tolerated by this virus. Passaging of virus with the truncated V3 loop resulted in the derivation of a virus strain that replicated with wild-type kinetics. This virus, termed TA1, retained the V3 loop truncation and acquired several adaptive changes in gp120 and gp41. TA1 could use CCR5 but not CXCR4 to infect cells, and was extremely sensitive to neutralization by HIV-1 positive human sera, and by antibodies to the CD4 binding site and to CD4-induced epitopes in the bridging sheet region of gp120. In addition, TA1 was completely resistant to CCR5 inhibitors, and was more dependent upon the N-terminal domain of CCR5, a region of the receptor that is thought to contact the bridging sheet of gp120 and the base of the V3 loop, and whose conformation may not be greatly affected by CCR5 inhibitors. These studies suggest that the V3 loop protects HIV from neutralization by antibodies prevalent in infected humans, that CCR5 inhibitors likely act by disrupting interactions between the V3 loop and the coreceptor, and that altered use of CCR5 by HIV-1 associated with increased sensitivity to changes in the N-terminal domain can be linked to high levels of resistance to these antiviral compounds.