Adaptation of a CCR5-Using, Primary Human Immunodeficiency Virus Type 1 Isolate for CD4-Independent Replication

The gp120 envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) promotes virus entry by sequentially binding CD4 and chemokine receptors on the target cell. Primary, clinical HIV-1 isolates require interaction with CD4 to allow gp120 to bind the CCR5 chemokine receptor efficiently. We adapted a primary HIV-1 isolate, ADA, to replicate in CD4-negative canine cells expressing human CCR5. The gp120 changes responsible for the adaptation were limited to alteration of glycosylation addition sites in the V2 loop-V1-V2 stem. The gp120 glycoproteins of the adapted viruses bound CCR5 directly, without prior interaction with CD4. Thus, a major function of CD4 binding in the entry of primary HIV-1 isolates can be bypassed by changes in the gp120 V1-V2 elements, which allow the envelope glycoproteins to assume a conformation competent for CCR5 binding.     

The crown and stem of the V3 loop play distinct roles in human immunodeficiency virus type 1 envelope glycoprotein interactions with the CCR5 coreceptor

Human immunodeficiency virus type 1 envelope glycoprotein gp120 interacts with CD4 and the CCR5 coreceptor in order to mediate viral entry. A CD4-induced surface on gp120, primarily composed of residues in the V3 loop and the C4 domain, interacts with CCR5. In the present study, we generated envelope glycoproteins comprising chimeric V3 loops and/or V3 loops with deletions and studied their binding to CCR5 amino-terminal domain (Nt)-based sulfopeptides and cell surface CCR5, as well as their ability to mediate viral entry. We thus delineated two functionally distinct domains of the V3 loop, the V3 stem and the V3 crown. The V3 stem alone mediates soluble gp120 binding to the CCR5 Nt. In contrast, both the V3 stem and crown are required for soluble gp120 binding to cell surface CCR5. Within the context of a virion, however, the V3 crown alone determines coreceptor usage. Our data support a two-site gp120-CCR5 binding model wherein the V3 crown and stem interact with distinct regions of CCR5 in order to mediate viral entry.     

An aptamer that neutralizes R5 strains of human immunodeficiency virus type 1 blocks gp120-CCR5 interaction

We recently described the isolation and structural characterization of 2'-fluoropyrimidine-substituted RNA aptamers that bind to gp120 of R5 strains of human immunodeficiency virus type 1 and thereby potently neutralize the infectivity of phylogenetically diverse R5 strains. Here we investigate the physical basis of their antiviral action. We show that both N-linked oligosaccharides and the variable loops V1/V2 and V3 are not required for binding of one aptamer, B40, to gp120. Using surface plasmon resonance binding analyses, we show that the aptamer binds to the CCR5-binding site on gp120 in a relatively CD4-independent manner, providing a mechanistic explanation for its neutralizing potency.     

Human immunodeficiency virus type 1 coreceptor switching: V1/V2 gain-of-fitness mutations compensate for V3 loss-of-fitness mutations

Human immunodeficiency virus type 1 (HIV-1) entry into target cells is mediated by the virus envelope binding to CD4 and the conformationally altered envelope subsequently binding to one of two chemokine receptors. HIV-1 envelope glycoprotein (gp120) has five variable loops, of which three (V1/V2 and V3) influence the binding of either CCR5 or CXCR4, the two primary coreceptors for virus entry. Minimal sequence changes in V3 are sufficient for changing coreceptor use from CCR5 to CXCR4 in some HIV-1 isolates, but more commonly additional mutations in V1/V2 are observed during coreceptor switching. We have modeled coreceptor switching by introducing most possible combinations of mutations in the variable loops that distinguish a previously identified group of CCR5- and CXCR4-using viruses. We found that V3 mutations entail high risk, ranging from major loss of entry fitness to lethality. Mutations in or near V1/V2 were able to compensate for the deleterious V3 mutations and may need to precede V3 mutations to permit virus survival. V1/V2 mutations in the absence of V3 mutations often increased the capacity of virus to utilize CCR5 but were unable to confer CXCR4 use. V3 mutations were thus necessary but not sufficient for coreceptor switching, and V1/V2 mutations were necessary for virus survival. HIV-1 envelope sequence evolution from CCR5 to CXCR4 use is constrained by relatively frequent lethal mutations, deep fitness valleys, and requirements to make the right amino acid substitution in the right place at the right time.     

Antibody 17b binding at the coreceptor site weakens the kinetics of the interaction of envelope glycoprotein gp120 with CD4

HIV-1 utilizes CD4 and the chemokine coreceptor for viral entry. The coreceptor CCR5 binding site on gp120 partially overlaps with the binding epitope of 17b, a neutralizing antibody of HIV-1. We designed a multicomponent biosensor assay to investigate the kinetic mechanism of interaction between gp120 and its receptors and the cooperative effect of the CCR5 binding site on the CD4 binding site, using 17b as a surrogate of CCR5. The Env gp120 proteins from four viral strains (JRFL, YU2, 89.6, and HXB2) and their corresponding C1-, V1/V2-, C5-deleted mutants (DeltaJRFL, DeltaYU2, Delta89.6, and DeltaHXB2) were tested in this study. We found that, across the primary and lab-adapted virus strains, 17b reduced the affinity of all four full-length Env gp120s for sCD4 by decreasing the on-rate and increasing the off-rate. This effect of 17b on full-length gp120 binding to sCD4 contrasts with the enhancing effect of sCD4 on gp120-17b interaction. For the corresponding loop-deleted mutants of Env gp120, the off-rates of the gp120-sCD4 interaction were greatly reduced in the presence of 17b, resulting in higher affinities (except for that of DeltaHXB2). The results suggest that, when 17b is prebound to full-length gp120, the V1/V2 loops may be relocated to a position that partially blocks the CD4-binding site, leading to weakening of the CD4 interaction. Given the fact that the 17b binding epitope partially overlaps with the binding site of CCR5, the kinetic results suggest that coreceptor CCR5 binding could have a similar "release" effect on the gp120-CD4 interaction by increasing the off-rate of the latter. The results also suggest that the neutralizing effect of 17b may arise not only from partially blocking the CCR5 binding site but also from reducing the CD4 binding affinity of gp120. This negative cooperative effect of 17b may provide insight into approaches to designing antagonists for viral entry.     

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.     

Involvement of the V1/V2 variable loop structure in the exposure of human immunodeficiency virus type 1 gp120 epitopes induced by receptor binding.

The binding of human immunodeficiency virus type 1 (HIV-1) to the cellular receptor CD4 has been suggested to induce conformational changes in the viral envelope glycoproteins that promote virus entry. Conserved, discontinuous epitopes on the HIV-1 gp120 glycoprotein recognized by the 17b, 48d, and A32 antibodies are preferentially exposed upon the binding of soluble CD4 (sCD4). The binding of the 17b and 48d antibodies to the gp120 glycoprotein can also be enhanced by the binding of the A32 antibody. Here we constructed HIV-1 gp120 mutants in which the variable segments of the V1/V2 and V3 structures were deleted, individually or in combination, while the 17b, 48d, and A32 epitopes were retained. The effects of the variable loop deletions on the function of the HIV-1 envelope glycoproteins and on the exposure of epitopes induced by sCD4 or A32 binding to the monomeric gp120 glycoprotein were examined. The variable-loop-deleted envelope glycoproteins were able to mediate virus entry, albeit at lower efficiencies than those of the wild-type glycoproteins. Thus, the V1/V2 and V3 variable sequences contribute to the efficiency of HIV-1 entry but are not absolutely required for the process. Neither the V1/V2 nor V3 loops were necessary for the increase in exposure of the 17b/48d epitopes induced by binding of the A32 monoclonal antibody. By contrast, induction of the 17b, 48d, and A32 epitopes by sCD4 binding apparently involves a movement of the V1/V2 loops, which in the absence of CD4 partially mask these epitopes on the native gp120 monomer. The results obtained with a mutant glycoprotein containing a deletion of the V1 loop alone indicated that the contribution of the V2 loop to these phenomena was more significant than that of the V1 sequences. These results suggest that the V1/V2 loops, which have been previously implicated in CD4-modulated, postattachment steps in HIV-1 entry, contribute to CD4-induced gp120 conformational changes detected by the 17b, 48d, and A32 antibodies.     

Identification of conserved and variable structures in the human immunodeficiency virus gp120 glycoprotein of importance for CXCR4 binding

CD4 and the chemokine receptors, CXCR4 and CCR5, serve as receptors for human immunodeficiency virus type 1 (HIV-1). Binding of the HIV-1 gp120 envelope glycoprotein to the chemokine receptors normally requires prior interaction with CD4. Mapping the determinants on gp120 for the low-affinity interaction with CXCR4 has been difficult due to the nonspecific binding of this viral glycoprotein to cell surfaces. Here we examine the binding of a panel of gp120 mutants to paramagnetic proteoliposomes displaying CXCR4 on their surfaces. We show that the gp120 beta19 strand and third variable (V3) loop contain residues important for CXCR4 interaction. Basic residues from both elements, as well as a conserved hydrophobic residue at the V3 tip, contribute to CXCR4 binding. Removal of the gp120 V1/V2 variable loops allows the envelope glycoprotein to bind CXCR4 in a CD4-independent manner. These results indicate that although some variable gp120 residues contribute to the specific binding to CCR5 or CXCR4, gp120 elements common to CXCR4- or CCR5-using strains are involved in the interaction with both coreceptors.     

A novel human antibody against human immunodeficiency virus type 1 gp120 is V1, V2, and V3 loop dependent and helps delimit the epitope of the broadly neutralizing antibody immunoglobulin G1 b12

The V1/V2 and V3 loops are proximal to the CD4 binding site (CD4bs) of human immunodeficiency virus type 1 (HIV-1) gp120 and undergo conformational change upon CD4 receptor engagement by the HIV-1 envelope spike. Nearly all of the reported monoclonal antibodies (MAbs) against the CD4bs exhibit a very limited capacity to neutralize HIV-1. However, one such human MAb, immunoglobulin G1 (IgG1) b12, is uniquely able to neutralize primary isolates across subtypes with considerable potency. The molecular basis for the anti-HIV-1 activity of b12 is not fully understood but is relevant to vaccine design. Here we describe a novel human MAb, 4KG5, whose binding to monomeric gp120 is moderately enhanced by IgG1 b12. In sharp contrast, 4KG5 binding to gp120 is inhibited by soluble CD4 (sCD4) and by all other (n = 14) anti-CD4bs MAbs tested. 4KG5 is unable to recognize gp120 in which either V1, V2, or V3 has been deleted, and MAbs against the V2 or V3 loops inhibit the binding of 4KG5 to gp120. Moreover, 4KG5 is able to inhibit the binding of the CD4-induced MAbs 17b and X5 in the absence of sCD4, whereas 17b and X5 only weakly inhibit the binding of 4KG5 to gp120. Mutagenesis of gp120 provides further evidence of a discontinuous epitope of 4KG5 that is formed by the V1/V2 loop, the V3 loop, and a portion of the bridging sheet (C4). 4KG5 was isolated as a single-chain Fv from a phage display library constructed from the bone marrow of an HIV-1-seropositive subject (FDA2) whose serum neutralizes HIV-1 across subtypes. Despite its source, we observed no significant neutralization with 4KG5 against the autologous (R2) virus and several other strains of HIV-1. The results suggest a model in which antibody access to the CD4bs on the envelope spike of HIV-1 is restricted by the orientation and/or dynamics of the V1/V2 and V3 loops, and b12 avoids these restrictions.     

Structure and Dynamics of the gp120 V3 Loop That Confers Noncompetitive Resistance in R5 HIV-1JR-FL to Maraviroc

Maraviroc, an (HIV-1) entry inhibitor, binds to CCR5 and efficiently prevents R5 human immunodeficiency virus type 1 (HIV-1) from using CCR5 as a coreceptor for entry into CD4⁺ cells. However, HIV-1 can elude maraviroc by using the drug-bound form of CCR5 as a coreceptor. This property is known as noncompetitive resistance. HIV-1_V3-M5 derived from HIV-1_JR-FLan is a noncompetitive-resistant virus that contains five mutations (I304V/F312W/T314A/E317D/I318V) in the gp120 V3 loop alone. To obtain genetic and structural insights into maraviroc resistance in HIV-1, we performed here mutagenesis and computer-assisted structural study. A series of site-directed mutagenesis experiments demonstrated that combinations of V3 mutations are required for HIV-1_JR-FLan to replicate in the presence of 1 µM maraviroc, and that a T199K mutation in the C2 region increases viral fitness in combination with V3 mutations. Molecular dynamic (MD) simulations of the gp120 outer domain V3 loop with or without the five mutations showed that the V3 mutations induced (i) changes in V3 configuration on the gp120 outer domain, (ii) reduction of an anti-parallel β-sheet in the V3 stem region, (iii) reduction in fluctuations of the V3 tip and stem regions, and (iv) a shift of the fluctuation site at the V3 base region. These results suggest that the HIV-1 gp120 V3 mutations that confer maraviroc resistance alter structure and dynamics of the V3 loop on the gp120 outer domain, and enable interactions between gp120 and the drug-bound form of CCR5.     

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.     

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.     

Atomic insight into the CD4 binding-induced conformational changes in HIV-1 gp120

The entry of HIV-1 into a target cell requires gp120 and receptor CD4 as well as coreceptor CCR5/CXCR4 recognition events associated with conformational changes of the involved proteins. The binding of CD4 to gp120 is the initiation step of the whole process involving structural rearrangements that are crucial for subsequent pathways. Despite the wealth of knowledge about the gp120/CD4 interactions, details of the conformational changes occurring at this stage remain elusive. We have performed molecular dynamics simulations in explicit solvent based on the gp120/CD4/CD4i crystal structure in conjunction with modeled V3 and V4 loops to gain insight into the dynamics of the binding process. Three differentiated interaction modes between CD4 and gp120 were found, which involve electrostatics, hydrogen bond and van der Waals networks. A "binding funnel" model is proposed based on the dynamical nature of the binding interface together with a CD4-attraction gradient centered in gp120 at the CD4-Phe43-binding cavity. Distinct dynamical behaviors of free and CD4-bound gp120 were monitored, which likely represent the ground and pre-fusogenic states, respectively. The transition between these states revealed concerted motions in gp120 leading to: i) loop contractions around the CD4-Phe43-insertion cavity; ii) stabilization of the four-stranded "bridging sheet" structure; and iii) translocation and clustering of the V3 loop and the bridging sheet leading to the formation of the coreceptor binding site. Our results provide new insight into the dynamic of the underlying molecular recognition mechanism that complements the biochemical and structural studies.     

Critical Amino Acids within the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein V4 N- and C-Terminals Contribute to Virus Entry

The importance of the fourth variable (V4) region of the human immunodeficiency virus 1 (HIV-1) envelope glycoprotein (Env) in virus infection has not been well clarified, though the polymorphism of this region has been found to be associated with disease progression to acquired immunodeficiency syndrome (AIDS). In the present work, we focused on the correlation between HIV-1 gp120 V4 region polymorphism and the function of the region on virus entry, and the possible mechanisms for how the V4 region contributes to virus infectivity. Therefore, we analyzed the differences in V4 sequences along with coreceptor usage preference from CCR5 to CXCR4 and examined the importance of the amino acids within the V4 region for CCR5- and CXCR4-tropic virus entry. In addition, we determined the influence of the V4 amino acids on Env expression and gp160 processing intracellularly, as well as the amount of Env on the pseudovirus surface. The results indicated that V4 tended to have a shorter length, fewer potential N-linked glycosylation sites (PNGS), greater evolutionary distance, and a lower negative net charge when HIV-1 isolates switched from a coreceptor usage preference for CCR5 to CXCR4. The N- and C-terminals of the HIV-1 V4 region are highly conserved and critical to maintain virus entry ability, but only the mutation at position 417 in the context of ADA (a R5-tropic HIV-1 strain) resulted in the ability to utilize CXCR4. In addition, 390L, 391F, 414I, and 416L are critical to maintain gp160 processing and maturation. It is likely that the hydrophobic properties and the electrostatic surface potential of gp120, rather than the conformational structure, greatly contribute to this V4 functionality. The findings provide information to aid in the understanding of the functions of V4 in HIV-1 entry and offer a potential target to aid in the development of entry inhibitors.     

Mapping the determinants of the CCR5 amino-terminal sulfopeptide interaction with soluble human immunodeficiency virus type 1 gp120-CD4 complexes

CD4 and CCR5 mediate fusion and entry of R5 human immunodeficiency virus type 1 (HIV-1) strains. Sulfotyrosine and other negatively charged residues in the CCR5 amino-terminal domain (Nt) are crucial for gp120 binding and viral entry. We previously showed that a soluble gp120-CD4 complex specifically binds to a peptide corresponding to CCR5 Nt residues 2 to 18, with sulfotyrosines in positions 10 and 14. This sulfopeptide also inhibits soluble gp120-CD4 binding to cell surface CCR5 as well as infection by an R5 virus. Here we show that residues 10 to 18 constitute the minimal domain of the CCR5 Nt that is able to specifically interact with soluble gp120-CD4 complexes. In addition to sulfotyrosines in positions 10 and 14, negatively charged residues in positions 11 and 18 participate in this interaction. Furthermore, the CCR5 Nt binds to a CD4-induced surface on gp120 that is composed of conserved residues in the V3 loop stem and the C4 domain. Binding of gp120 to cell surface CCR5 is further influenced by residues in the crown of the V3 loop, C1, C2, and C3. Our data suggest that gp120 docking to CCR5 is a multistep process involving several independent regions of the envelope glycoprotein and the coreceptor.     

Functional mimicry of a human immunodeficiency virus type 1 coreceptor by a neutralizing monoclonal antibody

Interaction of the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein with the primary receptor, CD4, promotes binding to a chemokine receptor, either CCR5 or CXCR4. The chemokine receptor-binding site on gp120 elicits CD4-induced (CD4i) antibodies in some HIV-1-infected individuals. Like CCR5 itself, the CD4i antibody 412d exhibits a preference for CCR5-using HIV-1 strains and utilizes sulfated tyrosines to achieve binding to gp120. Here, we show that 412d binding requires the gp120 beta19 strand and the base of the V3 loop, elements that are important for the binding of the CCR5 N terminus. Two gp120 residues in the V3 loop base determined 412d preference for CCR5-using HIV-1 strains. A chimeric molecule in which the 412d heavy-chain third complementarity-determining loop sequence replaces the CCR5 N terminus functioned as an efficient second receptor, selectively supporting the entry of CCR5-using HIV-1 strains. Sulfation of N-terminal tyrosines contributed to the function of this chimeric receptor. These results emphasize the close mimicry of the CCR5 N terminus by the gp120-interactive region of a naturally elicited CD4i antibody.     

N-linked glycosylation sites adjacent to and within the V1/V2 and the V3 loops of dualtropic human immunodeficiency virus type 1 isolate DH12 gp120 affect coreceptor usage and cellular tropism

The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is extensively glycosylated, containing approximately 23 asparagine (N)-linked glycosylation sites on its gp120 subunit. In this study, specific glycosylation sites on gp120 of a dualtropic primary HIV-1 isolate, DH12, were eliminated by site-directed mutagenesis and the properties of the resulting mutant envelopes were evaluated using a recombinant vaccinia virus-based cell-to-cell fusion assay alone or in the context of viral infections. Of the glycosylation sites that were evaluated, those proximal to the V1/V2 loops (N135, N141, N156, N160) and the V3 loops (N301) of gp120 were functionally critical. The glycosylation site mutations near the V1/V2 loop compromised the use of CCR5 and CXCR4 equally. In contrast, a mutation within the V3 loop preferentially inhibited the usage of CCR5; although this mutant protein completely lost its CCR5-dependent fusion activity, it retained 50% of the wild-type fusion activity with CXCR4. The replication of a virus containing this mutation was severely compromised in peripheral blood mononuclear cells, MT-4 cells, and primary monocyte-derived macrophages. A revertant virus, which acquired second site changes in the V3 loop that resulted in an increase in net positive charge, was isolated. The revertant virus fully recovered the usage of CXCR4 but not of CCR5, thereby altering the tropism of the parental virus from dualtropic to T-tropic. These results suggest that carbohydrate moieties near the V1/V2 and the V3 loops play critical roles in maintaining proper conformation of the variable loops for optimal interaction with receptors. Our results, combined with those of previously reported studies, further demonstrate that the function of individual glycans may be virus isolate dependent.     

Determinants of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Activation by Soluble CD4 and Monoclonal Antibodies

Infection by some human immunodeficiency virus type 1 (HIV-1) isolates is enhanced by the binding of subneutralizing concentrations of soluble receptor, soluble CD4 (sCD4), or monoclonal antibodies directed against the viral envelope glycoproteins. In this work, we studied the abilities of different antibodies to mediate activation of the envelope glycoproteins of a primary HIV-1 isolate, YU2, and identified the regions of gp120 envelope glycoprotein contributing to activation. Binding of antibodies to a variety of epitopes on gp120, including the CD4 binding site, the third variable (V3) loop, and CD4-induced epitopes, enhanced the entry of viruses containing YU2 envelope glycoproteins. Fab fragments of antibodies directed against either the CD4 binding site or V3 loop also activated YU2 virus infection. The activation phenotype was conferred on the envelope glycoproteins of a laboratory-adapted HIV-1 isolate (HXBc2) by replacing the gp120 V3 loop or V1/V2 and V3 loops with those of the YU2 virus. Infection by the YU2 virus in the presence of activating antibodies remained inhibitable by macrophage inhibitory protein 1β, indicating dependence on the CCR5 coreceptor on the target cells. Thus, antibody enhancement of YU2 entry involves neither Fc receptor binding nor envelope glycoprotein cross-linking, is determined by the same variable loops that dictate enhancement by sCD4, and probably proceeds by a process fundamentally similar to the receptor-activated virus entry pathway.     

Discovery of small-molecule human immunodeficiency virus type 1 entry inhibitors that target the gp120-binding domain of CD4

The interaction between human immunodeficiency virus type 1 (HIV-1) gp120 and the CD4 receptor is highly specific and involves relatively small contact surfaces on both proteins according to crystal structure analysis. This molecularly conserved interaction presents an excellent opportunity for antiviral targeting. Here we report a group of pentavalent antimony-containing small molecule compounds, NSC 13778 (molecular weight, 319) and its analogs, which exert a potent anti-HIV activity. These compounds block the entry of X4-, R5-, and X4/R5-tropic HIV-1 strains into CD4(+) cells but show little or no activity in CD4-negative cells or against vesicular stomatitis virus-G pseudotyped virions. The compounds compete with gp120 for binding to CD4: either immobilized on a solid phase (soluble CD4) or on the T-cell surface (native CD4 receptor) as determined by a competitive gp120 capture enzyme-linked immunosorbent assay or flow cytometry. NSC 13778 binds to an N-terminal two-domain CD4 protein, D1/D2 CD4, immobilized on a surface plasmon resonance sensor chip, and dose dependently reduces the emission intensity of intrinsic tryptophan fluorescence of D1/D2 CD4, which contains two of the three tryptophan residues in the gp120-binding domain. Furthermore, T cells incubated with the compounds alone show decreased reactivity to anti-CD4 monoclonal antibodies known to recognize the gp120-binding site. In contrast to gp120-binders that inhibit gp120-CD4 interaction by binding to gp120, these compounds appear to disrupt gp120-CD4 contact by targeting the specific gp120-binding domain of CD4. NSC 13778 may represent a prototype of a new class of HIV-1 entry inhibitors that can break into the gp120-CD4 interface and mask the gp120-binding site on the CD4 molecules, effectively repelling incoming virions.