Disulfide Reduction in CD4 Domain 1 or 2 Is Essential for Interaction with HIV Glycoprotein 120 (gp120), which Impairs Thioredoxin-driven CD4 Dimerization

Human CD4 is a membrane-bound glycoprotein expressed on the surface of certain leukocytes, where it plays a key role in the activation of immunostimulatory T cells and acts as the primary receptor for human immunodeficiency virus (HIV) glycoprotein (gp120). Although growing evidence suggests that redox exchange reactions involving CD4 disulfides, potentially catalyzed by cell surface-secreted oxidoreductases such as thioredoxin (Trx) and protein disulfide isomerase, play an essential role in regulating the activity of CD4, their mechanism(s) and biological utility remain incompletely understood. To gain more insights in this regard, we generated a panel of recombinant 2-domain CD4 proteins (2dCD4), including wild-type and Cys/Ala variants, and used these to show that while protein disulfide isomerase has little capacity for 2dCD4 reduction, Trx reduces 2dCD4 highly efficiently, catalyzing the formation of conformationally distinct monomeric 2dCD4 isomers, and a stable, disulfide-linked 2dCD4 dimer. Moreover, we show that HIV gp120 is incapable of binding a fully oxidized, monomeric 2dCD4 in which both domain 1 and 2 disulfides are intact, but binds robustly to reduced counterparts that are the ostensible products of Trx-mediated isomerization. Finally, we demonstrate that Trx-driven dimerization of CD4, a process believed to be critical for the establishment of functional MHCII-TCR-CD4 antigen presentation complexes, is impaired when CD4 is bound to gp120. These observations reinforce the importance of cell surface redox activity for HIV entry and posit the intriguing possibility that one of the many pathogenic effects of HIV may be related to gp120-mediated inhibition of oxidoreductive CD4 isomerization.     

Stabilization of HIV-1 gp120-CD4 Receptor Complex through Targeted Interchain Disulfide Exchange

HIV-1 enters cells via interaction between the trimeric envelope (Env) glycoprotein gp120/gp41 and the host cell surface receptor molecule CD4. The requirement of CD4 for viral entry has rationalized the development of recombinant CD4-based proteins as competitive viral attachment inhibitors and immunotherapeutic agents. In this study, we describe a novel recombinant CD4 protein designed to bind gp120 through a targeted disulfide-exchange mechanism. According to structural models of the gp120-CD4 receptor complex, substitution of Ser⁶⁰ on the CD4 domain 1 α-helix with Cys positions a thiol in proximity of the gp120 V1/V2 loop disulfide (Cys¹²⁶–Cys¹⁹⁶), satisfying the stereochemical and geometric conditions for redox exchange between CD4 Cys⁶⁰ and gp120 Cys¹²⁶, and the consequent formation of an interchain disulfide bond. In this study, we provide experimental evidence for this effect by describing the expression, purification, refolding, receptor binding and antiviral activity analysis of a recombinant two-domain CD4 variant containing the S60C mutation (2dCD4-S60C). We show that 2dCD4-S60C binds HIV-1 gp120 with a significantly higher affinity than wild-type protein under conditions that facilitate disulfide exchange and that this translates into a corresponding increase in the efficacy of CD4-mediated viral entry inhibition. We propose that targeted redox exchange between conserved gp120 disulfides and nucleophilic moieties positioned strategically on CD4 (or CD4-like scaffolds) conceptualizes a new strategy in the development of high affinity HIV-1 Env ligands, with important implications for therapy and vaccine development. More generally, this chalcogen substitution approach provides a general means of stabilizing receptor-ligand complexes where the structural and biophysical conditions for disulfide exchange are satisfied.     

Inhibitors of protein-disulfide isomerase prevent cleavage of disulfide bonds in receptor-bound glycoprotein 120 and prevent HIV-1 entry

We previously reported that monoclonal antibodies to protein-disulfide isomerase (PDI) and other membrane-impermeant PDI inhibitors prevented HIV-1 infection. PDI is present at the surface of HIV-1 target cells and reduces disulfide bonds in a model peptide attached to the cell membrane. Here we show that soluble PDI cleaves disulfide bonds in recombinant envelope glycoprotein gp120 and that gp120 bound to the surface receptor CD4 undergoes a disulfide reduction that is prevented by PDI inhibitors. Concentrations of inhibitors that prevent this reduction and inhibit the cleavage of surface-bound disulfide conjugate prevent infection at the level of HIV-1 entry. The entry of HIV-1 strains differing in their coreceptor specificities is similarly inhibited, and so is the reduction of gp120 bound to CD4 of coreceptor-negative cells. PDI inhibitors also prevent HIV envelope-mediated cell-cell fusion but have no effect on the entry of HIV-1 pseudo-typed with murine leukemia virus envelope. Importantly, PDI coprecipitates with both soluble and cellular CD4. We propose that a PDI.CD4 association at the cell surface enables PDI to reach CD4-bound virus and to reduce disulfide bonds present in the domain of gp120 that binds to CD4. Conformational changes resulting from the opening of gp120-disulfide loops may drive the processes of virus-cell and cell-cell fusion. The biochemical events described identify new potential targets for anti-HIV agents.     

The Cellular Thioredoxin-1/Thioredoxin Reductase-1 Driven Oxidoreduction Represents a Chemotherapeutic Target for HIV-1 Entry Inhibition

Background

The entry of HIV into its host cell is an interesting target for chemotherapeutic intervention in the life-cycle of the virus. During entry, reduction of disulfide bridges in the viral envelope glycoprotein gp120 by cellular oxidoreductases is crucial. The cellular thioredoxin reductase-1 plays an important role in this oxidoreduction process by recycling electrons to thioredoxin-1. Therefore, thioredoxin reductase-1 inhibitors may inhibit gp120 reduction during HIV-1 entry. In this present study, tellurium-based thioredoxin reductase-1 inhibitors were investigated as potential inhibitors of HIV entry.

Results

The organotellurium compounds inhibited HIV-1 and HIV-2 replication in cell culture at low micromolar concentrations by targeting an early event in the viral infection cycle. Time-of-drug-addition studies pointed to virus entry as the drug target, more specifically: the organotellurium compound TE-2 showed a profile similar or close to that of the fusion inhibitor enfuvirtide (T-20). Surface plasmon resonance-based interaction studies revealed that the compounds do not directly interact with the HIV envelope glycoproteins gp120 and gp41, nor with soluble CD4, but instead, dose-dependently bind to thioredoxin reductase-1. By inhibiting the thioredoxin-1/thioredoxin reductase-1-directed oxidoreduction of gp120, the organotellurium compounds prevent conformational changes in the viral glycoprotein which are necessary during viral entry.

Conclusion

Our findings revealed that thioredoxin-1/thioredoxin reductase-1 acts as a cellular target for the inhibition of HIV entry.     

Structural and functional characterization of the human CCR5 receptor in complex with HIV gp120 envelope glycoprotein and CD4 receptor by molecular modeling studies

The entry of human immunodeficiency virus (HIV) into cells depends on a sequential interaction of the gp120 envelope glycoprotein with the cellular receptors CD4 and members of the chemokine receptor family. The CC chemokine receptor CCR5 is such a receptor for several chemokines and a major coreceptor for the entry of R5 HIV type-1 (HIV-1) into cells. Although many studies focus on the interaction of CCR5 with HIV-1, the corresponding interaction sites in CCR5 and gp120 have not been matched. Here we used an approach combining protein structure modeling, docking and molecular dynamics simulation to build a series of structural models of the CCR5 in complexes with gp120 and CD4. Interactions such as hydrogen bonds, salt bridges and van der Waals contacts between CCR5 and gp120 were investigated. Three snapshots of CCR5-gp120-CD4 models revealed that the initial interactions of CCR5 with gp120 are involved in the negatively charged N-terminus (Nt) region of CCR5 and positively charged bridging sheet region of gp120. Further interactions occurred between extracellular loop2 (ECL2) of CCR5 and the base of V3 loop regions of gp120. These interactions may induce the conformational changes in gp120 and lead to the final entry of HIV into the cell. These results not only strongly support the two-step gp120-CCR5 binding mechanism, but also rationalize extensive biological data about the role of CCR5 in HIV-1 gp120 binding and entry, and may guide efforts to design novel inhibitors.     

Human immunodeficiency virus type 1 envelope gp120 is cleaved after incubation with recombinant soluble CD4.

Human immunodeficiency virus type 1 (HIV-1) infects human CD4+ cells by a high-affinity interaction between its envelope glycoprotein gp120 and the CD4 molecule on the cell surface. Subsequent virus entry into the cells involves other steps, one of which could be cleavage of the gp120 followed by virus-cell fusion. The envelope gp120 is highly variable among different HIV-1 isolates, but conserved amino acid sequence motifs that contain potential proteolytic cleavage sites can be found. Following incubation with a soluble form of CD4, we demonstrate that gp120 of highly purified HIV-1 preparations is, without addition of exogenous proteinase, cleaved most likely in the V3 loop, yielding two proteins of 50 and 70 kDa. The extent of gp120 proteolysis is HIV-1 strain dependent and correlates with the recombinant soluble CD4 sensitivity to neutralization of the particular strain. The origin of the proteolytic activity in the virus preparations remains unclear. The results support the hypothesis that cleavage of gp120 is required for HIV infection of cells.     

Thioredoxin-1 and protein disulfide isomerase catalyze the reduction of similar disulfides in HIV gp120

HIV-1 enters cells via interaction of the viral glycoprotein gp120, the host cell surface receptor CD4 and the co-receptors CCR5 or CXCR4. For entry, gp120 undergoes conformational changes that depend on the reduction of one or more disulfides. Previous studies indicate that protein disulfide isomerase (PDI), thioredoxin-1 (Trx1), and glutaredoxin-1 (Grx1) catalyze gp120 reduction, but their specific disulfide targets are not known. Here, it was demonstrated that PDI and Trx1 have similar gp120 disulfide targets as determined by labeling after reduction, but with some pattern differences, including overall stronger labeling with Trx1 than with PDI. Furthermore, uneven labeling of the residues of a disulfide may reflect altered accessibility by conformational changes upon the reduction process. Since both PDI and Trx1 may be involved in viral entry, compounds that target the host redox system or the viral gp120 were tested in vitro to investigate whether redox regulation is a target for anti-HIV therapy. Carbohydrate binding agents (CBAs), previously shown to bind gp120 and inhibit HIV entry, were now demonstrated to inhibit gp120 disulfide reduction. Auranofin, an inhibitor of thioredoxin reductase 1 (TrxR1), also showed inhibitory activity towards HIV infection, although close to its cytotoxic concentration. Our results demonstrate that both the host redox system and the viral surface glycoproteins are of interest for the development of new generations of anti-HIV therapeutics.     

Neutralizing antibodies against the V3 loop of human immunodeficiency virus type 1 gp120 block the CD4-dependent and -independent binding of virus to cells.

The CD4 molecule is an essential receptor for human immunodeficiency virus type 1 (HIV-1) through high-affinity interactions with the viral external envelope glycoprotein gp120. Previously, neutralizing monoclonal antibodies (MAbs) specific to the third hypervariable domain of gp120 (the V3 loop) have been thought to block HIV infection without affecting the binding of HIV particles to CD4-expressing human cells. However, here we demonstrate that this conclusion was not correct and was due to the use of soluble gp120 instead of HIV particles. Indeed, neutralizing anti-V3 loop MAbs inhibited completely the binding and entry of HIV particles into CD4+ human cells. In contrast, the binding of virus was only partially inhibited by neutralizing anti-CD4 MAbs against the gp120 binding site in CD4, which, like the anti-V3 loop MAbs, completely inhibited HIV entry and infection. Nonneutralizing control MAbs against either the V3 loop or the N or C terminus of gp120 had no significant effect on HIV binding and entry. HIV-1 particles were also found to bind human and murine cells expressing or not expressing the human CD4 molecule. Interestingly, the binding of HIV to CD4+ murine cells was inhibited by both anti-V3 and anti-CD4 MAbs, whereas the binding to human and murine CD4- cells was affected only by anti-V3 loop MAbs. The effect of anti-V3 loop neutralizing MAbs on the HIV binding to cells appears not to be the direct consequence of gp120 shedding from HIV particles or of a decreased affinity of CD4 or gp120 for binding to its surface counterpart. Taken together, our results suggest the existence of CD4-dependent and -independent binding events involved in the attachment of HIV particles to cells; in both of these events, the V3 loop plays a critical role. As murine cells lack the specific cofactor CXCR4 for HIV-1 entry, other cell surface molecules besides CD4 might be implicated in stable binding of HIV particles to cells.     

Binding of soluble recombinant HIV envelope glycoprotein, rgp120, induces conformational changes in the cellular membrane-anchored CD4 molecule

During HIV entry or resulting cell to cell fusion, the envelope glycoprotein gp120 binds first to the CD4 membrane distal domain and second to a chemokine receptor as coreceptor. Taking into consideration the relative length of these two molecules' extracellular parts, structural modulations of CD4 would be required to make the second interaction possible. In this work, we assessed the effect of gp120 binding on the conformation of CD4 expressed on cell surface. We demonstrated that following gp120 binding the avidity of some, but not all, monoclonal antibodies specific to epitopes, outside of the gp120-binding site, in D1, D3 and D4 domains of CD4 was decreased dramatically. This finding demonstrates that the gp120-CD4 interaction induces local and specific conformational changes of CD4 and constitutes functional evidence for hinge regions that could confer to this molecule the flexibility required for its various functions.     

Human glutaredoxin-1 catalyzes the reduction of HIV-1 gp120 and CD4 disulfides and its inhibition reduces HIV-1 replication

Reduction of intramolecular disulfides in the HIV-1 envelope protein gp120 occurs after its binding to the CD4 receptor. Protein disulfide isomerase (PDI) catalyzes the disulfide reduction in vitro and inhibition of this enzyme blocks viral entry. PDI belongs to the thioredoxin protein superfamily that also includes human glutaredoxin-1 (Grx1). Grx1 is secreted from cells and the protein has also been found within the HIV-1 virion. We show that Grx1 efficiently catalyzes gp120, and CD4 disulfide reduction in vitro, even at low plasma levels of glutathione. Grx1 catalyzes the reduction of two disulfide bridges in gp120 in a similar manner as PDI. Purified anti-Grx1 antibodies were shown to inhibit the Grx1 activity in vitro and block HIV-1 replication in cultured peripheral blood mononuclear cells. Also, the polyanion PRO2000, that was previously shown to prevent HIV entry, inhibits the Grx1- and PDI-dependent reduction of gp120 disulfides. Our findings suggest that Grx1 activity is important for HIV-1 entry and that Grx1 and the gp120 intramolecular disulfides are novel pharmacological targets for rational drug development.     

Identification of the principal neutralizing determinant of human immunodeficiency virus type 1 as a fusion domain.

The V3 loop, located near the middle of the surface envelope glycoprotein gp120, is the major neutralizing domain of human immunodeficiency virus type 1 (HIV-1). Although the majority of the V3 loop is highly variable between different strains of HIV-1, a Gly-Pro-Gly-Arg motif at the tip of the loop is highly conserved. To determine whether this region plays a role in fusion mediated by the HIV-1 envelope glycoproteins, we introduced seven single-amino-acid changes in the V3 loop. The mutant envelope glycoproteins were expressed from an HIV-1 envelope expression vector and analyzed for their ability to induce cell fusion in the absence of virus replication. Our results indicated that single-amino-acid changes in the V3 loop were capable of completely abolishing or greatly reducing the ability of the HIV-1 envelope glycoproteins to induce cell fusion, thereby identifying the V3 loop as a fusion domain of HIV-1. Mutations in the highly conserved tip of the loop or in a nonconserved region flanking the highly conserved tip had no effect on envelope glycoprotein synthesis, processing, transport, or binding to the CD4 receptor molecule. Mutation of the putative disulfide bridge-forming Cys at residue 336 blocked gp160 cleavage and CD4 binding.     

Reduced monomeric CD4 is the preferred receptor for HIV

CD4 is a co-receptor for binding of T cells to antigen-presenting cells and the primary receptor for the human immunodeficiency virus type 1 (HIV). CD4 exists in three different forms on the cell surface defined by the state of the domain 2 cysteine residues: an oxidized monomer, a reduced monomer, and a covalent dimer linked through the domain 2 cysteines. The disulfide-linked dimer is the preferred immune co-receptor. The form of CD4 that is preferred by HIV was examined in this study. HIV entry and envelope-mediated cell-cell fusion were tested using cells expressing comparable levels of wild-type or disulfide bond mutant CD4 in which the domain 2 cysteines were mutated to alanine. Eliminating the domain 2 disulfide bond increased entry of HIV reporter viruses and enhanced HIV envelope-mediated cell-cell fusion 2-4-fold. These observations suggest that HIV enters susceptible cells preferably through monomeric reduced CD4, whereas dimeric CD4 is the preferred receptor for binding to antigen-presenting cells. Cleavage of the domain 2 disulfide bond is possibly involved in the conformational change in CD4 associated with fusion of the HIV and cell membranes.     

Determinants of CD4 independence for a human immunodeficiency virus type 1 variant map outside regions required for coreceptor specificity

Although infection by human immunodeficiency virus (HIV) typically requires an interaction between the viral envelope glycoprotein (Env), CD4, and a chemokine receptor, CD4-independent isolates of HIV and simian immunodeficiency virus have been described. The structural basis and underlying mechanisms for this phenotype are unknown. We have derived a variant of HIV-1/IIIB, termed IIIBx, that acquired the ability to utilize CXCR4 without CD4. This virus infected CD4-negative T and B cells and fused with murine 3T3 cells that expressed human CXCR4 alone. A functional IIIBx env clone exhibited several mutations compared to the CD4-dependent HXBc2 env, including the striking loss of five glycosylation sites. By constructing env chimeras with HXBc2, the determinants for CD4 independence were shown to map outside the V1/V2 and V3 hypervariable loops, which determine chemokine receptor specificity, and at least partly within an area on the gp120 core that has been implicated in forming a conserved chemokine receptor binding site. We also identified a point mutation in the C4 domain that could render the IIIBx env clone completely CD4 dependent. Mutations in the transmembrane protein (TM) were also required for CD4 independence. Remarkably, when the V3 loop of a CCR5-tropic Env was substituted for the IIIBx Env, the resulting chimera was found to utilize CCR5 but remained CD4 independent. These findings show that Env determinants for chemokine receptor specificity are distinct from those that mediate CD4-independent use of that receptor for cell fusion and provide functional evidence for multiple steps in the interaction of Env with chemokine receptors. Combined with our observation that the conserved chemokine receptor binding site on gp120 is more exposed on the IIIBx gp120 (T. L. Hoffman, C. C. LaBranche, W. Zhang, G. Canziani, J. Robinson, I. Chaiken, J. A. Hoxie, and R. W. Doms, Proc. Natl. Acad. Sci. USA 96:6359-6364, 1999), the findings from this study suggest novel approaches to derive and design Envs with exposed chemokine receptor binding sites for vaccine purposes.     

HIV coreceptors: role of structure,posttranslational modifications,and internalization in viral-cell fusion and as targets for entry inhibitors

The human immunodeficiency virus (HIV) envelope glycoprotein forms trimers on the virion surface, with each monomer consisting of two subunits, gp120 and gp41. The gp120 envelope component binds to CD4 on target cells and undergoes conformational changes that allow gp120 to interact with certain G-protein-coupled receptors (GPCRs) on the same target membranes. The GPCRs that function as HIV coreceptors were found to be chemokine receptors. The primary coreceptors are CCR5 and CXCR4, but several other chemokine receptors were identified as "minor coreceptors", indicating their ability support entry of some HIV strains in tissue cultures. Formation of the tri-molecular complexes stabilizes virus binding and triggers a series of conformational changes in gp41 that facilitate membrane fusion and viral cell entry. Concerted efforts are underway to decipher the specific interactions between gp120/CD4, gp120/coreceptors, and their contributions to the subsequent membrane fusion process. It is hoped that some of the transient conformational intermediates in gp120 and gp41 would serve as targets for entry inhibitors. In addition, the CD4 and coreceptors are primary targets for several classes of inhibitors currently under testing. Our review summarizes the current knowledge on the interactions of HIV gp120 with its receptor and coreceptors, and the important properties of the chemokine receptors and their regulation in primary target cells. We also summarize the classes of coreceptor inhibitors under development.     

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.     

Is the V3 loop involved in HIV binding to CD4?

The entry of the human immunodeficiency virus into cells requires the interaction of the viral envelope glycoprotein gp120 with CD4 and a chemokine receptor. The gp120 binding site has been previously mapped to the Ig-CDR2-like region of CD4 first domain. A second area of this domain (Ig-CDR3-like region) is involved in gp120-CD4 interactions, but its gp120 counterpart remained so far unknown. Using a photoaffinity labeling experiment, we demonstrate that a peptide, mapping the (307-330)m region of HIV-MN-gp120 V3 loop, binds a sequence including a part of the Ig-CDR3-like region. These results may contribute to explain the complex mechanism of human immunodeficiency virus penetration, helping the development of new therapeutic agents.     

HIV coreceptors: role of structure, posttranslational modifications, and internalization in viral-cell fusion and as targets for entry inhibitors

The human immunodeficiency virus (HIV) envelope glycoprotein forms trimers on the virion surface, with each monomer consisting of two subunits, gp120 and gp41. The gp120 envelope component binds to CD4 on target cells and undergoes conformational changes that allow gp120 to interact with certain G-protein-coupled receptors (GPCRs) on the same target membranes. The GPCRs that function as HIV coreceptors were found to be chemokine receptors. The primary coreceptors are CCR5 and CXCR4, but several other chemokine receptors were identified as “minor coreceptors”, indicating their ability support entry of some HIV strains in tissue cultures. Formation of the tri-molecular complexes stabilizes virus binding and triggers a series of conformational changes in gp41 that facilitate membrane fusion and viral cell entry. Concerted efforts are underway to decipher the specific interactions between gp120/CD4, gp120/coreceptors, and their contributions to the subsequent membrane fusion process. It is hoped that some of the transient conformational intermediates in gp120 and gp41 would serve as targets for entry inhibitors. In addition, the CD4 and coreceptors are primary targets for several classes of inhibitors currently under testing. Our review summarizes the current knowledge on the interactions of HIV gp120 with its receptor and coreceptors, and the important properties of the chemokine receptors and their regulation in primary target cells. We also summarize the classes of coreceptor inhibitors under development.     

Replication-competent variants of human immunodeficiency virus type 2 lacking the V3 loop exhibit resistance to chemokine receptor antagonists

Entry of human immunodeficiency virus type 1 (HIV-1) and HIV-2 requires interactions between the envelope glycoprotein (Env) on the virus and CD4 and a chemokine receptor, either CCR5 or CXCR4, on the cell surface. The V3 loop of the HIV gp120 glycoprotein plays a critical role in this process, determining tropism for CCR5- or CXCR4-expressing cells, but details of how V3 interacts with these receptors have not been defined. Using an iterative process of deletion mutagenesis and in vitro adaptation of infectious viruses, variants of HIV-2 were derived that could replicate without V3, either with or without a deletion of the V1/V2 variable loops. The generation of these functional but markedly minimized Envs required adaptive changes on the gp120 core and gp41 transmembrane glycoprotein. V3-deleted Envs exhibited tropism for both CCR5- and CXCR4-expressing cells, suggesting that domains on the gp120 core were mediating interactions with determinants shared by both coreceptors. Remarkably, HIV-2 Envs with V3 deletions became resistant to small-molecule inhibitors of CCR5 and CXCR4, suggesting that these drugs inhibit wild-type viruses by disrupting a specific V3 interaction with the coreceptor. This study represents a proof of concept that HIV Envs lacking V3 alone or in combination with V1/V2 that retain functional domains required for viral entry can be derived. Such minimized Envs may be useful in understanding Env function, screening for new inhibitors of gp120 core interactions with chemokine receptors, and designing novel immunogens for vaccines.     

Role of V3 independent domains on a dualtropic human immunodeficiency virus type 1 (HIV-1) envelope gp120 in CCR5 coreceptor utilization and viral infectivity

The molecular mechanism of human immunodeficiency virus type 1 (HIV-1) entry into cells involves specific interactions between the viral envelope glycoprotein gp120 and two target cell proteins, CD4 and either CCR5 or CXCR4 chemokine receptors. In order to delineate the functional role of HIV-1 gp120 subdomains of dualtropic strains in CCR5 coreceptor usage, we used a panel of chimeric viruses in which the V1/V2 and V3 domains of gp120 from the dualtropic HIV-1(KMT) isolate were introduced either alone or in combination into the T-tropic HIV-1(NL4-3) background. These chimeric constructs were employed in cell-cell fusion and cell-free virus infectivity assays using cell lines expressing CD4 and the CCR5 chemokine receptor. In both assays, the V3 domain of HIV-1(KMT) but not the V1/V2 domain proved to be the principal determinant of CCR5 coreceptor usage. However, in the cell-free viral infectivity assay although a chimeric virus with a combined V1/V2 and V3 domains of HIV-1(KMT) efficiently fused with coreceptor expressing cells, yet its infectivity was markedly diminished in CCR5 as well as CXCR4 expressing cells. Restoring a comparable level of infection of such chimeric virus required the C3-V5 domain from HIV-1(KMT) to be introduced. Our present findings confirmed that the V3 domain is the major determinant of fusion activity and cellular tropism, and demonstrated a dispensable role for the V1/V2 domain. In addition the C3-V5 domain appeared to play an important role in viral infectivity when the corresponding V1/V2 and V3 domains are present.