CCR5 Utilization Research Articles

P-D4 Tyrosine-sulfated peptides from the gp120 V2 domain block HIV-1 entry through CCR5 Mimicry

We recently identified 2 sulfated tyrosines, Tys173 and Tys177, in the second variable (V2) region of HIV-1 gp120 and shown that they play a role in stabilizing the pre-fusion conformation of the envelope trimer. The sulfated region of V2 has a high similarity with the N-terminal domain of CCR5, which also contains sulfated tyrosines and interacts with V3-loop base of gp120, suggesting a potential intramolecular interaction mode between V2 and V3. Here, we employed a tyrosine- sulfated V2-derived peptide, pV2alpha-Tys, to investigate binding to gp120 and its functional consequences. A direct interaction was observed between peptide pV2alpha-Tys and the base of V3, with Tys177 playing a dominant role. Surface plasmon resonance showed that binding of the pV2alpha-Tys is enhanced by CD4-induced conformational changes in gp120, and the effect is even more significant with a soluble trimer. Functionally, pV2alpha-Tys inhibits HIV-1 entry and fusion by preventing CCR5 utilization, with a potency comparable to that of a sulfated CCR5 N-terminal peptide, pCCR5-Tys. These studies characterize the structural and functional features of the V2–V3 intramolecular interaction, providing new leads for the design of therapeutic and vaccine strategies for the control of HIV/AIDS.

CCR5 Conformations Are Dynamic and Modulated by Localization, Trafficking and G Protein Association

CCR5 acts as the principal coreceptor during HIV-1 transmission and early stages of infection. Efficient HIV-1 entry requires a series of processes, many dependent on the conformational state of both viral envelope protein and cellular receptor. Monoclonal antibodies (MAbs) are able to identify different CCR5 conformations, allowing for their use as probes to distinguish CCR5 populations. Not all CCR5 MAbs are able to reduce HIV-1 infection, suggesting the use of select CCR5 populations for entry. In the U87.CD4.CCR5-GFP cell line, we used such HIV-1-restricting MAbs to probe the relation between localization, trafficking and G protein association for individual CCR5 conformations. We find that CCR5 conformations not only exhibit different localization and abundance patterns throughout the cell, but that they also display distinct sensitivities to endocytosis inhibition. Using chemokine analogs that vary in their HIV-1 inhibitory mechanisms, we also illustrate that responses to ligand engagement are conformation-specific. Additionally, we provide supporting evidence for the select sensitivity of conformations to G protein association. Characterizing the link between the function and dynamics of CCR5 populations has implications for understanding their selective targeting by HIV-1 and for the development of inhibitors that will block CCR5 utilization by the virus.

Characterization of a dual-tropic Human immunodeficiency virus (HIV-1) strain derived from the prototypical X4 isolate HXBc2

Human immunodeficiency virus type 1 (HIV-1) coreceptor usage and tropism can be modulated by the V3 loop sequence of the gp120 exterior envelope glycoprotein. For coreceptors, R5 viruses use CCR5, X4 viruses use CXCR4, and dual-tropic (R5X4) viruses use either CCR5 or CXCR4. To understand the requirements for dual tropism, we derived and analyzed a dual-tropic variant of an X4 virus. Changes in the V3 base, which allow gp120 to interact with the tyrosine-sulfated CCR5 N-terminus, and deletion of residues 310/311 in the V3 tip were necessary for efficient CCR5 binding and utilization. Thus, both sets of V3 changes allowed CCR5 utilization with retention of the ability to use CXCR4. We also found that the stable association of gp120 with the trimeric envelope glycoprotein complex in R5X4 viruses, as in X4 viruses, is less sensitive to V3 loop changes than gp120-trimer association in R5 viruses.

Comparison of Viral Env Proteins from Acute and Chronic Infections with Subtype C Human Immunodeficiency Virus Type 1 Identifies Differences in Glycosylation and CCR5 Utilization and Suggests a New Strategy for Immunogen Design

Comparison of Viral Env Proteins from Acute and Chronic Infections with Subtype C Human Immunodeficiency Virus Type 1 Identifies Differences in Glycosylation and CCR5 Utilization and Suggests a New Strategy for Immunogen Design

The Puzzling Role of CXCR4 in Human Immunodeficiency Virus Infection

The human immunodeficiency virus type-1 (HIV-1) is the etiological agent of the acquired immunodeficiency syndrome (AIDS), a disease highly lethal in the absence of combination antiretroviral therapy. HIV infects CD4+ cells of the immune system (T cells, monocyte-macrophages and dendritic cells) via interaction with a universal primary receptor, the CD4 molecule, followed by a mandatory interaction with a second receptor (co-receptor) belonging to the chemokine receptor family. Apart from some rare cases, two chemokine receptors have been evolutionarily selected to accomplish this need for HIV-1: CCR5 and CXCR4. Yet, usage of these two receptors appears to be neither casual nor simply explained by their levels of cell surface expression. While CCR5 use is the universal rule at the start of every infection regardless of the transmission route (blood-related, sexual or mother to child), CXCR4 utilization emerges later in disease coinciding with the immunological deficient phase of infection. Moreover, in most instances CXCR4 use as viral entry co-receptor is associated with maintenance of CCR5 use. Since antiviral agents preventing CCR5 utilization by the virus are already in use, while others targeting either CCR5 or CXCR4 (or both) are under investigation, understanding the biological correlates of this “asymmetrical” utilization of HIV entry co-receptors bears relevance for the clinical choice of which therapeutics should be administered to infected individuals. We will here summarize the basic knowledge and the hypotheses underlying the puzzling and yet unequivocal role of CXCR4 in HIV-1 infection.

Efficiency of CCR5 coreceptor utilization by the HIV quasispecies increases over time, but is not associated with disease progression.

CCR5 is the primary coreceptor for HIV entry. Early after infection, the HIV viral population diversifies rapidly into a quasispecies. It is not known whether the initial efficiency of the viral quasispecies to utilize CCR5 is associated with HIV disease progression or if it changes in an infected individual over time. The CCR5 and CXCR4 utilization efficiencies (R5-UE and X4-UE) of the HIV quasispecies were examined using a pseudovirus, single-round infection assay for samples obtained from known seroconverters from Rakai district, Uganda (n=88). Initial and longitudinal R5-UE values were examined to assess the association of R5-UE with HIV disease progression using multivariate Cox proportional hazard models. Longitudinal samples were analyzed for 35 seroconverters who had samples available from multiple time points. There was no association between initial or longitudinal changes in R5-UE and the hazard of HIV disease progression (p=0.225 and p=0.942, respectively). In addition, R5-UE increased significantly over time after HIV seroconversion (p<0.001), regardless of HIV subtype or the emergence of CXCR4-tropic virus. These data demonstrate that the R5-UE of the viral quasispecies early in HIV infection is not associated with disease progression, and that R5-UE levels increase in HIV-infected individuals over time.

Phenotypic and Immunologic Comparison of Clade B Transmitted/Founder and Chronic HIV-1 Envelope Glycoproteins

Sexual transmission of human immunodeficiency virus type 1 (HIV-1) across mucosal barriers is responsible for the vast majority of new infections. This relatively inefficient process results in the transmission of a single transmitted/founder (T/F) virus, from a diverse viral swarm in the donor, in approximately 80% of cases. Here we compared the biological activities of 24 clade B T/F envelopes (Envs) with those from 17 chronic controls to determine whether the genetic bottleneck that occurs during transmission is linked to a particular Env phenotype. To maximize the likelihood of an intact mucosal barrier in the recipients and to enhance the sensitivity of detecting phenotypic differences, only T/F Envs from individuals infected with a single T/F variant were selected. Using pseudotyping to assess Env function in single-round infectivity assays, we compared coreceptor tropism, CCR5 utilization efficiencies, primary CD4(+) T cell subset tropism, dendritic cell trans-infections, fusion kinetics, and neutralization sensitivities. T/F and chronic Envs were phenotypically equivalent in most assays; however, T/F Envs were modestly more sensitive to CD4 binding site antibodies b12 and VRC01, as well as pooled human HIV Ig. This finding was independently validated with a panel of 14 additional chronic HIV-1 Env controls. Moreover, the enhanced neutralization sensitivity was associated with more efficient binding of b12 and VRC01 to T/F Env trimers. These data suggest that there are subtle but significant structural differences between T/F and chronic clade B Envs that may have implications for HIV-1 transmission and the design of effective vaccines.

Heterosexual Transmission of Human Immunodeficiency Virus Type 1 Subtype C: Macrophage Tropism, Alternative Coreceptor Use, and the Molecular Anatomy of CCR5 Utilization

Human immunodeficiency virus type 1 transmission selects for virus variants with genetic characteristics distinct from those of donor quasispecies, but the biological factors favoring their transmission or establishment in new hosts are poorly understood. We compared primary target cell tropisms and entry coreceptor utilizations of donor and recipient subtype C Envs obtained near the time of acute infection from Zambian heterosexual transmission pairs. Both donor and recipient Envs demonstrated only modest macrophage tropism, and there was no overall difference between groups in macrophage or CD4 T-cell infection efficiency. Several individual pairs showed donor/recipient differences in primary cell infection, but these were not consistent between pairs. Envs had surprisingly broad uses of GPR15, CXCR6, and APJ, but little or no use of CCR2b, CCR3, CCR8, GPR1, and CXCR4. Donors overall used GPR15 better than did recipients. However, while several individual pairs showed donor/recipient differences for GPR15 and/or other coreceptors, the direction of the differences was inconsistent, and several pairs had unique alternative coreceptor patterns that were conserved across the transmission barrier. CCR5/CCR2b chimeras revealed that recipients as a group were more sensitive than were donors to replacement of the CCR5 extracellular loops with corresponding regions of CCR2b, but significant differences in this direction were not consistent within pairs. These data show that sexual transmission does not select for enhanced macrophage tropism, nor for preferential use of any alternative coreceptor. Recipient Envs are somewhat more constrained than are donors in flexibility of CCR5 use, but this pattern is not universal for all pairs, indicating that it is not an absolute requirement.

Conserved Changes in Envelope Function during Human Immunodeficiency Virus Type 1 Coreceptor Switching

We studied the evolution of human immunodeficiency virus type 1 (HIV-1) envelope function during the process of coreceptor switching from CCR5 to CXCR4. Site-directed mutagenesis was used to introduce most of the possible intermediate mutations in the envelope for four distinct coreceptor switch mutants, each with a unique pattern of CCR5 and CXCR4 utilization that extended from highly efficient use of both coreceptors to sole use of CXCR4. Mutated envelopes with some preservation of entry function on either CCR5- or CXCR4-expressing target cells were further characterized for their sensitivity to CCR5 or CXCR4 inhibitors, soluble CD4, and the neutralizing antibodies b12-IgG and 4E10. A subset of mutated envelopes was also studied in direct CD4 or CCR5 binding assays and in envelope-mediated fusion reactions. Coreceptor switch intermediates displayed increased sensitivity to CCR5 inhibitors (except for a few envelopes with mutations in V2 or C2) that correlated with a loss in CCR5 binding. As use of CXCR4 improved, infection mediated by the mutated envelopes became more resistant to soluble CD4 inhibition and direct binding to CD4 increased. These changes were accompanied by increasing resistance to the CXCR4 inhibitor AMD3100. Sensitivity to neutralizing antibody was more variable, although infection of CXCR4-expressing targets was generally more sensitive to neutralization by both b12-IgG and 4E10 than infection of CCR5-expressing target cells. These changes in envelope function were uniform in all four series of envelope mutations and thus were independent of the final use of CCR5 and CXCR4. Decreased CCR5 and increased CD4 binding appear to be common features of coreceptor switch intermediates.

Mutations in the V3 stem versus the V3 crown and C4 region have different effects on the binding and fusion steps of human immunodeficiency virus type 1 gp120 interaction with the CCR5 coreceptor

The current model for HIV-1 envelope–coreceptor interaction depicts the V3 stem and bridging sheet binding to the CCR5 N-terminus while the V3 crown interacts with the second extracellular loop, which is the coreceptor domain that appears to be relatively more important for fusion and infection. Our prediction based on this model is that mutations in the V3 crown might consequently have more effects on cell–cell fusion and virus entry than mutations introduced in the V3 stem and C4 region. We performed alanine-scanning of the V3 loop and selected C4 residues in the JRFL envelope and tested the capacity of the resulting mutants for CCR5 binding, cell–cell fusion, and virus infection. Our cross comparison analysis revealed that residues in C4 and in both the V3 stem and crown were important for CCR5 binding of gp120 subunits. Contrary to our prediction, mutations in the V3 crown had less effect on membrane fusion than mutations in the V3 stem. The V3 stem thus appears to be the most important region for CCR5 utilization since it affected both coreceptor binding and subsequent fusion and viral entry. Our data raises the possibility that some residues in the V3 crown and in C4 may play distinct roles in the binding and fusion steps of envelope–coreceptor interaction.

Restricted Variable Residues in the C-terminal Segment of HIV-1 V3 Loop Regulate the Molecular Anatomy of CCR5 Utilization

The V3 loop of the HIV-1 envelope glycoprotein (Env) is the major determinant for coreceptor utilization, but the structural basis for this specificity remains to be defined. By characterizing a set of naturally occurring R5 Env variants, we demonstrate that Asp324 in the conserved IIGDIR motif of the V3 loop (CTRPN(300)NNTRKSIHIGP(311)GRAFYTTGEIIGD(324)IRQAHC) C-terminal segment regulates the molecular anatomy of CCR5 utilization. Whereas gp120 subunits with Asp or Asn at position 324 were fusogenic with coreceptor chimeras containing either the N-terminal domain or the body of CCR5, substitution of charged (Glu, Lys) or small hydrophobic (Gly, Ala) residues resulted in complete loss of fusogenic activity with the N terminus and markedly reduced utilization of the body of CCR5, although their ability to use wild-type CCR5 was unchanged. This phenotypic conversion was confirmed in both gain and loss of function experiments using Env from multiple subtypes. Alignment of sequences of R5 V3 loops (n=599) from the HIV database revealed that the mutation of Asp324 in the conserved IIGDIR motif is restricted to Asn324, with proportions of 71.5% and 28%, respectively. Infection of primary CD4(+)T cells demonstrated that Env bearing Asp324 was less sensitive to RANTES, suggesting that Asp or Asn in this position may be crucial for viral fitness. The CD4-dependent gp120 binding to CCR5 was decreased when Asp324 was replaced with a charged or hydrophobic residue, but unchanged when replaced with Asn. Molecular modeling analyses predicted that Asp/Asn324 forms a critical H-bond with Asn300. These findings indicate that Asp or Asn at position 324 of the V3 stem stabilizes the conformation of V3 loop and hence influences the intensities of interaction between CD4-activated gp120 and CCR5 which results in viral entry.

Effect of amino acid substitution of the V3 and bridging sheet residues in human immunodeficiency virus type 1 subtype C gp120 on CCR5 utilization.

The V3 loop and the bridging sheet domain of human immunodeficiency virus type 1 (HIV-1) subtype B envelope glycoprotein gp120 have been implicated in CCR5 coreceptor utilization. In this study, mutant envelope glycoproteins of a subtype C isolate containing substitutions in the V3 or C4 region were generated to determine which are required for efficient CCR5-dependent cell fusion and viral entry. We found that the V3 crown and C4 residues are relatively dispensable for cell-cell fusion, although some residues may be involved in the regulation of early postentry steps in viral replication. In contrast, seven highly conserved residues located in the V3 stem are critical for CCR5 utilization, which can explain the apparent paradox that the functional convergence in CCR5 usage by genetically divergent HIV-1 strains involves a variable region. The finding that C4 residues do not have a critical role may appear to contradict the current model that bridging sheet residues are involved in the gp120-CCR5 interaction. However, a plausible interpretation is that these C4 residues may have a distinct role in the binding and fusion steps of the gp120-CCR5 interaction.

N-Linked Glycosylation of the HIV Type-1 gp120 Envelope Glycoprotein as a Major Determinant of CCR5 and CXCR4 Coreceptor Utilization

The variable V1V2 and V3 regions of the human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein (gp120) can influence viral coreceptor usage. To substantiate this we generated isogenic HIV-1 molecularly cloned viruses that were composed of the HxB2 envelope backbone containing the V1V2 and V3 regions from viruses isolated from a patient progressing to disease. We show that the V3 amino acid charge per se had little influence on altering the virus coreceptor phenotype. The V1V2 region and its N-linked glycosylation degree were shown to confer CXCR4 usage and provide the virus with rapid replication kinetics. Loss of an N-linked glycosylation site within the V3 region had a major influence on the virus switching from the R5 to X4 phenotype in a V3 charge-dependent manner. The loss of this V3 N-linked glycosylation site was also linked with the broadening of the coreceptor repertoire to incorporate CCR3. By comparing the amino acid sequences of primary HIV-1 isolates, we identified a strong association between high V3 charge and the loss of this V3 N-linked glycosylation site. These results demonstrate that the N-linked glycosylation pattern of the HIV-1 envelope can strongly influence viral coreceptor utilization and the R5 to X4 switch.

Interaction between HIV type 1 glycoprotein 120 and CXCR4 coreceptor involves a highly conserved arginine residue in hypervariable region 3.

Several seven-transmembrane chemokine receptors are known to function as entry coreceptors for human immunodeficiency virus type 1. CCR5 and CXCR4 are the major coreceptors for non-syncytium-inducing (NSI) and syncytium-inducing (SI) viruses, respectively. During the natural course of infection, the emergence of variants with a phenotypic transition from NSI to SI and rapid disease progression is associated with expanded coreceptor usage to CXCR4. Characteristic amino acids at several positions in the hypervariable region 3 (V3) of gp120 have been linked to CXCR4 utilization. Previously, we reported that a highly conserved arginine residue of V3 played an important role in CCR5 utilization. In this study, the possible involvement of the same arginine residue in CXCR4 utilization was investigated. Amino acid substitutions introduced to this arginine on R5X4 viruses were found to have a significant effect on their utilization of CXCR4. These results, taken together with those reported previously, suggest that this highly conserved arginine may contribute to the functional convergence of chemokine coreceptor utilization by human immunodeficiency viruses and may represent a unique target for future antiviral design.

Identification of ENV determinants in V3 that influence the molecular anatomy of CCR5 utilization

The V3 loop of the ENV glycoprotein exerts a dominant influence on the interaction of gp120 with coreceptors. Primary env genes cloned from sequential isolates from two seroconverters revealed Pro→Ala conversion in the conserved GPG motif of the V3 crown in seven of 17 R5 ENV. ENV containing the GPG motif in the V3 crown had fusogenic activity with chimeric receptors containing either the N terminus or loops of CCR5, whereas those with the GAG variant utilized only the former. Site-directed mutagenesis of multiple primary and prototypic R5 env genes demonstrated that the GPG motif was necessary for dual utilization of the N terminus and body of CCR5 in both gain and loss-of-function experiments. All ENV containing the GPG V3 crown showed CCR5 binding in the presence of soluble CD4, whereas it was not detected with the GAG variants. Molecular dynamic simulations of a V3 peptide predicts that the Pro→Ala substitution results in a conformational change with loss of the crown structure. These studies demonstrate that sequences in the third hypervariable region determine the specificity of coreceptor utilization for fusion, and that a conserved motif in the crown directly influences the molecular anatomy of the interaction between gp120 and CCR5.

Co-receptor usage was more predictive than NSI/SI phenotype for HIV replication in macrophages: is NSI/SI phenotyping sufficient?

A monocyte-derived macrophage (MDM) culture assay was used to define the replication kinetics of HIV isolates. Ten-day-old MDMs were infected with HIV. Supernatants were collected and assayed for HIV p24 on days 3, 7, 10, and 14 post-infection (PI). In this assay, SF162 (macrophage tropic, NSI) produced increasing amounts of HIV p24 antigen with increasing time in culture. BRU (nonmacrophage tropic, SI) infection resulted in low levels of HIV p24 antigen with no increase in production during the culture period. A panel of 12 clinical isolates was evaluated. All isolates produced detectable levels of HIV p24 antigen in MDMs. However, the NSI viruses had significantly higher log10 HIV p24 antigen values at all times PI (P < 0.01). Co-receptor usage was determined for all 12 isolates (8 NSI and 4 SI). All SI isolates used CXCR4 for entry; two used CXCR4 only, one used CXCR4, CCR5, and CCR3, and one was a mixture of two isolates using CXCR4 and CCR5. None of the NSI viruses used CXCR4 for entry. All used CCR5 as their predominant co-receptor. Of the eight NSI isolates, three used CCR5 only, two used CCR5 and CCR2b, one used CCR5 and CCR3, and one used CCR5, CCR3, and CCR2b. Log10 HIV p24 antigen production on day 14 PI for viruses that used CCR5+CCR3 (3.79 + 1.40) was greater than for viruses that used CCR5+CCR2b (3.22 + 1.55) or CCR5 (3.32 + 1.49), and all were greater than those that used CXCR4 only (1.69 + 0.28), regardless of SI phenotype (P < 0.05). Thus, in these primary isolates, macrophage tropism and replication kinetics were closely linked to CCR5 utilization, whereas SI capacity was closely linked to CXCR4 utilization. Furthermore, viruses, which could use CCR5 and CCR3 for entry, had a replication advantage in macrophages, regardless of SI phenotype.

Persistent CCR5 utilization and enhanced macrophage tropism by primary blood human immunodeficiency virus type 1 isolates from advanced stages of disease and comparison to tissue-derived isolates.

Viral phenotype, tropism, coreceptor usage, and envelope gene diversity were examined in blood isolates collected from 27 individuals at different stages of human immunodeficiency virus type 1 (HIV-1) disease and tissue derived isolates from 10 individuals with AIDS. The majority (89%) of blood and all tissue HIV-1 isolates from all stages of infection were non-syncytium inducing and macrophage (M) tropic. Tropism and productive infection by HIV isolates in both monocytes and monocyte-derived macrophages (MDM) increased in advanced disease (HIV tropism for monocytes, 1 of 6 from categories I and II versus 11 of 21 [P = 0.05] from category IV and II [CD4 < 250]; and high-level replication in MDM, 1 of 6 from categories I and II versus 16 of 21 from categories IV and II [P = 0. 015]). There was a high level of replication of blood and tissue isolates in T lymphocytes without restriction at any stage. Overall, the level of replication in MDM was 5- to 10-fold greater than in monocytes, with restriction in the latter occurring mainly at entry and later stages of replication. Only three blood isolates were identified as syncytium inducing, and all had a dualtropic phenotype. There was a significant increase of HIV envelope gene diversity, as shown by a heteroduplex mobility assay, in advanced disease; this may partly underlie the increase of HIV replication in MDM. Unlike blood isolates (even those from patients with advanced disease), tissue isolates displayed greater similarities (90%) in productive infection between MDM and monocytes. The majority (87%) of all isolates, including those from patients with advanced disease, used CCR5, and only 5 of 37 isolates showed expanded coreceptor usage. These results indicate that in the late stage of disease with increasing viral load and diversity, CCR5 utilization and M-tropism persist in blood and tissue and the replicative ability in macrophages increases. This suggests that these characteristics are advantageous to HIV and are important to disease progression.

Relationship between Productive HIV-1 Infection of Macrophages and CCR5 Utilization

HIV-1 isolates exhibit specificity for infection of immortalized T-cell lines and macrophages. The distinct cellular tropisms have been attributed to expression of coreceptors CXCR4 or CCR5, respectively. However, it is unclear whether or not other tissue-specific determinants regulate entry. The current study uses a panel of viruses to analyze the relationship between CCR5 utilization and macrophage infection. Only chimeric viruses with the entire V3 loop from macrophage-tropic isolates, ADA or SF162, were able to infect macrophages. In contrast, chimeric viruses with smaller portions of the ADA V3 loop or the V3 loop of SF2, sufficient to allow CCR5 use, were insufficient for macrophage infection. PCR analysis showed that the defect in macrophage infection of the latter viruses was due to a defect in entry. Moreover, strains capable of infecting macrophages showed relative resistance to neutralization by anti-CCR5 antibody, 2D7, compared to strains which utilize CCR5 but are incapable of macrophage infection.

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.

CCR5 coreceptor utilization involves a highly conserved arginine residue of HIV type 1 gp120.

The seven-transmembrane CCR5 was recently found to double as a coreceptor for a genetically diverse family of human and nonhuman primate lentiviruses. Paradoxically, the main region of the envelope protein believed to be involved in CCR5 utilization was mapped to hypervariable region 3, or V3, of the envelope glycoprotein gp120. In this study, we addressed the question of whether functional convergence in CCR5 utilization is mediated by certain V3 residues that are highly conserved among HIV type 1 (HIV-1), HIV type 2, and simian immunodeficiency virus. Site-directed mutagenesis carried out on three such V3 residues revealed that the Arg-298 of HIV-1 gp120 has an important role in CCR5 utilization. In contrast, no effect was observed for the other residues we tested. The inability of Arg-298 mutants to use CCR5 was not attributed to global alteration of gp120 conformation. Neither the expression, processing, and incorporation of mutant envelope proteins into virions, nor CD4 binding were significantly affected by the mutations. This interpretation is further supported by the finding that alanine substitutions of five residues immediately adjacent to the arginine residue had no effect on CCR5 utilization. Taken together, our data strongly suggests that the highly conserved Arg-298 residue identified in the V3 of HIV-1 has a significant role in CCR5 utilization, and may represent an unusually conserved target for future anti-viral designs.