Length variation of glycoprotein 120 V2 region in relation to biological phenotypes and coreceptor usage of primary HIV type 1 isolates.

Conflicting data have been published concerning the correlation between the length of the second variable region (V2) in the HIV-1 envelope and the biological phenotype of the virus. Here the V2 region length of primary HIV-1 isolates was compared with biological phenotype and coreceptor usage. The V2 region variation was determined by DNA fragment length analysis, virus biological phenotype by the MT-2 cell assay, and coreceptor usage by infection of U87.CD4 cells expressing CCR3, CCR5, or CXCR4. Ninety-three primary virus isolates from 40 patients were analyzed. This panel of viruses included sequential isolates obtained from patients who progressed to AIDS with or without a virus phenotypic switch. We found that NSI MT-2-negative isolates had significantly shorter V2 regions than SI MT-2-positive isolates. However, when V2 region lengths of viruses were analyzed in more detail, we observed that NSI isolates obtained from patients shortly before the phenotypic switch had V2 region lengths similar to those of SI isolates. V2 regions of NSI isolates obtained from patients who progressed to AIDS without a virus phenotypic switch had, in contrast, shorter V2 region than isolates obtained just before virus phenotypic switch. Coreceptor analysis revealed that CCR5-using (R5) isolates generally had shorter V2 regions than virus isolates with the ability to enter CXCR4-expressing cells. Moreover, no significant difference in V2 region length was observed between monotropic SI isolates, that is, X4 isolates, and multitropic SI isolates, that is, R3R5X4 or R5X4 isolates. Thus, we conclude that R5 NSI isolates obtained from patients with stable virus phenotype through the whole disease course display shorter V2 regions than isolates obtained from patients at switch of virus phenotype, suggesting that V2 region length may influence virus coreceptor usage.     

Structural basis for coreceptor selectivity by the HIV type 1 V3 loop

The third variable region (V3) of the HIV-1 surface glycoprotein, gp120, plays a central role in the interaction of the virus envelope with the cell surface chemokine receptors, triggering membrane fusion and virus entry into human lymphocytes and macrophages. The CXCR4 and CCR5 chemokine receptors are used by "X4-tropic" and "R5-tropic" viruses, respectively. Recently, the crown of the V3 loop was shown to bear a close structural homology to the beta2-beta3 loop in the CXC and CC chemokines, the natural ligands of CXCR4 and CCR5, respectively. This homology can serve as the foundation for 3D molecular modeling of the V3 loops from primary isolates whose coreceptor usage was experimentally defined. The modeling revealed a charged "patch" on the surface of V3 that correlates with coreceptor usage. This V3 surface patch is positively charged in X4-tropic viruses and negatively charged or neutral in R5-tropic viruses, and is formed by two amino acids, at position 11 and at position 24 or 25; amino acids 11 and 24 or 11 and 25 contact each other in 3D space. Residues at positions 11 and 25 were known previously to influence coreceptor usage, and the charge of the residues at these two positions is often used to predict viral tropism. However, we found that the predictive value of using the charge of residues 11, 24, and 25 to identify X4 or R5 tropism was improved over using only the charge of residues 11 and 25. Thus, the data suggest a new " 11/24/25 rule" : a positively charged amino acid at position 11, 24, or 25 defines X4; otherwise R5. This rule gave an overall predictive value of 94% for 217 viruses whose tropism had been determined experimentally as either X4 or R5. The results have additional implications for the design of HIV therapeutics, vaccines, and strategies for monitoring disease progression.     

N-linked glycosylation in the V3 region of HIV type 1 surface antigen modulates coreceptor usage in viral infection.

The V3 hypervariable region of HIV-1 surface protein has been identified as a major determinant for viral tropism and coreceptor usage. However, the role of the highly conserved N-linked glycan at the V3 loop remains controversial. To further examine its role in viral infection, we introduced a conservative amino acid substitution (asparagine to glutamine) in the V3-proximal glycosylation motif (Asn-X-Ser/Thr) in the surface glycoprotein of a CXCR4-using virus (BRU), a CCR5-using virus (SF162), and a dual-tropic virus (89.6). The effect of the mutation was determined by complementation assays, and by infectivity on CEMx174 and U373-MAGI cells expressing either CXCR4 or CCR5. The mutation resulted in decreased CXCR4 usage by SHIV89.6, but increased usage by BRU. Similarly, it abrogated CCR5 usage by SHIV89.6, but had no effect on SF162. This effect was not dependent on the specific amino acid substitution used, because a threonine-toalanine mutation in the same motif in 89.6 Env yielded identical results as the asparagine-to-glutamine mutation. These findings support the notion that multiple factors, including glycosylation at V3, contribute to coreceptor usage and that the particular effects exerted by the N-linked glycan itself appear to be isolate dependent.     

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.     

Protection of neutralization epitopes in the V3 loop of oligomeric human immunodeficiency virus type 1 glycoprotein 120 by N-linked oligosaccharides in the V1 region

The V3 region of the human immunodeficiency virus type 1 envelope protein gp120 constitutes a potential neutralization target, but the oligosaccharide of one conserved N-glycosylation site in this region protects it from neutralizing antibodies. Here, we determined whether N-linked glycans of other gp120 domains were also involved in protection of V3 neutralization epitopes. Two molecular clones of HIV-1, one lacking three N-linked glycans of the V1 region (HIV-1(3N/V1)) and another lacking three N-linked glycans of the C2 region (HIV-1(3N/C2)), were created and characterized. gp120 from both mutated viral clones had higher electrophoretic mobilities than gp120 from wild-type virus, confirming loss of N-linked glycans. Wild-type virus and both mutant clones replicated equally well in established T cell lines and all three viruses were able to utilize CXCR4 but not CCR5 as a coreceptor. The induced mutations increased gp120 affinity for CXCR4 but caused no corresponding increase in viral ability to replicate in T cell lines. HIV-1(3N/V1) was neutralized at about 25 times lower concentrations of an antibody to the V3 region than were wild-type virus and HIV-1(3N/C2). Soluble, monomeric gp120 from HIV-1(3N/V1) and wild type virus had identical avidity for the V3 antibody, indicating that the V1 glycans were able to shield V3 only in oligomeric but not monomeric gp120. In conclusion, one or more N-linked glycans of gp120 V1 is engaged in protection of the V3 region from potential neutralizing antibodies, and this effect is dependent on the oligomeric organization of gp120/gp41.     

HIV type 1 glycoprotein 120 inhibits cardiac myocyte contraction

Cardiomyopathy is a common, life-threatening, but poorly understood complication of HIV infection. The purpose of the present study is to study the effects of an HIV surface envelope protein, glycoprotein 120 (gp120), on cell contraction and L-type Ca(2+) current in rabbit ventricular myocytes. Rabbit ventricular cells were isolated by an enzyme dissociation method. Cell contractions were induced by electric field stimulation. Whole cell L-type Ca(2+) channel currents were measured by the whole cell voltage-clamp technique. We found that perfusion with solution containing gp120 (0.1 microg/ml) derived from HIV-1(SF2) significantly inhibited field-stimulated contractions and L-type Ca(2+) current in rabbit ventricular myocytes as compared with perfusion with buffer alone. These results suggest that HIV-1 gp120 may directly contribute to cardiac dysfunction as seen in many HIV patients.     

Antigenicity and immunogenicity of the V3 domain of HIV type 1 glycoprotein 120 expressed on the surface of Streptococcus gordonii

Five different V3 domains of HIV-1 gp120 were expressed on the surface of the gram-positive bacterium Streptococcus gordonii, a model live vector for vaccine delivery. Sera of HIV-1-infected individuals and human monoclonal antibodies specifically recognized the gp120 sequences on the bacterial surface. Recombinant V3 from the reference HIV-1 strain MN was also shown to retain a conformation that allowed reaction with a conformation-specific monoclonal antibody. A V3-specific serum antibody response was detected in mice immunized both by subcutaneous injection and by vaginal colonization. V3-specific IgG2a antibodies, suggestive of a Th1 response, were found in the sera of mice colonized by recombinant bacteria.     

Studies on the role of the V3 loop in human immunodeficiency virus type 1 envelope glycoprotein function.

Mutations within the principal neutralizing determinant (the V3 loop) of the HIV-1 surface envelope glycoprotein gp120 block or greatly reduce the ability of the HIV-1 envelope glycoprotein to induce cell fusion in CD4+ HeLa T4 cells while keeping its CD4 binding ability. However, when either cysteine or both cysteines forming the V3 disulfide bridge were mutated, the resultant glycoprotein could not mediate cell fusion, undergo proteolytic processing, or bind CD4. To investigate the role that the V3 loop plays in gp160 processing and CD4 binding, we deleted the entire V3 loop region of the HIV-1 env gene. The resultant glycoprotein could not mediate cell fusion in the HeLa T4 cell line and no proteolytic processing of gp160 or CD4 binding could be detected. To test whether any domain of the V3 loop is involved in attaining the proper envelope glycoprotein conformation required for proteolytic processing and CD4 binding, we introduced a series of deletions into the coding region of the V3 loop. Most of the residues within the V3 loop could be removed while retaining gp160 processing and CD4 binding. Our results indicate that the cysteines that form the V3 loop or the disulfide bond itself are important for proper envelope glycoprotein folding and processing. Because many of the mutants constructed in this study do not contain the type-specific neutralizing determinant of HIV-1, they may be potential reagents to bind group-specific neutralizing antibodies or to elicit a group-specific neutralizing response against HIV-1.     

The N-terminal of the V3 loop in HIV type 1 gp120 is responsible for its conformation-dependent interaction with cell surface molecules

The V3 loop of HIV-1 gp120 plays an important role in the interaction of the viral envelope with cellular coreceptors and/or with other cell surface molecules. To clarify this interaction we used a panel of monoclonal antibodies (MAbs) against V3 loop and synthetic looped V3 peptides V3-BH10, V3-ADA, and V3-89.6, derived from the V3 regions of the BH10 clone of IIIB (X4-tropic), ADA (R5-tropic), and 89.6 (R5X4-tropic), respectively. A linear mutant peptide, V3-BH10/CA, was also synthesized as a control. Biotinylated V3-BH10, -BH10/CA, and-ADA were also made. The binding abilities of the biotinylated and nonbiotinylated peptides to various types of cells were investigated by using flow cytometry. Subsequently, the principal region of the V3 loop involved in cell surface binding was analyzed by using MAbs against the tip (447-52D and 694-98D), N-termini (IIIB-V3-21) or C-termini (IIIB-V3-01) of the V3 loop in flow cytometry and enzyme-linked immunoabsorbent assay. We demonstrate that looped V3 peptides of both X4 and R5X4 HIV (V3-BH10 and V3-89.6) can bind to various types of cells irrespective of their CD4 and/or coreceptor expression in a conformation-dependent manner. In contrast, the V3 loop of R5 HIV (V3-ADA) can scarcely bind to the cells. Using MAbs whose epitopes cover the entire V3 loop we found that MAb IIIB-V3-21 can react with platebound but not cell-bound peptides, and the MAb blocked biotin-V3-BH10 binding suggesting that the N-terminal of the V3 loop interacts directly with cell surface molecule(s).     

Genotypic analysis of human immunodeficiency virus type 1 env V3 loop sequences: bioinformatics prediction of coreceptor usage among 28 infected mother-infant pairs in a drug-naive population

We sought to predict virus coreceptor utilization using a simple bioinformatics method based on genotypic analysis of human immunodeficiency virus types 1 (HIV-1) env V3 loop sequences of 28 infected but drug-naive women during pregnancy and their infected infants and to better understand coreceptor usage in vertical transmission dynamics. The HIV-1 env V3 loop was sequenced from plasma samples and analyzed for viral coreceptor usage and subtype in a cohort of HIV-1-infected pregnant women. Predicted maternal frequencies of the X4, R5X4, and R5 genotypes were 7%, 11%, and 82%, respectively. Antenatal plasma viral load was higher, with a mean log(10) (SD) of 4.8 (1.6) and 3.6 (1.2) for women with the X4 and R5 genotypes, respectively, p?=?0.078. Amino acid substitution from the conserved V3 loop crown motif GPGQ to GPGR and lymphadenopathy were associated with the X4 genotype, p?=?0.031 and 0.043, respectively. The maternal viral coreceptor genotype was generally preserved in vertical transmission and was predictive of the newborn's viral genotype. Infants born to mothers with X4 genotypes were more likely to have lower birth weights relative to those born to mothers with the R5 genotype, with a mean weight (SD) of 2870 (±332) and 3069 (±300) g, respectively. These data show that at least in HIV-1 subtype C, R5 coreceptor usage is the most predominant genotype, which is generally preserved following vertical transmission and is associated with the V3 GPGQ crown motif. Therefore, antiretroviral-naive pregnant women and their infants can benefit from ARV combination therapies that include R5 entry inhibitors following prediction of their coreceptor genotype using simple bioinformatics methods.     

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.     

Baseline HIV type 1 coreceptor tropism predicts disease progression.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) coreceptor tropism, the ability of the virus to enter cells via CCR5 or CXCR4, is a viral characteristic mediated by the envelope gene. The impact of coreceptor tropism on the natural history of HIV-1 infection has not been fully explored. METHODS: Coreceptor tropism was measured using a recombinant virus single-cycle assay on plasma specimens obtained at baseline from 126 children and adolescents in the Hemophilia Growth and Development Study cohort who were enrolled from 1989 through 1990 and underwent follow-up through 1997. RESULTS: Detectable CXCR4-using virus at baseline was associated with a lower baseline CD4(+) T cell count and a higher plasma HIV-1 RNA level. In addition, it independently predicted a greater decrease in CD4(+) T cell count over time (P<.001) and was associated with a 3.8-fold increased risk of progression to clinical AIDS. CONCLUSIONS: This study demonstrates that coreceptor tropism, as assessed by this single-cycle assay, independently influences the natural history of HIV-1 disease.     

The ability of HIV type 1 to use CCR-3 as a coreceptor is controlled by envelope V1/V2 sequences acting in conjunction with a CCR-5 tropic V3 loop

Although infection by primary HIV type 1 (HIV-1) isolates normally requires the functional interaction of the viral envelope protein with both CD4 and the CCR-5 coreceptor, a subset of such isolates also are able to use the distinct CCR-3 receptor. By analyzing the ability of a series of wild-type and chimeric HIV-1 envelope proteins to mediate CCR-3-dependent infection, we have determined that CCR-3 tropism maps to the V1 and V2 variable region of envelope. Although substitution of the V1/V2 region of a CCR-3 tropic envelope into the context of a CCR-5 tropic envelope is both necessary and sufficient to confer CCR-3 tropism, this same substitution has no phenotypic effect when inserted into a CXCR-4 tropic HIV-1 envelope context. However, this latter chimera acquires both CCR-3 and CCR-5 tropism when a CCR-5 tropic V3 loop sequence also is introduced. These data demonstrate that the V1/2 region of envelope can, like the V3 loop region, encode a particular coreceptor requirement and suggest that a functional envelope:CCR-3 interaction may depend on the cooperative interaction of CCR-3 with both the V1/V2 and the V3 region of envelope.     

Hypervariable region 3 residues of HIV type 1 gp120 involved in CCR5 coreceptor utilization: therapeutic and prophylactic implications

Crystallographic characterization of a ternary complex containing a monomeric gp120 core, parts of CD4, and a mAb, revealed a region that bridges the inner and outer domains of gp120. In a related genetic study, several residues conserved among primate lentiviruses were found to play important roles in CC-chemokine receptor 5 (CCR5) coreceptor utilization, and all but one were mapped to the bridging domain. To reconcile this finding with previous reports that the hypervariable region 3 (V3) of gp120 plays an important role in chemokine coreceptor utilization, elucidating the roles of various V3 residues in this critical part of the HIV type 1 (HIV-1) life cycle is essential. Alanine-scanning mutagenesis was carried out to identify V3 residues critical for CCR5 utilization. Our findings demonstrated that several residues in V3 were critical to CCR5 utilization. Furthermore, these residues included not only those conserved across HIV-1 subtypes, but also those that varied among HIV-1 subtypes. Although the highly conserved V3 residues may represent unique targets for antiviral designs, the involvement of variable residues raises the possibility that antigenic variation in the coreceptor binding domain could further complicate HIV-1 vaccine design.     

Salivary agglutinin inhibits HIV type 1 infectivity through interaction with viral glycoprotein 120

Salivary agglutinin (SAG) is a high molecular mass glycoprotein (340 kDa) that plays important roles in innate immunity. SAG has been found to specifically inhibit HIV-1 infectivity and to bind to virus through the envelope protein gp120. Although SAG binds to gp120 of the virus, the exact nature of this binding has not been characterized. Using surface plasmon resonance technology, we have found that SAG interacts with recombinant envelopes derived from diverse HIV-1 isolates with K(D) values ranging from 10(-7) to 10(-10) M, comparable to gp120-sCD4 binding. Furthermore, SAG binding to gp120 is Ca(2+) dependent. sCD4 prebound to gp120 failed to abrogate SAG binding, suggesting a distinct mechanism for SAG inhibition of HIV-1 infectivity. Inhibition by monoclonal antibodies specific for carbohydrates also implicates the involvement of carbohydrates in the interaction between SAG and gp120. These results argue that the anti-HIV-1 activity of SAG is due to carbohydrate-mediated binding to gp120. A demonstration that SAG is related to lung scavenger receptor, gp-340, further suggests the roles of SAG in preventing pathogen invasion at the entry portal and raises its potential as an anti-HIV-1 drug candidate.