Intrapatient alterations in the human immunodeficiency virus type 1 gp120 V1V2 and V3 regions differentially modulate coreceptor usage, virus inhibition by CC/CXC chemokines, soluble CD4, and the b12 and 2G12 monoclonal antibodies

We studied human immunodeficiency virus type 1 (HIV-1) chimeric viruses altering in their gp120 V1V2 and V3 envelope regions to better map which genetic alterations are associated with specific virus phenotypes associated with HIV-1 disease progression. The V1V2 and V3 regions studied were based on viruses isolated from an individual with progressing HIV-1 disease. Higher V3 charges were linked with CXCR4 usage, but only when considered within a specific V1V2 and V3 N-linked glycosylation context. When the virus gained R5X4 dual tropism, irrespective of its V3 charge, it became highly resistant to inhibition by RANTES and highly sensitive to inhibition by SDF-1alpha. R5 viruses with higher positive V3 charges were more sensitive to inhibition by RANTES, while R5X4 dualtropic viruses with higher positive V3 charges were more resistant to inhibition by SDF-1alpha. Loss of the V3 N-linked glycosylation event rendered the virus more resistant to inhibition by SDF-1alpha. The same alterations in the V1V2 and V3 regions influenced the extent to which the viruses were neutralized with soluble CD4, as well as monoclonal antibodies b12 and 2G12, but not monoclonal antibody 2F5. These results further identify a complex set of alterations within the V1V2 and V3 regions of HIV-1 that can be selected in the host via alterations of coreceptor usage, CC/CXC chemokine inhibition, CD4 binding, and antibody neutralization.     

Variants of human immunodeficiency virus type 1 that efficiently use CCR5 lacking the tyrosine-sulfated amino terminus have adaptive mutations in gp120, including loss of a functional N-glycan

By selecting the R5 human immunodeficiency virus type 1 (HIV-1) strain JR-CSF for efficient use of a CCR5 coreceptor with a badly damaged amino terminus [i.e., CCR5(Y14N)], we previously isolated variants that weakly utilize CCR5(Delta18), a low-affinity mutant lacking the normal tyrosine sulfate-containing amino-terminal region of the coreceptor. These previously isolated HIV-1(JR-CSF) variants contained adaptive mutations situated exclusively in the V3 loop of their gp120 envelope glycoproteins. We now have weaned the virus from all dependency on the CCR5 amino terminus by performing additional selections with HeLa-CD4 cells that express only a low concentration of CCR5(Delta18). The adapted variants had additional mutations in their V3 loops, as well as one in the V2 stem (S193N) and four alternative mutations in the V4 loop that eliminated the same N-linked oligosaccharide from position N403. Assays using pseudotyped viruses suggested that these new gp120 mutations all made strong contributions to use of CCR5(Delta18) by accelerating a rate-limiting CCR5-dependent conformational change in gp41 rather than by increasing viral affinity for this damaged coreceptor. Consistent with this interpretation, loss of the V4 N-glycan at position N403 also enhanced HIV-1 use of a different low-affinity CCR5 coreceptor with a mutation in extracellular loop 2 (ECL2) [i.e., CCR5(G163R)], whereas the double mutant CCR5(Delta18,G163R) was inactive. We conclude that loss of the N-glycan at position N403 helps to convert the HIV-1 envelope into a hair-trigger form that no longer requires strong interactions with both the CCR5 amino terminus and ECL2 but efficiently uses either site alone. These results demonstrate a novel functional role for a gp120 N-linked oligosaccharide and a high degree of adaptability in coreceptor usage by HIV-1.     

Frequent intrapatient recombination between human immunodeficiency virus type 1 R5 and X4 envelopes: implications for coreceptor switch

Emergence of human immunodeficiency virus type 1 (HIV-1) populations that switch or broaden coreceptor usage from CCR5 to CXCR4 is intimately coupled to CD4+ cell depletion and disease progression toward AIDS. To better understand the molecular mechanisms involved in the coreceptor switch, we determined the nucleotide sequences of 253 V1 to V3 env clones from 27 sequential HIV-1 subtype B isolates from four patients with virus populations that switch coreceptor usage. Coreceptor usage of clones from dualtropic R5X4 isolates was characterized experimentally. Sequence analysis revealed that 9% of the clones from CXCR4-using isolates had originated by recombination events between R5 and X4 viruses. The majority (73%) of the recombinants used CXCR4. Furthermore, coreceptor usage of the recombinants was determined by a small region of the envelope, including V3. This is the first report demonstrating that intrapatient recombination between viruses with distinct coreceptor usage occurs frequently. It has been proposed that X4 viruses are more easily suppressed by the immune system than R5 viruses. We hypothesize that recombination between circulating R5 viruses and X4 viruses can result in chimeric viruses with the potential to both evade the immune system and infect CXCR4-expressing cells. The broadening in cell tropism of the viral population to include CXCR4-expressing cells would gradually impair the immune system and eventually allow the X4 population to expand. In conclusion, intrapatient recombination between viruses with distinct coreceptor usage may contribute to the emergence of X4 viruses in later stages of infection.     

Phenotypic and genotypic comparisons of CCR5- and CXCR4-tropic human immunodeficiency virus type 1 biological clones isolated from subtype C-infected individuals

Individuals infected with human immunodeficiency virus type 1 (HIV-1) subtype C infrequently harbour X4 viruses. We studied R5 and X4 biological clones generated from HIV-1 subtype C-infected individuals. All subtype C R5 viruses demonstrated slower profiles of replication on CD4⁺ lymphocytes in comparison to subtype B viruses, whereas subtype C X4 viruses replicated with comparable efficiency to subtype B X4 viruses. No differences were identified in CC or CXC chemokine inhibitions (RANTES and SDF-1α, respectively) between subtype C and subtype B viruses. Immature dendritic cells were shown in coculture experiments to similarly enhance the infection of subtype C and subtype B R5 as well as X4 viruses. By amino acid sequence analysis, we showed that the R5 and X4 subtype C gp120 envelope gene alterations were similar to those for a switching subtype B virus, specifically with respect to the V3 charge and envelope N-linked glycosylation patterns. By phylogenetic analysis, we showed that one patient was infected with HIV-1 C′ and the other was infected with HIV-1 C" and that one of the patients harbored a virus that was a recombinant in the gp120 env gene between an R5 and an X4 virus, with the resultant virus being R5. No differences were identified between the long terminal repeat regions of the subtype C R5 and X4 biological clones. These results indicate that even though R5 subtype C viruses are restrictive for virus replication, the R5-to-X4 phenotype switch can occur and does so in a manner similar to that of subtype B viruses.     

HIV-1的表型及其感染的细胞嗜性

HIV-1的表型分为合胞体诱导型(syncytium-inducing,SI)和非合胞体诱导型(non-syncytium-inducing,NSI)。依据所用辅助受体和感染靶细胞的不同,HIV-1又被分为R5、X4和R5X4型。R5和X4型病毒分别利用CCR5和CXCR4作为辅助受体,而R5X4型病毒可利用这两种辅助受体。在病毒的复制力、细胞嗜性以及合胞体诱导能力上,SI型与X4型病毒一致,NSI型与R5型病毒一致。在HIV-1感染过程中,疾病的发展伴随着病毒从NSI型向SI型、及R5型向X4型的转变。HIV-1的表型影响和决定着HIV-1的感染、传播及AIDS的疾病进程。HIV-1的表型和细胞嗜性主要由病毒gp120的V3区(特别是第11和25位的氨基酸)决定。V3区的氨基酸序列信息,将为预测HIV-1的表型,以及病毒感染后的疾病进程提供生物信息学的依据。     

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

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

Improved coreceptor usage prediction and genotypic monitoring of R5-to-X4 transition by motif analysis of human immunodeficiency virus type 1 env V3 loop sequences

Early in infection, human immunodeficiency virus type 1 (HIV-1) generally uses the CCR5 chemokine receptor (along with CD4) for cellular entry. In many HIV-1-infected individuals, viral genotypic changes arise that allow the virus to use CXCR4 (either in addition to CCR5 or alone) as an entry coreceptor. This switch has been associated with an acceleration of both CD3(+) T-cell decline and progression to AIDS. While it is well known that the V3 loop of gp120 largely determines coreceptor usage and that positively charged residues in V3 play an important role, the process of genetic change in V3 leading to altered coreceptor usage is not well understood. Further, the methods for biological phenotyping of virus for research or clinical purposes are laborious, depend on sample availability, and present biosafety concerns, so reliable methods for sequence-based "virtual phenotyping" are desirable. We introduce a simple bioinformatic method of scoring V3 amino acid sequences that reliably predicts CXCR4 usage (sensitivity, 84%; specificity, 96%). This score (as determined on the basis of position-specific scoring matrices [PSSM]) can be interpreted as revealing a propensity to use CXCR4 as follows: known R5 viruses had low scores, R5X4 viruses had intermediate scores, and X4 viruses had high scores. Application of the PSSM scoring method to reconstructed virus phylogenies of 11 longitudinally sampled individuals revealed that the development of X4 viruses was generally gradual and involved the accumulation of multiple amino acid changes in V3. We found that X4 viruses were lost in two ways: by the dying off of an established X4 lineage or by mutation back to low-scoring V3 loops.     

Phenotypic studies on recombinant human immunodeficiency virus type 1 (HIV-1) containing CRF01_AE env gene derived from HIV-1-infected patient,residing in central Thailand

Human immunodeficiency virus type 1 (HIV-1) env genes were cloned from blood samples of HIV-1-infected Thai patients, and 35 infectious CRF01_AE envelope glycoprotein (Env)-recombinant viruses were established. In this report, we examined the neutralization susceptibility of these viruses to human monoclonal antibodies, 2G12, IgG1 b12, 2F5 and 4E10, pooled patient plasma, coreceptor antagonists and fusion inhibitor, T-20. The neutralization susceptibility of CRF01_AE Env-recombinant viruses to 2F5, 4E10, patient plasma, coreceptor antagonists and T-20 varied, while most viruses showed low susceptibility to 2G12 and IgG1 b12. Several dual-tropic viruses showed lower susceptibility to 2F5 and 4E10 than CXCR4- or CCR5-tropic viruses. Neutralization susceptibility of the CRF01_AE Env-recombinant virus to pooled patient plasma was negatively correlated with the length of the V1/V2 region or the number of potential N-linked glycosylation sites in conserved regions of gp120. No correlation was found between the coreceptor usage and neutralization susceptibility of the virus to T-20, whereas several dual-tropic viruses showed higher susceptibility to coreceptor antagonists than CXCR4- or CCR5-tropic viruses. We propose that these CRF01_AE Env-recombinant viruses are useful to further study the molecular mechanism of the susceptibility of CRF01_AE Env to neutralizing antibodies and viral entry inhibitors.     

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.     

Isolation of TAK-779-resistant HIV-1 from an R5 HIV-1 GP120 V3 loop library

The human immunodeficiency virus (HIV-1) envelope glycoprotein (GP) 120 interacts with CD4 and the CCR5 coreceptor for viral entry. The V3 loop in GP120 is a crucial region for determining coreceptor usage during viral entry, and a variety of amino acid substitutions has been observed in clinical isolates. To construct an HIV-1 V3 loop library, we chose 10 amino acid positions in the V3 loop and incorporated random combinations (27,648 possibilities) of the amino acid substitutions derived from 31 R5 viruses into the V3 loop of HIV-1(JR-FL) proviral DNA. The constructed HIV-1 library contained 6.6 x 10(6) independent clones containing a set of 0-10 amino acid substitutions in the V3 loop. To address whether restricted steric alteration in the V3 loop could confer resistance to an entry inhibitor, TAK-779, we selected entry inhibitor-resistant HIV-1 by increasing the concentration of TAK-779 from 0.10 to 0.30 microM in PM1-CCR5 cells with high expression of CCR5. The selected viruses at passage 8 contained five amino acid substitutions in the V3 loop without any other mutations in GP120 and showed 15-fold resistance compared with the parental virus. These results indicated that a certain structure of the V3 loop containing amino acid substitutions derived from 31 R5 viruses can contribute to the acquisition of resistance to entry inhibitors binding to CCR5. Taken together, this type of HIV-1 V3 loop library is useful for isolating and analyzing the specific biological features of HIV-1 with respect to alterations of the V3 loop structure.     

Interaction of HIV-1 with dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin-expressing cells is influenced by gp120 envelope modifications associated with disease progression

Dendritic cells can enhance the replication of HIV-1 in CD4(+) lymphocytes through the interaction of the gp120 envelope protein with such molecules as dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin. The variable loops of gp120 have previously been shown to modulate the interaction of HIV-1 with its principal receptor CD4 and its various coreceptors, namely CCR5 and CXCR4. Here, we utilized a panel of molecular cloned viruses to identify whether gp120 modifications can influence the virus interaction with immature dendritic cells or a cell line expressing dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (Raji-DC-SIGN). The viruses encompass the R5, R5X4 and X4 phenotypes, and are based upon V1V2 and V3 sequences from a patient with disease progression. We found that dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin enhancement of virus replication can be modulated by the V1V2 length, the overall V3 charge and N-linked glycosylation patterns; similar results were observed with immature dendritic cells. Viruses with higher V3 charges are more readily transferred to CD4(+) lymphocytes when the V1V2 region is longer and contains an additional N-linked glycosylation site, whereas transfer of viruses with lower V3 charges is greater when the V1V2 region is shorter. Viruses differing in the V1V2 and V3 regions also demonstrated differential capture by Raji-DC-SIGN cells in the presence of mannan. These results indicate that the interaction between HIV-1 and immature dendritic cells via such molecules as dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin may have a role in selecting viruses undergoing transmission and evolution during disease progression.     

Relationships between CD4 independence, neutralization sensitivity, and exposure of a CD4-induced epitope in a human immunodeficiency virus type 1 envelope protein

A CD4-independent version of the X4 human immunodeficiency virus type 1 (HIV-1) HXBc2 envelope (Env) protein, termed 8x, mediates infection of CD4-negative, CXCR4-positive cells, binds directly to CXCR4 in the absence of CD4 due to constitutive exposure of a conserved coreceptor binding site in the gp120 subunit, and is more sensitive to antibody-mediated neutralization. To study the relationships between CD4 independence, neutralization sensitivity, and exposure of CD4-induced epitopes associated with the coreceptor binding site, we generated a large panel of Env mutants and chimeras between 8x and its CD4-dependent parent, HXBc2. We found that a frameshift mutation just proximal to the gp41 cytoplasmic domain in 8x Env was necessary but not sufficient for CD4 independence and led to increased exposure of the coreceptor binding site. In the presence of this altered cytoplasmic domain, single amino acid changes in either the 8x V3 (V320I) or V4/C4 (N386K) regions imparted CD4 independence, with other changes playing a modulatory role. The N386K mutation resulted in loss of an N-linked glycosylation site, but additional mutagenesis showed that it was the presence of a lysine rather than loss of the glycosylation site that contributed to CD4 independence. However, loss of the glycosylation site alone was sufficient to render Env neutralization sensitive, providing additional evidence that carbohydrate structures shield important neutralization determinants. Exposure of the CD4-induced epitope recognized by monoclonal antibody 17b and which overlaps the coreceptor binding site was highly sensitive to an R298K mutation at the base of the V3 loop and was often but not always associated with CD4 independence. Finally, while not all neutralization-sensitive Envs were CD4 independent, all CD4-independent Envs exhibited enhanced sensitivity to neutralization by HIV-1-positive human sera, indicating that the humoral immune response can exert strong selective pressure against the CD4-independent phenotype in vivo. Whether this can be used to advantage in designing more effective immunogens remains to be seen.     

HIV-1 envelope subregion length variation during disease progression

The V3 loop of the HIV-1 Env protein is the primary determinant of viral coreceptor usage, whereas the V1V2 loop region is thought to influence coreceptor binding and participate in shielding of neutralization-sensitive regions of the Env glycoprotein gp120 from antibody responses. The functional properties and antigenicity of V1V2 are influenced by changes in amino acid sequence, sequence length and patterns of N-linked glycosylation. However, how these polymorphisms relate to HIV pathogenesis is not fully understood. We examined 5185 HIV-1 gp120 nucleotide sequence fragments and clinical data from 154 individuals (152 were infected with HIV-1 Subtype B). Sequences were aligned, translated, manually edited and separated into V1V2, C2, V3, C3, V4, C4 and V5 subregions. V1-V5 and subregion lengths were calculated, and potential N-linked glycosylation sites (PNLGS) counted. Loop lengths and PNLGS were examined as a function of time since infection, CD4 count, viral load, and calendar year in cross-sectional and longitudinal analyses. V1V2 length and PNLGS increased significantly through chronic infection before declining in late-stage infection. In cross-sectional analyses, V1V2 length also increased by calendar year between 1984 and 2004 in subjects with early and mid-stage illness. Our observations suggest that there is little selection for loop length at the time of transmission; following infection, HIV-1 adapts to host immune responses through increased V1V2 length and/or addition of carbohydrate moieties at N-linked glycosylation sites. V1V2 shortening during early and late-stage infection may reflect ineffective host immunity. Transmission from donors with chronic illness may have caused the modest increase in V1V2 length observed during the course of the pandemic.     

Cryptic nature of a conserved, CD4-inducible V3 loop neutralization epitope in the native envelope glycoprotein oligomer of CCR5-restricted, but not CXCR4-using, primary human immunodeficiency virus type 1 strains

The external subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env), gp120, contains conserved regions that mediate sequential interactions with two cellular receptor molecules, CD4 and a chemokine receptor, most commonly CCR5 or CXCR4. However, antibody accessibility to such regions is hindered by diverse protective mechanisms, including shielding by variable loops, conformational flexibility and extensive glycosylation. For the conserved neutralization epitopes hitherto described, antibody accessibility is reportedly unrelated to the viral coreceptor usage phenotype. Here, we characterize a novel, conserved gp120 neutralization epitope, recognized by a murine monoclonal antibody (MAb), D19, which is differentially accessible in the native HIV-1 Env according to its coreceptor specificity. The D19 epitope is contained within the third variable (V3) domain of gp120 and is distinct from those recognized by other V3-specific MAbs. To study the reactivity of MAb D19 with the native oligomeric Env, we generated a panel of PM1 cells persistently infected with diverse primary HIV-1 strains. The D19 epitope was conserved in the majority (23/29; 79.3%) of the subtype-B strains tested, as well as in selected strains from other genetic subtypes. Strikingly, in CCR5-restricted (R5) isolates, the D19 epitope was invariably cryptic, although it could be exposed by addition of soluble CD4 (sCD4); epitope masking was dependent on the native oligomeric structure of Env, since it was not observed with the corresponding monomeric gp120 molecules. By contrast, in CXCR4-using strains (X4 and R5X4), the epitope was constitutively accessible. In accordance with these results, R5 isolates were resistant to neutralization by MAb D19, becoming sensitive only upon addition of sCD4, whereas CXCR4-using isolates were neutralized regardless of the presence of sCD4. Other V3 epitopes examined did not display a similar divergence in accessibility based on coreceptor usage phenotype. These results provide the first evidence of a correlation between HIV-1 biological phenotype and neutralization sensitivity, raising the possibility that the in vivo evolution of HIV-1 coreceptor usage may be influenced by the selective pressure of specific host antibodies.     

R5X4 viruses are evolutionary, functional, and antigenic intermediates in the pathway of a simian-human immunodeficiency virus coreceptor switch

To examine the pathway of the coreceptor switching of CCR5-using (R5) virus to CXCR4-using (X4) virus in simian-human immunodeficiency virus SHIV(SF162P3N)-infected rhesus macaque BR24, analysis was performed on variants present at 20 weeks postinfection, the time when the signature gp120 V3 loop sequence of the X4 switch variant was first detected by PCR. Unexpectedly, circulating and tissue variants with His/Ile instead of the signature X4 V3 His/Arg insertions predominated at this time point. Phylogenetic analysis of the sequences of the C2 conserved region to the V5 variable loop of the envelope (Env) protein showed that viruses bearing HI insertions represented evolutionary intermediates between the parental SHIV(SF162P3N) and the final X4 HR switch variant. Functional analyses demonstrated that the HI variants were phenotypic intermediates as well, capable of using both CCR5 and CXCR4 for entry. However, the R5X4 intermediate virus entered CCR5-expressing target cells less efficiently than the parental R5 strain and was more sensitive to both CCR5 and CXCR4 inhibitors than either the parental R5 or the final X4 virus. It was also more sensitive than the parental R5 virus to antibody neutralization, especially to agents directed against the CD4 binding site, but not as sensitive as the late X4 virus. Significantly, the V3 loop sequence that determined CXCR4 use also conferred soluble CD4 neutralization sensitivity. Collectively, the data illustrate that, similar to human immunodeficiency virus type 1 (HIV-1) infection in individuals, the evolution from CCR5 to CXCR4 usage in BR24 transitions through an intermediate phase with reduced virus entry and coreceptor usage efficiencies. The data further support a model linking an open envelope gp120 conformation, better CD4 binding, and expansion to CXCR4 usage.     

Replacement of the V3 region of gp120 with SDF-1 preserves the infectivity of T-cell line-tropic human immunodeficiency virus type 1

Interaction between the human immunodeficiency virus type 1 (HIV-1) envelope and the relevant chemokine receptors is crucial for subsequent membrane fusion and viral entry. Although the V3 region of gp120 is known to determine the cell tropism as well as the coreceptor usage, the significance of the binding of the V3 region to the chemokine receptor has not been fully understood. To address this issue, we adopted the pseudotyped virus infection assay in which the V3 region of the T-cell line-tropic (T-tropic) NL4-3 envelope was replaced with a portion of stromal cell-derived factor 1 (SDF-1), the ligand of CXCR4. The V3 region of the NL4-3 envelope expression vector was replaced with three different stretches of SDF-1 cDNA. Expression of each chimeric envelope protein was confirmed by immunoprecipitation and Western blotting. Luciferase reporter viruses were prepared by cotransfection of the pNL4-3.Luc.E(-)R(-) vector and each chimeric envelope expression vector, and the infection assay was then carried out. We showed that pseudotyped viruses with one of the chimeric envelopes, NL4-3/SDF1-51, could infect U87.CD4.CXCR4 but not U87.CD4 or U87.CXCR4 cells and that this infection was inhibited by the ligand of CXCR4, SDF-1beta, by anti-human SDF-1 antibody, or by an anti-CD4 antibody, Leu3a, in a dose-dependent manner. Furthermore, chimeric NL4-3/SDF1-51 gp120 significantly inhibited binding of labeled SDF-1 to CXCR4. It was suggested that replacement of the V3 region of the NL4-3 envelope with SDF-1 preserved the CD4-dependent infectivity of T-tropic HIV-1. These results indicate that binding between the V3 region and the relevant coreceptor is important for viral entry, whether its amino acid sequence is indigenous to the virus or not.     

Improved prediction of coreceptor usage and phenotype of HIV-1 based on combined features of V3 loop sequence using random forest

HIV-1 coreceptor usage and phenotype mainly determined by V3 loop are associated with the disease progression of AIDS. Predicting HIV-1 coreceptor usage and phenotype facilitates the monitoring of R5-to-X4 switch and treatment decision-making. In this study, we employed random forest to predict HIV-1 biological phenotype, based on 37 random features of V3 loop. In comparison with PSSM method, our RF predictor obtained higher prediction accuracy (95.1% for coreceptor usage and 92.1% for phenotype), especially for non-B non-C HIV-1 subtypes (96.6% for coreceptor usage and 95.3% for phenotype). The net charge, polarity of V3 loop and five V3 sites are seven most important features for predicting HIV-1 coreceptor usage or phenotype. Among these features, V3 polarity and four V3 sites (22, 12, 18 and 13) are first reported to have high contribution to HIV-1 biological phenotype prediction.     

The CCR5 and CXCR4 coreceptors are both used by human immunodeficiency virus type 1 primary isolates from subtype C

Human immunodeficiency virus type 1 (HIV-1) subtype C viruses with different coreceptor usage profiles were isolated from 29 South African patients with advanced AIDS. All 24 R5 isolates were inhibited by the CCR5-specific agents, PRO 140 and RANTES, while the two X4 viruses and the three R5X4 viruses were sensitive to the CXCR4-specific inhibitor, AMD3100. The five X4 or R5X4 viruses were all able to replicate in peripheral blood mononuclear cells that did not express CCR5. When tested using coreceptor-transfected cell lines, one R5 virus was also able to use CXCR6, and another R5X4 virus could use CCR3, BOB/GPR15, and CXCR6. The R5X4 and X4 viruses contained more-diverse V3 loop sequences, with a higher overall positive charge, than the R5 viruses. Hence, some HIV-1 subtype C viruses are able to use CCR5, CXCR4, or both CXCR4 and CCR5 for entry, and they are sensitive to specific inhibitors of entry via these coreceptors. These observations are relevant to understanding the rapid spread of HIV-1 subtype C in the developing world and to the design of intervention and treatment strategies.     

Gp120 V3-dependent impairment of R5 HIV-1 infectivity due to virion-incorporated CCR5

Entry of R5 human immunodeficiency virus type 1 (HIV-1) into target cells requires sequential interactions of the envelope glycoprotein gp120 with the receptor CD4 and the coreceptor CCR5. We investigated replication of 45 R5 viral clones derived from the HIV-1JR-FLan library carrying 0-10 random amino acid substitutions in the gp120 V3 loop. It was found that 6.7% (3/45) of the viruses revealed >or=10-fold replication suppression in PM1/CCR5 cells expressing high levels of CCR5 compared with PM1 cells expressing low levels of CCR5. In HIV-1V3L#08, suppression of replication was not associated with entry events and viral production but with a marked decrease in infectivity of nascent progeny virus. HIV-1V3L#08, generated from infected PM1/CCR5 cells, was 98% immunoprecipitated by anti-CCR5 monoclonal antibody T21/8, whereas the other infectious viruses were only partially precipitated, suggesting that incorporation of larger amounts of CCR5 into the virions caused impairment of viral infectivity in HIV-1V3L#08. The results demonstrate the implications of an alternative influence of CCR5 on HIV-1 replication.     

Human immunodeficiency virus type 1 V1-V2 envelope loop sequences expand and add glycosylation sites over the course of infection, and these modifications affect antibody neutralization sensitivity

Over the course of infection, human immunodeficiency virus type 1 (HIV-1) continuously adapts to evade the evolving host neutralizing antibody responses. Changes in the envelope variable loop sequences, particularly the extent of glycosylation, have been implicated in antibody escape. To document modifications that potentially influence antibody susceptibility, we compared envelope variable loops 1 and 2 (V1-V2) from multiple sequences isolated at the primary phase of infection to those isolated around 2 to 3 years into the chronic phase of infection in nine women with HIV-1 subtype A. HIV-1 sequences isolated during chronic infection had significantly longer V1-V2 loops, with a significantly higher number of potential N-linked glycosylation sites, than the sequences isolated early in infection. To assess the effects of these V1-V2 changes on antibody neutralization and infectivity, we created chimeric envelope sequences, which incorporated a subject's V1-V2 sequences into a common subtype A envelope backbone and then used them to generate pseudotyped viruses. Compared to the parent virus, the introduction of a subject's early-infection V1-V2 envelope variable loops rendered the chimeric envelope more sensitive to that subject's plasma samples but only to plasma samples collected >6 months after the sequences were isolated. Neutralization was not detected with the same plasma when the early-infection V1-V2 sequences were replaced with chronic-infection V1-V2 sequences, suggesting that changes in V1-V2 contribute to antibody escape. Pseudotyped viruses with V1-V2 segments from different times in infection, however, showed no significant difference in neutralization sensitivity to heterologous pooled plasma, suggesting that viruses with V1-V2 loops from early in infection were not inherently more neutralization sensitive. Pseudotyped viruses bearing chimeric envelopes with early-infection V1-V2 sequences showed a trend in infecting cells with low CD4 concentrations more efficiently, while engineered viruses with V1-V2 sequences isolated during chronic infection were moderately better at infecting cells with low CCR5 concentrations. These studies suggest that changes within the V1-V2 envelope domains over the course of an infection influence sensitivity to autologous neutralizing antibodies and may also impact host receptor/coreceptor interactions.