Indian primary HIV-2 isolates and relationship between V3 genotype, biological phenotype and coreceptor usage

The chemokine coreceptors play a significant role in HIV entry and pathogenesis. The V3 region of HIV envelope glycoprotein is considered as a principal determinant for viral phenotype and tropism. The present study describes lack of association between the V3 genotype and viral phenotype of 18 Indian HIV-2 isolates. The viruses were isolated, confirmed by PCR and the HIV subtypes were determined by sequencing V3 region of the env gene. The coreceptor usage and syncytium inducing (SI) capacity of isolates was determined. Our study indicated that CCR5 coreceptor usage and NSI phenotype is predominant among Indian HIV-2 isolates obtained from patients in the early stage of infection. Two of the four HIV-2 isolates obtained from the late stage patients were SI and dual tropic. Phylogenetic analysis of these isolates revealed close relatedness to the isolates from western and southern India.     

Coreceptor usage and RANTES sensitivity of non-syncytium-inducing HIV-1 isolates obtained from patients with AIDS

OBJECTIVES: The biologic phenotype of HIV-1 primary isolates obtained from approximately 50% of patients who progress to AIDS switches from non-syncytium-inducing (NSI) to syncytium-inducing (SI). We evaluated possible associations between virus coreceptor usage, sensitivity to inhibition by beta-chemokines, and disease progression of patients who continue to yield NSI isolates after developing AIDS. STUDY DESIGN/METHODS: Sequential virus isolates were analyzed for biologic phenotype using the MT-2 cell assay, for sensitivity to beta-chemokines using RANTES inhibition, and for coreceptor usage using U87.CD4 and GHOST.CD4 cells expressing different chemokine/orphan receptors or donor peripheral blood mononuclear cells (PBMC) defective in CCR5 expression. In addition, the env V3 region was sequenced and the length of the V2 region determined. RESULTS: All NSI isolates, regardless of patient status at time of isolation, were dependent on CCR5 expression for cell entry. Furthermore, there was no indication of broadened coreceptor usage of NSI isolates obtained from persons with late-stage AIDS. A majority of NSI isolates remained RANTES sensitive; however, virus variants with reduced sensitivity were observed. The V2 lengths and the V3 sequences exhibited no or minor changes at analysis of sequential NSI isolates. CONCLUSIONS: Our data suggest that NSI isolates obtained from AIDS patients remain CCR5 dependent (ie, R5) and, in many cases, also remain sensitive to RANTES inhibition. However, virus variants with decreased sensitivity to RANTES inhibition may evolve during disease progression, not only as a result of a switch from NSI to SI but also in patients who develop AIDS while continuing to maintain R5 isolates.     

Molecular identification of 13 new enterovirus types, EV79-88, EV97, and EV100-101, members of the species Human Enterovirus B

Paired PBMCs and plasma samples from 34 HIV-infected patients were studied to verify the relationship between coreceptor use based on genotyping of V3 region of HIV-1 envelope gp120 and biological phenotype with virus isolation and subsequent correlation to clinical characteristics. The “11/25” rule, geno2pheno and PSSM were compared. All SI patients were HIV-1 subtype B (p = 0.04) and had a lower CD4 count than NSI patients (p = 0.01), while no differences were observed in mean HIV-RNA _log (p = 0.6). SI phenotype was not associated with AIDS-defining events (p = 0.1) or with concurrent antiretroviral therapy (p = 0.4). With geno2pheno, which shows the highest sensibility (83%), an X4 or X4/R5 genotype in PBMC DNA was also associated to B-subtype and lower CD4 count (p = 0.01) compared to R5 isolates. Based on plasma RNA sequences, the predicted coreceptor usage agreed with PBMC DNA in 79% of cases with the “11/25” rule, 82% with geno2pheno, and 82% with PSSM. A X4 virus in plasma (but not in PBMCs) was significantly associated with HAART in all three methods (p = 0.01 for “11/25” rule, p = 0.01 for geno2pheno and p = 0.03 for PSSM). Due to viral mixtures and/or difficulties in genotype interpretation, current V3 sequence-based methods cannot accurately predict HIV-1 coreceptor use.     

Functional complementation of the envelope hypervariable V3 loop of human immunodeficiency virus type 1 subtype B by the subtype E V3 loop.

The hypervariable V3 loop within gp120 of human immunodeficiency virus type 1 (HIV-1) is the major determinant of cell tropism and the entry coreceptor usage of the virus. However, the information obtained thus far has been from only subtype B from North America and Europe, and little is known about other subtypes whose V3 amino acids differ by as much as 50% from subtype B V3. In this study, we examined the functional potential of the V3 element of the HIV-1 subtype E, the most crucial variant causing the AIDS epidemic throughout southeast Asia. A panel of HIV-1LAI recombinants was constructed by the overlap extension method, by which the LAI V3 loop was precisely replaced by that of the subtype E nonsyncytium-inducing (NSI) or syncytium-inducing (SI) variant. All of the recombinant viruses infected peripheral blood mononuclear cells, whereas only those with SI V3 infected MT2 cells, a CD4(+) T cell line. Consistently, the SI V3 recombinants used CXCR4, while the NSI V3 recombinants used CCR5 for infection of HOS-CD4(+) cells. Finally, only the NSI V3 sequence conferred CC-chemokine sensitivity on the parental virus. The data support the notion that the HIV-1 V3 loop consists of a relatively independent domain in gp120 and suggest that the subtype E V3 loop indeed contains the functional element to dictate the cell tropism, coreceptor preference, and chemokine sensitivity of the virus. These findings are of immediate importance in understanding V3 structure-function relationship and for examining phenotypic evolution of HIV-1 subtype E.     

Genotypic and phenotypic analysis of the env gene from South African HIV-1 subtype B and C isolates

The objective of the study was to assess the genotypic and phenotypic properties of 18 viral strains from human immunodeficiency virus-1 (HIV-1) positive patients and to identify subtype C isolates for vaccine design strategies. All the isolates were non-syncytium-inducing (NSI) in both the primary and MT-2 cell cultures. The amino acid charge of the V3 loop correlated with the NSI phenotype of the strains. The V3 competitive peptide enzyme immunoassay and DNA sequencing of the partial gp120 region gave concordant results on the 15 subtype C strains, whereas the three B genotypes gave a positive to B, a nonreactive to B, and a dual reaction to the B-D peptides, respectively. Sixteen of the isolates used only CCR5 as coreceptor whereas two isolates made use of additional coreceptors including CXCR4. In summary, all our subtype C isolates are NSI phenotypically and almost all of them use CCR5 exclusively as their coreceptor.     

Simple determination of human immunodeficiency virus type 1 syncytium-inducing V3 genotype by PCR.

Human immunodeficiency virus type 1 (HIV-1) phenotype variability plays an important role in the pathogenesis of AIDS. The presence of syncytium-inducing (SI) HIV-1 isolates in infected individuals is associated with a rapid decline of CD4+ T cells, rapid disease progression, and reduced survival time after AIDS diagnosis. The strong association between the SI capacity of HIV-1 and the presence of positively charged amino acid residues at positions 306 and/or 320 in the third variable domain (V3) of gp120 could here be confirmed in 97% of 402 primary HIV-1 isolates, indicating that the V3 genotype may be useful for prediction of the viral phenotype. The V3 DNA sequences revealed a remarkably limited codon usage for the amino acid residues that are responsible for virus phenotype. On the basis of this limited SI-specific DNA sequence variation, four SI-specific oligonucleotides were designed for selective amplification of V3 from SI but not non-SI HIV-1 isolates. This PCR analysis allowed the prediction of the biological phenotype of HIV-1 isolates on the basis of the V3 genotype and may prove to be useful for monitoring SI capacity of HIV-1 isolates in infected individuals.     

Role of HIV-1 phenotype in viral pathogenesis and its relation to viral load and CD4+ T-cell count

The predictive value of HIV-1 phenotype in peripheral blood mononuclear cell (PBMC) coculture and the relation among viral phenotype, viral load, and CD4+ T-cell count were examined in two studies. In study A, 132 HIV-1–infected individuals were examined retrospectively for the relation between the result of their initial HIV cultivation in PBMC coculture and survival rate 6 years later. In study B, 176 patients were examined since 1994 for markers of HIV disease progression. HIV-1 phenotype was determined by PBMC cocultivation, viral load by NASBA HIV RNA QT System, and CD4+ T-cell count by flow cytometry. In study A, the percentage of survival for patients with initial negative virus culture was significantly higher (95%) than in patients with nonsyncytia-inducing (NSI) isolates (78%) and syncytia-inducing (SI) isolates (21%) (P < 0.05 and P < 0.0001, respectively). When SI phenotype was subdivided into moderately cytopathogenic and highly cytopathogenic, significant differences in the rate of survival between these subgroups could be observed (45% vs. 14%; P < 0.05). In study B, progression from negative virus culture to the isolation of NSI variants was associated with increasing viral load (P < 0.0001) but did not affect CD4+ T-cell count significantly (P > 0.07), whereas the switch from NSI to SI virus was accompanied by significant decline of CD4+ T-cells (P < 0.0001) but no change in viral load (P > 0.21). Thus, isolation and phenotyping of HIV represents an additional striking predictive marker for progression of HIV infection. J. Med. Virol. 56:259–263, 1998. © 1998 Wiley-Liss, Inc.     

Diversity of HIV-1 subtype E in semen and cervicovaginal secretion

OBJECTIVES: To characterize human immunodeficiency virus type 1 (HIV-1) subtype E variants in blood and genital fluid of infected Thai couples. STUDY DESIGN/METHODS: Blood and genital fluid were collected from 30 asymptomatic healthy HIV-1 subtype E infected couples from Bangkok, Thailand from 1995 to 1998. RESULTS: All 60 viruses in blood samples were identified as subtype E by heteroduplex mobility assay. The biotype of viruses founded in blood was syncytium-inducing (SI), whereas M-tropic and non-syncytium-inducing (NSI) isolates were predominantly detected in genital fluid. HIV-1 proviral DNA was detected in 43.33% and 56.67%, and viral RNA was detected in 93.33% and 56.67%, of semen (n = 30) and cervicovaginal secretion (n = 30) samples tested, respectively. A higher intersample genetic distance and more positive charge of the V3 loop were found in blood strains composed of genital fluid strains (22.30 +/- 5.92% and 17.96 +/- 6.3%), which was statistically significant (P = 0.003). The env V1-V4 intraperson variation of the HIV-1 subtype E in the blood and genital fluid of each individual was in the range 3.0%-5.7%. We also determined the intrasample variation of HIV-1 from blood and genital fluid by heteroduplex mobility assay. The mean heteroduplex mobility of the HIV-1, V1-V4 region of env gene, in blood (n = 8) and genital fluid (n = 8) was 0.59 +/- 0.06 and 0.74 +/- 0.11 (t test, p = 0.001), respectively. CONCLUSIONS: There was genetic and phenotypic compartmentalization of HIV-1 subtype E in blood and genital fluid with the presence of SI and NSI phenotypic variants as a common property of subtype E isolates from blood and genital fluid, respectively.     

Five human immunodeficiency virus type 1 phenotypic variants with different MT-2 cell tropisms correlate with prognostic markers of disease

OBJECTIVES: We correlated virologic and immunologic parameters of disease progression with cytopathogenicity of HIV isolates. STUDY DESIGN/METHODS: Human immunodeficiency virus type 1 (HIV-1) isolates from 207 patients with CD4+ cell counts < 500/mm3 were examined for biologic phenotype in MT-2 cells. We used a cross-sectional study design. RESULTS: Three subtypes of syncytium-inducing (SI) strains with different times of appearance of syncytia formation in cell culture and two subtypes of non-syncytium-inducing (NSI) isolates, with (NSI/MT2+) or without (NSI/MT2-) replicative capacity in MT-2 cells, were identified. Early SI strains were associated with the lowest CD4+ cell counts and the highest levels of viral load, and NSI/MT2- isolates correlated with the highest CD4+ cell counts and the lowest viral loads. Patients with late SI and NSI/MT2+ strains showed minimal differences in immunologic and virologic markers. CONCLUSIONS: Five HIV phenotypic variants that correlate significantly (P < 0.001) with markers of disease progression were identified.     

HIV colonizing peripheral blood monocytes follows lymphocytic isolates in shifting from NSI to SI genotype

Summary Non-syncytium inducing (NSI) and syncytium inducing (SI) variants of human immunodeficiency virus (HIV) isolated from peripheral blood mononuclear cells (PBMC) could be definitely typed by sequence analysis of the env-gene V3 region. It was thus possible to compare the genotypes of viral variants isolated from PBMC and accompanying monocyte cultures and those derived directly from the patients' blood cells prior to cultivation. Within the investigated group of patients it was shown that HIV variants colonizing monocytes displayed a similar shift from NSI to SI as observed previously for PBMC, i.e. lymphocyte derived isolates. Lymphocytic SI variants could be isolated from the blood of patients, while simultaneously the predominant provirus in both blood and monocytic isolate was NSI. Consequently, we observed a delayed switch in the predominant provirus genotype found in blood which was associated with a synchronous change in the genotype of the corresponding monocytic isolate. The results show that monocytes/macrophages can be colonized by heterogeneous HIV variants in vivo and can therefore also function as carriers for the spread of highly virulent SI variants into the tissues.     

Predicting HIV-1 coreceptor usage with sequence analysis

Bioinformatics approaches are increasingly being used to identify and understand the genetic variation underlying changes in HIV-1 biological phenotype. The variable regions of the viral envelope are the major determinant of virus coreceptor usage and cell tropism. Specifically, amino acids 11 and 25 in the 3rd variable (V3) loop have been found to strongly influence viral syncytium inducing capacity and coreceptor usage. Many additional V3 loop changes, however, as well as changes elsewhere in Env, are thought to contribute to phenotype. In this review we describe several recently developed methods to analyze this variability and their use to predict biological phenotype based on sequence information. These approaches have identified changes in the V3 loop, in addition to the known changes at positions 11 and 25, that affect phenotype and significantly enhance our ability to predict phenotype from genotype. Besides improving phenotype prediction, methods that score V3 sequences on a continuous scale can also assist in the interpretation of evolutionary information about shifts in phenotype, and the relationship between that evolution and pathogenesis. Several examples and potential practical applications of this scoring are discussed. We conclude that advances in computational approaches have enhanced both our ability to predict and to understand HIV-1 biological phenotype evolution. Further development of these methods, by extending analysis to regions outside the V3 loop and to clades beyond subtype B, will extend our understanding of HIV-1 pathogenesis and inform treatment strategies.     

Determining human immunodeficiency virus coreceptor use in a clinical setting: degree of correlation between two phenotypic assays and a bioinformatic model

Two recombinant phenotypic assays for human immunodeficiency virus (HIV) coreceptor usage and an HIV envelope genotypic predictor were employed on a set of clinically derived HIV type 1 (HIV-1) samples in order to evaluate the concordance between measures. Previously genotyped HIV-1 samples derived from antiretroviral-na?ve individuals were tested for coreceptor usage using two independent phenotyping methods. Phenotypes were determined by validated recombinant assays that incorporate either an approximately 2,500-bp ("Trofile" assay) or an approximately 900-bp (TRT assay) fragment of the HIV envelope gp120. Population-based HIV envelope V3 loop sequences ( approximately 105 bp) were derived by automated sequence analysis. Genotypic coreceptor predictions were performed using a support vector machine model trained on a separate genotype-Trofile phenotype data set. HIV coreceptor usage was obtained from both phenotypic assays for 74 samples, with an overall 85.1% concordance. There was no evidence of a difference in sensitivity between the two phenotypic assays. A bioinformatic algorithm based on a support vector machine using HIV V3 genotype data was able to achieve 86.5% and 79.7% concordance with the Trofile and TRT assays, respectively, approaching the degree of agreement between the two phenotype assays. In most cases, the phenotype assays and the bioinformatic approach gave similar results. However, in cases where there were differences in the tropism results, it was not clear which of the assays was "correct." X4 (CXCR4-using) minority species in clinically derived samples likely complicate the interpretation of both phenotypic and genotypic assessments of HIV tropism.     

Basic amino acid residues in the V3 loop of simian immunodeficiency virus envelope alter viral coreceptor tropism and infectivity but do not allow efficient utilization of CXCR4 as entry cofactor

In contrast to human immunodeficiency viruses type 1 and type 2 (HIV-1 and HIV-2, respectively), simian immunodeficiency virus (SIVmac) rarely uses CXCR4 (X4) for efficient entry into target cells. Basic amino acid residues in the V3 loop of HIV Env allow efficient coreceptor utilization of X4. Therefore, we investigated if similar changes in the SIVmac Env protein also mediate a coreceptor switch from CCR5 (R5) to X4. Functional analysis revealed that none of eight SIVmac variants, containing V3 regions with an overall charge between +4 and +10, efficiently utilized X4 as entry cofactor. Nonetheless, these alterations had differential effects on SIV coreceptor tropism and on Env expression levels. A single amino acid substitution of L328R, located near the tip of the V3 loop, resulted in grossly reduced Env expression levels and impaired viral infectivity. Notably, additional basic residues restored efficient Env expression and virion incorporation but not infectivity. In comparison to the L328R mutation, changes of P334K and D337K had little disruptive effects on SIVmac entry and replication. Interestingly, mutation of L320K and P321R disrupted coreceptor usage of GPR15 but not R5. These changes also impaired SIVmac replication in peripheral blood mononuclear cells (PBMC) derived from a Delta32/Delta32 donor but not in R5-expressing human or simian PBMC. Our results show that positively charged amino acid residues in the V3 loop affect SIVmac coreceptor tropism and infectivity but do not allow efficient utilization of X4.     

Biological properties of primary HIV-1 isolates with varying serotype]

Russian HIV variants with common (rapid/high SI and slow/low NSI) and rare (slow/low SI and rapid/high NSI) phenotypes are described. SI variants demonstrate a higher p24 concentration level than serotype-independent NSI. The majority of SI variants belonging to A, B, and C serotypes were isolated from patients with mainly stage B3. At stages B2 and C2, when the majority of viruses are characterized by NSI phenotype, serotype D isolates are characterized only by SI phenotype. The spectrum of cell tropism was wide for all rapid/high strains and narrow for all slow/low ones.     

Improved success of phenotype prediction of the human immunodeficiency virus type 1 from envelope variable loop 3 sequence using neural networks.

We have assembled two sets of HIV-1 V3 sequences with defined epidemiologic relationships associated with experimentally determined coreceptor usage or MT-2 cell tropism. These data sets were used for three purposes. First, they were employed to test existing methods for predicting coreceptor usage and MT-2 cell tropism. Of these methods, the presence of one basic amino acid at position 11 or 25 proved to be most reliable for both phenotypic classifications, although its predictive power for the X4 phenotype was less than 50%. Second, we used the sequence sets to train neural networks to infer coreceptor usage from V3 genotype with better success than the best available motif-based method, and with a predictive power equal to that of the best motif-based method for MT-2 cell tropism. Third, we used the sequence sets to reexamine patterns of variability associated with the different phenotypes, and we showed that the phenotype-associated sequence patterns could be reproduced from large sets of V3 sequences using phenotypes predicted by the trained neural network.     

Stable coreceptor usage of HIV in patients with ongoing treatment failure on HAART.

BACKGROUND: Disease progression in HIV infection has been associated with switch of viral coreceptor usage from CCR5 to CXCR4. OBJECTIVES: To investigate the relationship between HIV-coreceptor tropism and clinical and virological outcome in 40 heavily pretreated patients over time. METHODS: Coreceptor phenotype was predicted after sequencing the V3 loop of the HIV glycoprotein 120. RESULTS: Coreceptor use was stable during observation time in 87% of patients, and CCR5 tropism was predominant. Viral mutations in the pol gene and clinical parameters were not predictive for coreceptor switching. CONCLUSIONS: Even in patients with repeated HAART failure, CCR5 antagonists might be a valuable treatment option.     

CXCR4 mediates entry and productive infection of syncytia-inducing (X4) HIV-1 strains in primary macrophages

CCR5 and CXCR4 are the main coreceptors for non-syncytia-inducing (NSI) and syncytia-inducing (SI) HIV-1 strains, respectively. NSI HIV-1 isolates do not infect either human lymphoid or monocytoid cell lines, and this inability correlates with the absence of CCR5 expression in these cell types. The ability of SI HIV-1 isolates to infect human primary macrophages has been disputed. Here, we report that CXCR4 is expressed in primary blood-derived human mononuclear phagocytes at all stages of differentiation, although the maturation process correlates with downregulation of CXCR4 mRNA. Infection experiments with the SI molecular clone NL4-3 tagged with a mutant of the green fluorescent protein established that both monocytes and attached macrophages are susceptible to infection with CXCR4-restricted HIV-1 strains. NL4-3 entry into primary macrophages could be blocked by SDF-1alpha in a dose-dependent manner, or by the anti-CXCR4 monoclonal antibody 12G5. HIV-1 entry led to productive infection. No evidence of postentry defects or nuclear import delay for CXCR4-restricted HIV-1 strains was detected using a quantitative real-time PCR assay measuring HIV-1 DNA entry into the nucleus. Macrophages infected by HIV-1 and expressing virus were maintained in culture for long periods of time (up to 5 months). These results demonstrate that CXCR4 is the main HIV-1 SI coreceptor in human primary macrophages and underline the importance of the macrophage as a long-living viral reservoir for HIV-1.     

HIV-1 evolves into a nonsyncytium-inducing virus upon prolonged culture in vitro.

HIV-1 LAI is a syncytium-inducing (SI) virus with a broad host cell range. We previously isolated a LAI variant that improved replication in the SupT1 T cell line due to mutations within the C1 and C4 constant regions of the Env protein. We now report that this variant exhibits a severely restricted host cell range, as replication in other T cell lines and primary cells was abolished. Several Env-mediated functions were analyzed to provide a mechanistic explanation for this selective adaptation. The change in host cell tropism was not caused by a switch to a SupT1-specific coreceptor. Biosynthesis of the variant Env glycoprotein was not improved in SupT1 cells, and in fact a small defect in intracellular Env processing was observed. SupT1 infection assays did not reveal an improved Env function either, and a dramatic loss of infectivity was measured with other cell types. The Env-mutated HIV-1 reached an approximately fivefold higher level of virus production in SupT1 cells at the peak of infection. Unlike the LAI virus, the variant did not trigger the formation of syncytia. Our combined results suggest that the HIV-1 variant allows the infected host cell to survive longer, thus producing more viral progeny. The intricate virus-cell interaction results in a balance between optimal virus replication and host cell survival, causing a cytopathic SI isolate to evolve toward a nonsyncytium-inducing (NSI) phenotype in cell culture. These findings may help explain the absence of SI variants in the initial phase of HIV-1 infection, and the results dispute the notion that HIV-1 evolution should always go from the NSI to SI phenotype.     

The HIV-2 genotype and the HIV-1 syncytium-inducing phenotype are associated with a lower virus replication in dendritic cells

During sexual transmission, HIV infects the mucosal dendritic cells and is transferred to CD4 T cells. Whether HIV variants of a particular genetic (sub)type or phenotype selectively infect dendritic cells (DC) or are preferentially transferred to T cells remains highly controversial. To avoid the cumbersome use of primary dendritic cells, in vitro dendritic cell models were generated from precursors, either hematopoietic progenitor cells (HPC) or monocytes (MO). Productive infection in the dendritic cells and transfer of the virus to T cells was assessed for a range of HIV variants. HPC-derived dendritic cells (HPC-DC) were more susceptible to HIV-1 than to HIV-2 isolates. The HIV-1 group O strains were more productive in HPC-DC than group M, but amongst the latter, no subtype-related difference was observed. Both non-syncytium-inducing (NSI) and SI HIV isolates and lab strains could productively infect HPC-DC, albeit with a different efficiency. Adding blocking antibodies confirmed that both CCR-5 and CXCR-4 co-receptors were functional. Biological HIV-1 clones of the NSI/R5 phenotype infected more readily HPC-DC than SI/X4 clones. MO-derived dendritic cells were, however, more exclusive in their preference for NSI/R5 clones. Some HIV variants, that did not grow readily in HPC-DC alone, could be rescued by adding resting or pre-activated T cells. The present data show that HIV-2 isolates and SI clones replicate less in model-DC, but no preference for a particular HIV-1 subtype was evident. Co-culture with T cells could "correct" a limited growth in dendritic cells. Clearly, both intrinsic dendritic cell susceptibility and enhancement by T cells are explained only partly by HIV genotype and phenotype. The in vitro dendritic cell models seem useful tools to further unravel interactions between HIV, DC, and T cells.