Extreme genetic divergence is required for coreceptor switching in HIV-1 subtype C.

BACKGROUND: Coreceptor switching from CCR5 to CXCR4 is less common in subtype C HIV-1 infection than in subtype B for reasons that are unclear. We have examined sequential virus samples from a subtype C-infected child who had evidence of coreceptor switching. METHODS: To examine HIV-1 envelope evolution towards CXCR4 usage, env sequences were correlated with phenotypic characteristics determined by entry assays, as well as the ability to use alternative coreceptors such as FPRL1, CCR3, CCR8 and others. The value of a phenotype predictor based on V3 sequences was also assessed. RESULTS: Ninety-three sequences revealed 3 distinct coexistent virus lineages and only some members of one lineage evolved to use CXCR4. These lineages also had diverse alternative coreceptor patterns including the ability to use FPRL1, CCR3, CCR8, APJ, CMKLR1, RDC-1, CXCR6, CCR1, GPCR1, GPR15 and CCR6. Coreceptor switching was associated with extensive and rapid sequence divergence in the V1/V2 region in addition to V3 changes. Furthermore, interlineage recombination within the C2 region resulted in low predictability of a V3 sequence-based phenotype algorithm, and highlighted the importance of V1/V2 and V3 sequences in coreceptor usage. CONCLUSION: These results suggest that the evolution to coreceptor switching in subtype C infection requires more mutations than other subtypes, and this contributes to the reduced incidence of R5X4 viruses.

Longitudinal analysis of HIV type 1 subtype C envelope sequences from South Africa.

The envelope genes of 23 subtype C viral isolates from five individuals with early HIV-1 infection, followed for 2-4 years, were sequenced, analyzed, and correlated to coreceptor usage. Isolates from three participants used the CCR5 coreceptor at all time points, with no significant adaptations in the variable loop lengths, predicted N-linked glycosylation sites, or predicted change in sensitivity to monoclonal antibodies with disease progression. However, two individuals, Du151 and Du179, who had previously been shown to be dually infected with two phylogenetically distinct subtype C strains, were able to use CXCR4 with disease progression. The intraperson genetic diversity was 9% for Du151 and 3% for Du179 compared to <2% for participants who did not undergo a coreceptor switch. In both cases this coreceptor switch was associated with specific amino acid changes in the crown, an increased net amino acid charge in the V3 loop, and an increase in the length of the V1 region.

Genetic characteristics of the V3 region associated with CXCR4 usage in HIV-1 subtype C isolates.

CXCR4 coreceptor usage appears to occur less frequently among HIV-1 subtype C viruses. The aim of this study was to investigate the genetic determinants within the V3 region of subtype C isolates able to use CXCR4. Thirty-two subtype C isolates with known phenotypes (16 R5, 8 R5X4 and 8 X4 isolates) were assessed. A subtype C-specific V3 heteroduplex tracking assay (HTA) was used to determine sample complexity, and nucleotide sequencing analysis was used to compare characteristics associated with CCR5 and CXCR4-using isolates. There were sufficient genetic differences to discriminate between R5 viruses and those able to use CXCR4. In general, R5 isolates had an HTA mobility ratio >0.9 whereas CXCR4-using isolates were usually <0.9. Multiple bands were more frequently seen among the dualtropic isolates. Sequence analysis of the V3 region showed that CXCR4-using viruses were often associated with an increased positive amino acid charge, insertions and loss of a glycosylation site, similar to HIV-1 subtype B. In contrast, where subtype B consensus V3 has a GPGR crown motif irrespective of coreceptor usage, all 16 subtype C R5 viruses had a conserved GPGQ sequence at the tip of the loop, while 12 of the 16 (75%) CXCR4-using viruses had substitutions in this motif, most commonly arginine (R). These findings were confirmed using a larger published data set. We therefore suggest that changes within the crown motif of subtype C viruses might be an additional pathway to utilise CXCR4 and thus GPGQ may limit the potential for the development of X4 viruses.

High specificity of V3 serotyping among human immunodeficiency virus type-1 subtype C infected patients with varying disease status and viral phenotype.

V3 serotyping is a technique for determining HIV-1 genetic subtype based on the binding of antibodies from patient sera or plasma to synthetic V3 peptides derived from subtype consensus sequences. Variation in the performance of this assay has been attributed to V3 sequence heterogeneity, the degree of which varies with patient disease progression, virus co-receptor usage, and genetic subtype. This study assessed the performance of a competitive peptide enzyme immunoassay (cPEIA) in samples from HIV-1 subtype C infected patients with varying disease profiles, including those with syncytium (SI) and non-syncytium-inducing (NSI) viruses. Out of 90 sera tested, 94.4% reacted strongly against the subtype C peptide. There was no significant difference in assay sensitivity among samples from advanced AIDS patients in which humoral immune response may be lower, nor among SI viruses which carry changes in the V3 sequence. Four samples were found to be cross-reactive with other subtypes and one acutely infected patient sample was non-reactive due to low anti-gp120 antibody titers. A significantly higher number of samples showed secondary reactivity to subtype A, compared to other subtypes (P < 0.005). In conclusion, the assay was able to identify HIV-1 subtype C infection with a high level of sensitivity (94%) irrespective of the stage of disease and therefore provides a valuable resource for the large-scale epidemiological monitoring of the spread of HIV-1 subtypes in South Africa.

Silencing of HIV-1 subtype C primary isolates by expressed small hairpin RNAs targeted to gag.

Discovery of sequence-specific silencing by activating the RNA interference (RNAi) pathway has led to exciting new strategies for treating infection with human immunodeficiency virus type 1 (HIV-1). Of the HIV-1 subtypes, C is especially common in areas of the world that are worst affected. Although prone to mutation, genome plasticity of this subtype is limited in functionally important regions. We identified conserved sequences within the HIV-1 subtype C gag open reading frame and assessed whether they are suitable targets for inhibition of viral replication by RNA Pol III-driven small hairpin RNAs (shRNAs). Initially, the efficacy of each of a panel of 10 shRNAs against HIV-1 was determined using a reporter assay. shRNAs A and B, which targeted the 5 end of gag, were most effective and were used to assess inhibition of replication in cultured cells of two R5 isolates (Du151 and Du422) and one X4 virus (SW7). These shRNAs diminished intracellular HIV-1 gag RNA and HIV-1 protein concentrations as well as p24 secretion by up to 80% without inducing an interferon response. However, shRNA-mediated knockdown efficacy against each of these viral isolates varied slightly. These data show successful activation of RNAi to inhibit the replication of biologically distinct HIV-1 subtype C isolates. The effector shRNAs described here are potential candidates for gene therapy applications against the most common global subtype of HIV-1.

Genotypic and phenotypic characterization of viral isolates from HIV-1 subtype C-infected children with slow and rapid disease progression.

The genotypes and biological phenotypes of HIV-1 isolates obtained from 40 perinatally infected children in South Africa were analyzed. This included 15 infants who had HIV-related symptoms, most of whom died within 2 years of birth (rapid progressors), and 25 children who survived between 4 and 9 years with varying signs of disease (slow progressors). Heteroduplex mobility assays and sequence analysis confirmed that within the env and gag regions, all isolates were HIV-1 subtype C. Viral isolates from 14 of the 15 rapid progressors used the CCR5 coreceptor, whereas 1 (02ZARP1) used both the CXCR4 and CCR5 coreceptors. Among the 25 slow progressors, 22 isolates used CCR5 only, 2 used CXCR4 only, and 1 used both CCR5 and CXCR4. Two of the slow-progressing children who harbored CXCR4-using viruses had AIDS. All four CXCR4-using viruses had genotypic changes in the V3 region previously shown to be associated with CXCR4 usage. This cross-sectional study shows that HIV-1 subtype C viruses from both rapid- and slow-progressing perinatally infected children used predominantly CCR5. Similar to adults, CXCR4 usage was uncommon among HIV-1 subtype C isolates from pediatric infections.

In vitro generation of HIV type 1 subtype C isolates resistant to enfuvirtide.

Enfuvirtide (ENF) is the first in a new class of antiretroviral agents targeting the fusion process of the viral life cycle. ENF is a synthetic 36-amino acid peptide that binds to the HR-1 region of gp41 preventing fusion of viral and cellular membranes. With the introduction of ENF there are now four classes of antiretrovirals each with distinct and different resistance pathways. Resistance to ENF among subtype B HIV-1 isolates is associated with amino acid changes mainly in the HR-1 region, although other regions of envelope have also been implicated. To determine whether subtype C viruses developed resistance mutations similar to subtype B viruses, 11 subtype C and 4 subtype B viruses were passaged in the presence of increasing concentrations of ENF. The subtype C isolates showed varying levels of replication at 1 microg/ml ENF by day 18, but by day 29 all replicated efficiently at 10 microg/ml ENF. All subtype C isolates showed evidence of genotypic changes in gp41 HR-1 following exposure to ENF that included G36S/E/D, I37T, V38M/A/L/E, N/S42D, N43K, L45R/M, and A50T/V. Three subtype C viruses had compensatory changes in the HR-2 region, which corresponds to the ENF sequence, and two isolates had changes in the V3 region. Mutational patterns among the four subtype B viruses were similar to those for subtype C and those previously published in the literature. These data indicate that in vitro resistance to ENF develops rapidly among HIV-1 subtype C isolates. In general, mutational patterns for subtype C were similar to those described for subtype B, suggesting that the mechanism of action for ENF is similar for HIV-1 subtype B and C isolates.

Use of alternate coreceptors on primary cells by two HIV-1 isolates.

Two HIV-1 isolates (CM4 and CM9) able to use alternate HIV-1 coreceptors on transfected cell lines were tested for their sensitivity to inhibitors of HIV-1 entry on primary cells. CM4 was able to use CCR5 and Bob/GPR15 efficiently in transfected cells. The R5 isolate grew in Delta32/Delta32 CCR5 PBMC in the absence or presence of AMD3100, a CXCR4-specific inhibitor, indicating that it uses a receptor other than CCR5 or CXCR4 on primary cells. It was insensitive to the CCR5 entry inhibitors RANTES and PRO140, but was partially inhibited by vMIP-1, a chemokine that binds CCR3, CCR8, GPR15 and CXCR6. The coreceptor used by this isolate on primary cells is currently unknown. CM9 used CCR5, CXCR4, Bob/GPR15, CXCR6, CCR3, and CCR8 on transfected cells and was able to replicate in the absence or presence of AMD3100 in Delta32/Delta32 CCR5 PBMC. It was insensitive to eotaxin, vMIP-1 and I309 when tested individually, but was inhibited completely when vMIP-1 or I309 was combined with AMD3100. Both I309 and vMIP-1 bind CCR8, strongly suggesting that this isolate can use CCR8 on primary cells. Collectively, these data suggest that some HIV-1 isolates can use alternate coreceptors on primary cells, which may have implications for strategies that aim to block viral entry.

Characterization of human immunodeficiency virus type 1 from a previously unexplored region of South Africa with a high HIV prevalence.

HIV prevalence in the Limpopo Province has increased rapidly within the past 10 years, as in other parts of South Africa. Little is known about the genetic and biological properties of HIV circulating in this region including the baseline drug resistance profiles. We therefore collected blood samples from 42 HIV-1-infected patients residing in this region for analysis. All samples were shown to belong to HIV-1 subtype C by env and gag heteroduplex mobility assay (HMA). Viral isolates from 14 of these patients were shown to use the CCR5 coreceptor exclusively and had gp120 V3 loop sequences consistent with this phenotype. Sequence analysis of both protease and reverse transcriptase genes showed that none of 13 isolates harbored primary resistance mutations. These data suggest that HIV-1 subtype C is the predominant subtype circulating in the Limpopo Province, and that viral strains from this region are indistinguishable from those found in other parts of South Africa.

Predicted genotypic resistance to the novel entry inhibitor, BMS-378806, among HIV-1 isolates of subtypes A to G.

BMS-378806 targets virus entry by inhibiting the binding of HIV-1 gp120 to the CD4 receptor. Env sequences (n = 1226) of subtypes A-G were analysed to determine the frequency of mutations associated with resistance to BMS-378806. In line with reported sensitivity data, background genotypic resistance to BMS-378806 among non-B HIV-1 viruses was found to be higher than in subtype B. These data suggest that BMS-378806 may have reduced efficacy against non-B viruses.

Sensitivity of HIV type 1 subtype C isolates to the entry inhibitor T-20.

T-20 is the first in a new class of antiretroviral drugs targeting the entry stage of the virus life cycle. It is a 36 amino acid peptide that binds to the HR1 region of gp41 preventing gp41-mediated fusion with the host cell membrane. T-20 was designed based on the HR2 sequence of HIV-1 subtype B gp41, a region that shows significant genetic variation with HIV-1 subtype C sequences. In order to assess the efficacy of T-20 to inhibit subtype C isolates, a total of 23 isolates were tested for their ability to replicate in the presence of T-20. This included 15 isolates that used CCR5, five that used both CCR5 and CXCR4, and three that used CXCR4. Five of these were from patients failing other antiretroviral therapies. Sequence analysis of the HR2 region indicated that there were 10-16 amino acid changes in the region corresponding to T-20. However, all isolates were effectively inhibited by T-20 at 1 microg/ml. There were no significant differences between viruses that used CCR5 or CXCR4 to enter cells. All isolates, except one, had GIV at positions 36-38 in the HR1 region. One isolate had a GVV motif but this did not affect its sensitivity to T-20. Therefore, T-20 inhibited subtype C viruses despite significant genetic differences in the HR2 region and there was no evidence for baseline resistance to T-20. These data suggest that T-20 would be highly effective in patients with HIV-1 subtype C infection, including those failing existing antiretroviral drug regimens.

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.

Characterization and selection of HIV-1 subtype C isolates for use in vaccine development.

HIV-1 genetic diversity among circulating strains presents a major challenge for HIV-1 vaccine development, particularly for developing countries where less sequence information is available. To identify representative viruses for inclusion in candidate vaccines targeted for South Africa, we applied an efficient sequence survey strategy to samples from recently and chronically infected persons residing in potential vaccine trial sites. All 111 sequences were subtype C, including 30 partial gag, 26 partial pol, 27 V2-V3 env, and 28 V5-partial gp41 sequences. Of the 10 viruses cultured from recently infected individuals, 9 were R5 and 1 was R5X4. Two isolates, Du151 and Du422, collected within 2 months of infection, were selected as vaccine strains on the basis of their amino acid similarity to a derived South African consensus sequence The selection of recently transmitted R5 isolates for vaccine design may provide an advantage in a subtype C R5-dominant epidemic. The full-length Du422 gag and Du151 pol and env genes were cloned into the Venezuelan equine encephalitis (VEE) replicon particle (VRP) expression system. Du422 Gag protein expressed from the VRP accumulated to a high level and was immunogenic as demonstrated by cytotoxic T lymphocyte responses in mice vaccinated with gag-VRPs. Optimization of codon use for VRP expression in human cells did not enhance expression of the gag gene. The cloned Du151 env gene encoded a functional protein as demonstrated by fusion of VRP-infected cells with cells expressing CD4 and CCR5. Genes identified in this study have been incorporated into the VEE VRP candidate vaccines targeted for clinical trial in South Africa.

Coreceptor usage and biological phenotypes of HIV-1 isolates.

The discovery of chemokine receptors as HIV-1 entry molecules or "coreceptors" has lead to a greater understanding of how HIV-1 infects human cells. This has provided insight into the biological properties of HIV-1 isolates and unravelled the meaning of the syncytium-inducing and non-syncytium-inducing phenotypes. Understanding how HIV-1 exploits these coreceptors has given way to novel approaches to controlling HIV. As a result a new class of drugs has emerged that are being tested to prevent virus infection and to act as an alternative, or adjunct, to existing anti-retroviral drugs for HIV-infected individuals.

Full-length genome analysis of HIV-1 subtype C utilizing CXCR4 and intersubtype recombinants isolated in South Africa.

The HIV-1 epidemic in South Africa is largely due to subtype C viruses, which preferentially use CCR5 as a coreceptor for infection. We describe full-length genome sequences of two CXCR4-utilizing HIV-1 subtype C viruses and two intersubtype recombinants from South Africa. Three of the viruses (99ZACM4, 99ZACM9, and 99ZASW7) were isolated in 1999 from AIDS patients in Johannesburg, and a fourth virus (98ZADu178) was isolated in Durban in 1998 from an asymptomatic female sex worker. Isolates 99ZASW7 and 99ZACM9 from Johannesburg were subtype C throughout the genome, 99ZASW7 used the CXCR4 coreceptor, and 99ZACM9 used both CCR5 and CXCR4. Isolate 98ZADu178 from Durban was a novel recombinant between subsubtype A2 and subtype C. The third isolate from Johannesburg, 99ZACM4, was a complex, novel recombinant with multiple breakpoints and contained segments of subtypes A, C, D, G, and K. These results establish the presence of intersubtype recombinants in South Africa, indicating that ongoing surveillance for other subtypes and recombinants is necessary.

HIV-1 Subtype A, D, G, AG and unclassified sequences identified in South Africa.

HIV-1 subtype C accounts for the vast majority of infections in South Africa. However, increasingly non-C subtypes are being detected. Here we report 10 viruses that contain sequences that group with subtypes A, D, and G as well as CRF02_AG and 1 that could not be classified. Most of these individuals were from other countries in Africa. Some of these sequences were in combination with subtype C, possibly indicating local recombination events. Although there is no indication of endemic spread of these viruses, continued monitoring is warranted to track genetic changes, which may impact on diagnostic testing, therapeutic responses to antiretroviral therapies, and vaccine design.