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Wide variation in susceptibility of transmitted/founder HIV-1 subtype C Isolates to protease inhibitors and association with in vitro replication efficiency.

ABSTRACT: The gag gene is highly polymorphic across HIV-1 subtypes and contributes to susceptibility to protease inhibitors (PI), a critical class of antiretrovirals that will be used in up to 2 million individuals as second-line therapy in sub Saharan Africa by 2020. Given subtype C represents around half of all HIV-1 infections globally, we examined PI susceptibility in subtype C viruses from treatment-naïve individuals. PI susceptibility was measured in a single round infection assay of full-length, replication competent MJ4/gag chimeric viruses, encoding the gag gene and 142 nucleotides of pro derived from viruses in 20 patients in the Zambia-Emory HIV Research Project acute infection cohort. Ten-fold variation in susceptibility to PIs atazanavir and lopinavir was observed across 20 viruses, with EC50s ranging 0.71-6.95?nM for atazanvir and 0.64-8.54?nM for lopinavir. Ten amino acid residues in Gag correlated with lopinavir EC50 (p?

SUBMITTER: Sutherland KA

PROVIDER: S-EPMC5128871 | biostudies-other | 2016 Nov

REPOSITORIES: biostudies-other

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Wide variation in susceptibility of transmitted/founder HIV-1 subtype C Isolates to protease inhibitors and association with in vitro replication efficiency.

Sutherland Katherine A KA Collier Dami A DA Claiborne Daniel T DT Prince Jessica L JL Deymier Martin J MJ Goldstein Richard A RA Hunter Eric E Gupta Ravindra K RK

Scientific reports 20161130

The gag gene is highly polymorphic across HIV-1 subtypes and contributes to susceptibility to protease inhibitors (PI), a critical class of antiretrovirals that will be used in up to 2 million individuals as second-line therapy in sub Saharan Africa by 2020. Given subtype C represents around half of all HIV-1 infections globally, we examined PI susceptibility in subtype C viruses from treatment-naïve individuals. PI susceptibility was measured in a single round infection assay of full-length, re ...[more]

PMID: 27901085

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Project description:BackgroundThe HIV-1 epidemic in sub-Saharan Africa is heterogeneous with diverse unevenly distributed subtypes and regional differences in prevalence. Subtype-specific differences in disease progression rate and transmission efficiency have been reported, but the underlying biological mechanisms have not been fully characterized. Here, we tested the hypothesis that the subtypes prevalent in the East Africa, where adult prevalence rate is higher, have lower viral replication capacity (VRC) than their West African counterparts where adult prevalence rates are lower.ResultsGag-protease sequencing was performed on 213 and 160 antiretroviral-naïve chronically infected participants from West and East Africa respectively and bioinformatic tools were used to infer subtypes and recombination patterns. VRC of patient-derived gag-protease chimeric viruses from West (n = 178) and East (n = 114) Africa were determined using a green fluorescent protein reporter-based cell assay. Subtype and regional differences in VRC and amino acid variants impacting VRC were identified by statistical methods. CRF02_AG (65%, n = 139), other recombinants (14%, n = 30) and pure subtypes (21%, n = 44) were identified in West Africa. Subtypes A1 (64%, n = 103), D (22%, n = 35), or recombinants (14%, n = 22) were identified in East Africa. Viruses from West Africa had significantly higher VRC compared to those from East Africa (p < 0.0001), with subtype-specific differences found among strains within West and East Africa (p < 0.0001). Recombination patterns showed a preference for subtypes D, G or J rather than subtype A in the p6 region of gag, with evidence that subtype-specific differences in this region impact VRC. Furthermore, the Gag A83V polymorphism was associated with reduced VRC in CRF02_AG. HLA-A*23:01 (p = 0.0014) and HLA-C*07:01 (p = 0.002) were associated with lower VRC in subtype A infected individuals from East Africa.ConclusionsAlthough prevalent viruses from West Africa displayed higher VRC than those from East Africa consistent with the hypothesis that lower VRC is associated with higher population prevalence, the predominant CRF02_AG strain in West Africa displayed higher VRC than other prevalent strains suggesting that VRC alone does not explain population prevalence. The study identified viral and host genetic determinants of virus replication capacity for HIV-1 CRF02_AG and subtype A respectively, which may have relevance for vaccine strategies.

Project description:Adequate information on the precise molecular and biological composition of the viral strains that establish HIV infection in the human host will provide effective means of immunization against HIV infection. In an attempt to identify the transmitted founder (TF) virus and differentiate the biological properties and infectious potential of the TF virus from those of the population of the early transmitted viruses, 250 patient-derived gp120 envelope glycoproteins were cloned in pMN-K7-Luc-IRESs-NefΔgp120 to obtain chimeric viruses. Samples were obtained from eight infants who had recently become infected with HIV through mother-to-child transmission (MTCT) and two adults who acquired infection through the heterosexual route and were in the chronic stage of infection. Among the 250 clones tested, 65 chimeric viruses were infectious, and all belonged to HIV-1 subtype C. The 65 clones were analyzed for molecular features of the envelope, per-infectious-particle infectivity, coreceptor tropism, drug sensitivity, and sensitivity to broadly neutralizing antibodies. Based on genotypic and phenotypic analysis of the viral clones, we identified 10 TF viruses from the eight infants. The TF viruses were characterized by shorter V1V2 regions, a reduced number of potential N-linked glycosylation sites, and a higher infectivity titer compared to the virus variants from the adults in the chronic stage of infection. CXCR6 coreceptor usage, in addition to that of the CCR5 coreceptor, which was used by all 65 chimeric viruses, was identified in 13 viruses. The sensitivity of the TF variants to maraviroc and a standard panel of neutralizing monoclonal antibodies (VRC01, PG09, PG16, and PGT121) was found to be much lower than that of the virus variants from the adults in the chronic stage of infection.IMPORTANCE Tremendous progress has been made during the last three and half decades of HIV research, but some significant gaps continue to exist. One of the frontier areas of HIV research which has not seen a breakthrough yet is vaccine research, which is because of the enormous genetic diversity of HIV-1 and the unique infectious fitness of the virus. Among the repertoire of viral variants, the virus that establishes successful infection (transmitted founder [TF] virus) has not been well characterized yet. An insight into the salient features of the TF virus would go a long way toward helping with the design of an effective vaccine against HIV. Here we studied the biological properties of recently transmitted viruses isolated from infants who acquired infection from the mother and have come up with unique characterizations for the TF virus that establishes infection in the human host.

Project description:Chimeric Simian-Human Immunodeficiency Viruses (SHIVs) are an important tool for evaluating anti-HIV Env interventions in nonhuman primate (NHP) models. However, most unadapted SHIVs do not replicate well in vivo limiting their utility. Furthermore, adaptation in vivo often negatively impacts fundamental properties of the Env, including neutralization profiles. Transmitted/founder (T/F) viruses are particularly important to study since they represent viruses that initiated primary HIV-1 infections and may have unique attributes. Here we combined in vivo competition and rational design to develop novel subtype C SHIVs containing T/F envelopes. We successfully generated 19 new, infectious subtype C SHIVs, which were tested in multiple combinatorial pools in Indian-origin rhesus macaques. Infected animals attained peak viremia within 5 weeks ranging from 103 to 107 vRNA copies/mL. Sequence analysis during primary infection revealed 7 different SHIVs replicating in 8 productively infected animals with certain clones prominent in each animal. We then generated 5 variants each of 6 SHIV clones (3 that predominated and 3 undetectable after pooled in vivo inoculations), converting a serine at Env375 to methionine, tyrosine, histidine, tryptophan or phenylalanine. Overall, most Env375 mutants replicated better in vitro and in vivo than wild type with both higher and earlier peak viremia. In 4 of these SHIV clones (with and without Env375 mutations) we also created mutations at position 281 to include serine, alanine, valine, or threonine. Some Env281 mutations imparted in vitro replication dynamics similar to mutations at 375; however, clones with both mutations did not exhibit incremental benefit. Therefore, we identified unique subtype C T/F SHIVs that replicate in rhesus macaques with improved acute phase replication kinetics without altering phenotype. In vivo competition and rational design can produce functional SHIVs with globally relevant HIV-1 Envs to add to the growing number of SHIV clones for HIV-1 research in NHPs.

Project description:A genetic bottleneck is a hallmark of HIV-1 transmission such that only very few viral strains, termed transmitted/founder (T/F) variants establish infection in a newly infected host. Phenotypic characteristics of these variants may determine the subsequent course of disease. The HIV-1 5' long terminal repeat (LTR) promoter drives viral gene transcription and is genetically identical to the 3' LTR. We hypothesized that HIV-1 subtype C (HIV-1C) T/F virus LTR genetic variation is a determinant of transcriptional activation potential and clinical disease outcome. The 3'LTR was amplified from plasma samples of 41 study participants acutely infected with HIV-1C (Fiebig stages I and V/VI). Paired longitudinal samples were also available at one year post-infection for 31 of the 41 participants. 3' LTR amplicons were cloned into a pGL3-basic luciferase expression vector, and transfected alone or together with Transactivator of transcription (tat) into Jurkat cells in the absence or presence of cell activators (TNF-α, PMA, Prostratin and SAHA). Inter-patient T/F LTR sequence diversity was 5.7% (Renge: 2-12) with subsequent intrahost viral evolution observed in 48.4% of the participants analyzed at 12 months post-infection. T/F LTR variants exhibited differential basal transcriptional activity, with significantly higher Tat-mediated transcriptional activity compared to basal (p<0.001). Basal and Tat-mediated T/F LTR transcriptional activity showed significant positive correlation with contemporaneous viral loads and negative correlation with CD4 T cell counts (p<0.05) during acute infection respectively. Furthermore, Tat-mediated T/F LTR transcriptional activity significanly correlated positively with viral load set point and viral load; and negatively with CD4 T cell counts at one year post infection (all p<0.05). Lastly, PMA, Prostratin, TNF-α and SAHA cell stimulation resulted in enhanced yet heterologous transcriptional activation of different T/F LTR variants. Our data suggest that T/F LTR variants may influence viral transcriptional activity, disease outcomes and sensitivity to cell activation, with potential implications for therapeutic interventions.

Project description:Several APOBEC3 enzymes restrict HIV-1 by deaminating cytosine to form uracil in single-stranded proviral (-)DNA. However, HIV-1 Vif counteracts their activity by inducing their proteasomal degradation. This counteraction by Vif is incomplete, as evidenced by footprints of APOBEC3-mediated mutations within integrated proviral genomes of people living with HIV-1. The relative contributions of multiple APOBEC3s in HIV-1 restriction are not fully understood. Here, we investigated the activity of co-expressed APOBEC3F and APOBEC3G against HIV-1 Subtype B and Subtype C transmitted/founder viruses. We determined that APOBEC3F interacts with APOBEC3G through its N-terminal domain. We provide evidence that this results in protection of APOBEC3F from Vif-mediated degradation because the APOBEC3F N-terminal domain contains residues required for recognition by Vif. We also found that HIV-1 Subtype C Vifs, but not Subtype B Vifs, were less active against APOBEC3G when APOBEC3F and APOBEC3G were co-expressed. Consequently, when APOBEC3F and APOBEC3G were expressed together in a single cycle of HIV-1 replication, only HIV-1 Subtype C viruses showed a decrease in relative infectivity compared to when APOBEC3G was expressed alone. Inspection of Vif amino acid sequences revealed that differences in amino acids adjacent to conserved sequences influenced the Vif-mediated APOBEC3 degradation ability. Altogether, the data provide a possible mechanism for how combined expression of APOBEC3F and APOBEC3G could contribute to mutagenesis of HIV-1 proviral genomes despite the presence of Vif and provide evidence for variability in the Vif-mediated APOBEC3 degradation ability of transmitted/founder viruses.IMPORTANCEAPOBEC3 enzymes suppress HIV-1 infection by inducing cytosine deamination in proviral DNA but are hindered by HIV-1 Vif, which leads to APOBEC3 proteasomal degradation. Moving away from traditional studies that used lab-adapted HIV-1 Subtype B viruses and singular APOBEC3 enzymes, we examined how transmitted/founder isolates of HIV-1 replicated in the presence of APOBEC3F and APOBEC3G. We determined that APOBEC3F interacts with APOBEC3G through its N-terminal domain and that APOBEC3F, like APOBEC3G, has Vif-mediated degradation determinants in the N-terminal domain. This enabled APOBEC3F to be partially resistant to Vif-mediated degradation. We also demonstrated that Subtype C is more susceptible than Subtype B HIV-1 to combined APOBEC3F/APOBEC3G restriction and identified Vif variations influencing APOBEC3 degradation ability. Importantly, Vif amino acid variation outside of previously identified conserved regions mediated APOBEC3 degradation and HIV-1 Vif subtype-specific differences. Altogether, we identified factors that affect susceptibility to APOBEC3F/APOBEC3G restriction.

Dataset Information

Wide variation in susceptibility of transmitted/founder HIV-1 subtype C Isolates to protease inhibitors and association with in vitro replication efficiency.

Publications

Wide variation in susceptibility of transmitted/founder HIV-1 subtype C Isolates to protease inhibitors and association with in vitro replication efficiency.

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