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Identification of viral immune escape mutations that compromise HIV’s ability to replicate may aid rational attenuation-based vaccine design. Previously we reported amino acids associated with altered viral replication capacity (RC) from a sequence-function analysis of 487 patient-derived RT-integrase sequences. In this study, site-directed mutagenesis experiments were performed to validate the effect of these mutations on RC. Viral reverse transcripts were measured by quantitative PCR and structural modelling was performed to gain further insight into the effect of reverse transcriptase (RT) mutations on reverse transcription. RT-integrase variants in or flanking cytotoxic T cell epitopes in the RT palm (158S), RT thumb (241I and 257V) and integrase catalytic core domain (124N) were confirmed to significantly reduce RC. RT mutants showed a delayed initiation of viral DNA synthesis. Structural models provide insight into how these attenuating RT mutations may affect amino acid interactions in the helix clamp, primer grip and catalytic site regions.

OBJECTIVES: Elite controllers (EC) are a rare group of individuals living with HIV-1 who naturally control HIV-1 replication to levels below the limit of detection without antiretroviral therapy (ART) and rarely progress to AIDS. The mechanisms contributing to this control remain incompletely elucidated. In the present study, we have assessed whether cellular host factors could modulate HIV-1 replication post-entry in a controller-discordant couple living with HIV-1. METHODS: CD4 T cells from a controller-discordant couple, one partner being an EC and the other an HIV-1 progressor (PR), and healthy controls (HC) were isolated, activated and infected with VSV-G pseudotyped yellow fluorescent protein-encoding single-round HIV-1 virus (HIV-YFP). Viral reverse transcripts, 2-LTR circles and integrated proviral HIV-1 DNA were monitored by quantitative PCR (qPCR) and integration sites were analysed. We further measured LEDGF/p75 and p21 mRNA expression levels by qPCR. RESULTS: Infection of activated CD4 T cells with HIV-YFP was reduced in EC compared with the PR partner, and HC. Evaluation of viral DNA forms suggested a block after entry and during the early steps of HIV-1 reverse transcription in EC. The integration site distribution pattern in EC, PR and HC was similar. The p21 expression in CD4 T cells of EC was elevated compared with the PR or HC, in line with previous work. CONCLUSIONS: Our study suggests a reduced permissiveness to HIV-1 infection of CD4 T cells from EC due to a block of HIV-1 replication after entry and before integration that might contribute to the EC phenotype in our patient. ispartof: JOURNAL OF VIRUS ERADICATION vol:4 issue:3 pages:170-173 ispartof: location:England status: published

BACKGROUND Human cyclophilin A (CypA) encoded by peptidyl prolyl isomerase A gene (PPIA), enhances HIV-1 replication by aiding capsid uncoating. The association of genetic variation in the PPIA regulatory region with susceptibility to HIV-1 infection, disease progression, and gene expression among black South Africans at risk for infection or infected with HIV-1 is unknown. METHODS We genotyped 539 participants from 2 longitudinal study cohorts of black South Africans at high risk for infection or infected with HIV-1 for PPIA regulatory single nucleotide polymorphisms by polymerase chain reaction-restriction fragment length polymorphism. RESULTS Minor allele (G) of SNP rs6850 (rs6850 G) significantly associated with higher viral loads (mean 4.85 versus 4.46 log copies/mL, P = 0.0006) and lower CD4 T-cell counts (mean 506 versus 557 cells/μL, P = 0.0256) during the acute phase of infection in the Centre for the AIDS Programme of Research in South Africa (CAPRISA) 002 cohort. Consistently, rs6850 G significantly associated with higher viral loads (mean 4.49 versus 4.01 log copies/mL, P < 0.0001) and lower CD4 T-cell counts (mean 442 versus 494 cells/μL, P = 0.0002) during the early chronic phase of infection in the CAPRISA 002 cohort; rs6850 G further associated significantly with rapid CD4 T-cell decline in the CAPRISA 002 cohort (P = 0.0481) and Sinikithemba chronic infection cohort (P = 0.0156). Interestingly, rs6850 G significantly associated with elevated CypA mRNA levels in HIV-1-positive individuals (P = 0.0061). CONCLUSIONS These data suggest that rs6850 G enhances HIV-1 replication through upregulation of CypA expression following HIV-1 infection. The data support ongoing efforts to develop anti-HIV-1 drugs that block interaction of HIV-1 and cellular proteins.

Elite controllers (EC) are a rare group of individuals living with HIV-1 who naturally control HIV-1 replication to levels below the limit of detection without antiretroviral therapy (ART) and rarely progress to AIDS. The mechanisms contributing to this control remain incompletely elucidated. In the present study, we have assessed whether cellular host factors could modulate HIV-1 replication post-entry in a controller-discordant couple living with HIV-1.CD4 T cells from a controller-discordant couple, one partner being an EC and the other an HIV-1 progressor (PR), and healthy controls (HC) were isolated, activated and infected with VSV-G pseudotyped yellow fluorescent protein-encoding single-round HIV-1 virus (HIV-YFP). Viral reverse transcripts, 2-LTR circles and integrated proviral HIV-1 DNA were monitored by quantitative PCR (qPCR) and integration sites were analysed. We further measured LEDGF/p75 and p21 mRNA expression levels by qPCR.Infection of activated CD4 T cells with HIV-YFP was reduced in EC compared with the PR partner, and HC. Evaluation of viral DNA forms suggested a block after entry and during the early steps of HIV-1 reverse transcription in EC. The integration site distribution pattern in EC, PR and HC was similar. The p21 expression in CD4 T cells of EC was elevated compared with the PR or HC, in line with previous work.Our study suggests a reduced permissiveness to HIV-1 infection of CD4 T cells from EC due to a block of HIV-1 replication after entry and before integration that might contribute to the EC phenotype in our patient.

SummaryDistinct integration patterns of different retroviruses, including HIV-1, have puzzled virologists for over 20 years. A tetramer of the viral integrase (IN) assembles on the two viral cDNA ends, docks onto the target DNA (tDNA), and catalyzes viral genome insertion into the host chromatin. We identified the amino acids in HIV-1 IN that directly contact tDNA bases and affect local integration site sequence selection. These residues also determine the propensity of the virus to integrate into flexible tDNA sequences. Remarkably, natural polymorphisms INS119G and INR231G retarget viral integration away from gene-dense regions. Precisely these variants were associated with rapid disease progression in a chronic HIV-1 subtype C infection cohort. These findings link integration site selection to virulence and viral evolution, but also to the host immune response and antiretroviral therapy, since HIV-1 IN119 is under selection by HLA alleles and integrase inhibitors.

LEDGF/p75, encoded by the PSIP1 gene, interacts with HIV-1 integrase and targets HIV-1 integration into active genes. We investigated the influence of polymorphisms in PSIP1 on HIV-1 acquisition and disease progression in black South Africans.Integrase binding domain of LEDGF/p75 was sequenced in 126 participants. Four haplotype tagging SNPs rs2277191, rs1033056, rs12339417 and rs10283923 referred to as SNP1, SNP2, SNP3 and SNP4, respectively, and one exonic SNP rs61744944 (SNP5, Q472L) were genotyped in 195 HIV-1 seronegative, 52 primary and 403 chronically infected individuals using TaqMan assays. LEDGF/p75 expression was quantified by real-time RT-PCR. The impact of Q472L mutation on the interaction with HIV_1 IN was measured by AlphaScreen.rs2277191 (SNP1) A was more frequent among seropositives (P = 0.06, Fisher's exact test). Among individuals followed longitudinally SNP1A trended towards association with higher likelihood of HIV-1 acquisition [relative hazard (RH) = 2.21, P = 0.08; Cox model] and it was also associated with rapid disease progression (RH = 5.98, P = 0.04; Cox model) in the recently infected (primary infection) cohort. rs12339417 (SNP3)C was associated with slower decline of CD4(+) T cells (P = 0.02) and lower messenger RNA (mRNA) levels of LEDGF/p75 (P0.01). Seroconverters had higher preinfection mRNA levels of LEDGF/p75 (P0.01) and these levels decreased after HIV-1 infection (P = 0.02).Genetic variants of PSIP1 may affect HIV-1 outcomes. Further studies are needed to confirm the effect of genetic variation of PSIP1 on HIV-1 pathogenesis in different cohorts.

Stable integration in the host genome renders murine leukemia virus (MLV)-derived vectors attractive tools for gene therapy. Adverse events in otherwise successful clinical trials caused by proto-oncogene activation due to vector integration hamper their application. MLV and MLV-based vectors integrate near strong enhancers, active promoters, and transcription start sites (TSS) through specific interaction of MLV integrase (IN) with the bromodomain and extra-terminal (BET) family of proteins, accounting for insertional mutagenesis. We identified a BET-interaction motif in the C-terminal tail of MLV IN conserved among gammaretroviruses. By deletion of this motif or a single point mutation (INW390A), BET-independent MLV (BinMLV) were engineered. BinMLV vectors carrying INW390A integrate at wild-type efficiency, with an integration profile that no longer correlates with BET chromatin distribution nor with the traditional markers of MLV integration. In particular, BinMLV vector integration associated less with oncogene TSS compared to the MLV vectors currently used in clinical trials. Together, these findings open perspectives to increase the biosafety of gammaretroviral vectors for gene therapy. ispartof: Molecular therapy. Nucleic acids vol:3 issue:7 ispartof: location:United States status: published