CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

Results: Our preliminary analysis revealed that host genome DNA methylation profile is impacted by HIV-1 infection and highlighted several significantly differentially methylated sites (P<10 -7 ). Most genes where these differentially methylated sites are located have an immune-related function or were previously shown to interact with HIV-1 proteins ( MX1 , TNFAIP8 , PARP9 , and IFI44L genes in the top 5 hits). Conclusions: We established a unique collection of samples representing pre-infection and post-infection timepoints, which allows for detection of DNA methylation changes within an individual and between individuals following HIV infection and during the HIV-1 infection course. This first epigenome-wide association study conducted in HIV-infected subjects has identified targets of epigenetic modifications by HIV. Our report indicates that the exploration of host epigenetic mechanisms opens a new promising avenue for discovery of critical host factors interacting with the virus that might be leveraged for translation to drug or vaccine development. 198 IN Variants Retarget HIV-1 Integration and Are AssociatedWith Disease Progression Frauke Christ 1 ; Jonas Demeulemeester 1 ; Zeger Debyser 1 ; Rik Gijsbers 1 ; Jan De Rijck 1 ;Thumbi Ndung’u 2 ; Paradise Madlala 1 1 KU Leuven, Leuven, Belgium; 2 University of KwaZulu-Natal, Durban, South Africa Background: Distinct 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) to form the target capture complex (TCC) and catalyzes viral genome insertion into the host chromatin. Methods: We combined structural information on the Prototype Foamy Virus TCC with conservation in retroviral IN protein alignments to determine aa-tDNA base contacts. We generated HIV-1 variants based on the observed variability at these positions, assessed replication capacities and performed integration site sequencing to reveal their integration preferences. Finally, we examined their effect on disease progression in a chronic HIV-1 subtype C infection cohort. Results: We identified retroviral IN amino acids affecting molecular recognition in the TCC and resulting in distinct local tDNA nucleotide biases. These residues also determine the propensity of the virus to integrate into flexible tDNA sequences. Remarkably, natural polymorphisms IN S119G and IN R231G 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. Conclusions: Our findings reveal how polymorphisms at positions corresponding to HIV IN 119 and IN 231 affect local as well as global integration site targeting. Intriguingly, these findings link integration site selection to virulence and viral evolution but also to the host immune response and antiretroviral therapy, since HIV-1 IN 119 is under selection by HLA alleles and integrase inhibitors. 199 HIV-1 Integration Sites in Macrophages and CD4 + T Cells Are Distinct Yik Lim Kok ;ValentinaVongrad; Mohaned Shilaih; Herbert Kuster; Roger Kouyos; Huldrych F. Günthard; Karin J. Metzner University Hospital Zurich, Zurich, Switzerland Background: The host genetic surroundings of integrated HIV-1 provirus have great impact on the activity of the HIV-1 LTR . Thus, HIV-1 integration site (IS) studies will contribute to the understanding of how to reactivate latent HIV-1 provirus, a currently pursued approach to eradicate the latent reservoir. Analysis of in vivo HIV-1 IS in macrophages is not feasible as it is too invasive to obtain them from HIV-1 + patients. Moreover, IS analysis in monocyte-derived macrophages (MDMs) has only been done in vitro and was never compared to CD4 + T cells within the same experimental settings. In this study, we compared and characterized HIV-1 IS in treated HIV-1 + patients’ MDMs and activated CD4 + T cells infected ex vivo with the patients’ autologous primary HIV-1 isolates. Methods: 7 patients from the Zurich Primary HIV Infection study were selected based on the following inclusion criteria: (i) Successful viral suppression for ≥ 2 years and (ii) Efficient replication of autologous primary HIV-1 isolates in donors’ MDMs. Patients’ autologous HIV-1 and the clonal HIV-1 JR-FL strain were used. HIV-1 IS were amplified with optimized non-restrictive linear amplification–mediated PCR, and sequenced using next generation sequencing technology. Sequencing reads were subjected to high quality trimming and mapped to the human genome. Gene clustering was done using the online bioinformatics tool DAVID. Fischer’s exact test was used for statistical analysis. Results: A total of 1160 unique HIV-1 IS were analysed. As expected, HIV-1 favours integration into introns of genes. However, autologous HIV-1 was less likely to integrate into introns of genes in MDMs compared to CD4 + T cells (p<0.01). Significant difference was not observed between autologous HIV-1 and HIV-1 JR-FL in MDMs for all genetic features examined. Consistently, analysis with DAVID showed that 37.5% versus 15.8% of enriched gene clusters (DAVID enrichment score ≥ 1.3) in MDMs infected with HIV-1 JR-FL and CD4 + T cells infected with autologous HIV-1, respectively, overlap with those in MDMs infected with autologous HIV-1. Additionally, we have identified 12 genes in which at least 3 different HIV-1 IS are found between cell types and/or HIV-1 stains. 5 of these genes form an enriched cluster associated with catabolic processes and 3 have been found previously in clonally expanded CD4 + T cells in vivo . Conclusions: HIV-1 IS patterns between MDMs and CD4 + T cells are distinct. Nonetheless, MDMs and CD4 + T cells have common hotspots for HIV-1 integration. 200 A Screening for DNA Repair Enzymes That Affect HIV-1 Infection Noriyoshi Yoshinaga 1 ;Yusuke Matsui 1 ; Keisuke Shindo 1 ; shunichiTakeda 1 ; AkifumiTakaori-Kondo 1 1 Kyoto University, Kyoto-shi, Japan; 2 Kyoto University, Kyoto-shi, Japan Background: DNA repair enzymes might affect HIV-1 infection, as DNA intermediates of the virus play critical roles in the viral life cycle. Although several such molecules have been reported, interactions between HIV-1 and host DNA repair molecules have not been fully elucidated. Methods: To screen for DNA repair enzymes that affect HIV-1 infectivity, a set of 33 DNA-repair-deficient DT40 cell lines with a single DNA repair gene deletion was tested for infectivity of VSV-G pseudo-typed NL4-3 with a luciferase reporter. To elucidate which steps in the viral life cycle candidate molecules function, late reverse transcription products, 2-LTR circle DNA, and integrated provirus were measured by quantitative PCR. To ask whether human RAD18 suppresses HIV-1 infection in human cells, knockdown of RAD18 by siRNA was applied to Jurkat cells and the virus infectivity was tested. To identify responsible domain of RAD18, over-expression of RAD18 wild-type, C28F, D221A or L274P was applied to 293T cells, and the virus infectivity was tested. Results: HIV-1 infectivity in 8 of 33 DT40 cells was less than 50% of that in wild-type cells, suggesting the genes deficient in these cells might be required for efficient HIV-1 infection. On the other hand, the infectivity in Rad18 -/- cells was about 3-fold higher than that in wild type cells, suggesting Rad18 might inhibit HIV-1 infection. Quantitative PCR analyses showed that 4 of 8 DT40 cells with less infectivity accumulated 2-3 fold more 2-LTR circles and less than 50% integrated provirus, suggesting these genes might be required for efficient integration. In addition, Rad18 -/- cells accumulated about 5-fold more viral late RT product at 4h post-infection, suggesting Rad18 might inhibit reverse transcription. Jurkat cells that were transfected with siRNA for human RAD18 showed about 1.5-fold increase in HIV-1 infectivity when compared to cells transfected with a non- silencing control siRNA. Over-expression of RAD18 wild-typed, C28F RING domain mutant or C207F zinc-finger domain mutant in 293T cells inhibited HIV-1 infection, but that of L274P SAP domain mutant of RAD18 did not. Conclusions: We have identified several candidate DNA repair genes that might support HIV-1 infection. We confirmed that RAD18 suppresses HIV-1 infection, and found that substitution of a DNA-binding residue lost this suppression. Our data support a model in which RAD18 inhibit reverse transcription by directly binding to viral DNA intermediates.

Poster Abstracts

202

CROI 2015