CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

195 Reevaluation of the Role of the Small Host Cell GTPase Rab6 in the HIV-1 Replication Cycle Isaac Zentner ; Simon Cocklin Drexel University College of Medicine, Philadelphia, PA, US

Background: The HIV-1 genome consists of only 9 genes and thus, depends highly on host cellular factors to replicate. Recent studies, through genome-wide siRNA screens, have identified many possible “HIV-1 dependency factors” HDFs. However, there lies a large inconsistency between these analyses and the majority of potential hits have not been verified in primary human target cells. Therefore, the goal of this proposal is to validate a recently identified possible HIV-1 Env-mediated host factor, Rab6A GTPase. Methods: We utilized siRNA mediated knockdown in multiple cell lines and primary human monocyte-derived macrophages (MDMs) coupled with a HIV-1 single-round infection assay to define the mechanism by which this protein facilitates HIV-1 infection. Additionally, because Rab6A is ubiquitously expressed in two closely related isoforms, Rab6A and Rab6A’, that have similar but distinct roles in the cell, both isoforms were investigated. Given that Rab6A is always cycling between active and non-active states, we examined the activity state needed for HIV-1 infection through dominant and constitutively active mutants of Rab6A, as well as knocking down the Rab6A-specific guanine nucleotide exchange factor (GEF), Ric1/Rgp1. Results: siRNA knockdown of both isoforms of Rab6 in the U87 cell line confirmed its role in the viral replication cycle. Moreover, down regulation of both Rab6 isoforms in 293T cells used to generate virus demonstrated no effect on the overall production or infectivity of viruses, demonstrating that the protein facilitates processes only in the early stage of the replication. Contrary to previous reports, no dependency of the effect of knock down of Rab6 on the HIV-1 envelope was observed. Specific knock down of the individual isoforms, Rab6A and Rab6A’, demonstrated that both were utilized by the virus but HIV-1 had a greater dependency upon Rab6A. Similar results were obtained with knockdown studies using primary human monocyte-derived macrophages. Diminishing Ric1/Rgp1 decreased infectivity revealing this Rab6A-specific GEF as an additional host factor for HIV-1. Conclusions: Taken as a whole, these results demonstrate that Rab6 is co-opted by HIV-1 to facilitate a post entry step in the infection process and this is dependent on Ric1/Rgp1 activity. Further investigations may reveal a novel therapeutic pathway to combat HIV-1/AIDS, as well as help dissect the fundamental biological role of Rab6A(A/A’) in primary human macrophages. 2:30 pm– 4:00 pm Nucleus Entry, Integration, and Export 196 The Mutated Form of Transportin 3 From Patients With Limb-Girdle Muscular Dystrophy 1F Hijacks Wild-Type Transportin 3 and Interferes With CPSF6 Subcellular Localization, Impairing HIV-1 Nuclear Entry Sara Rodriguez-Mora 1 ; Mayte Coiras 1 ; Mercedes Bermejo 1 ; Elena Mateos 1 ; Ramon Marti 2 ; Juan JesusVilchez 3 ; Antonio Luis Andreu 2 ; Jose Alcami 1 On behalf of the AIDS Immunopathogenesis Unit 1 Instituto de Salud Carlos III, Madrid, Spain; 2 Vall d’Hebron Institut de Recerca, Barcelona, Spain; 3 Hospital Universitari i Politecnic La Fe, Valencia, Spain Background: Cells from patients with LGMD1F (limb-girdle muscular dystrophy 1F) show a point heterozygous mutation in transportin 3 ( TNPO3 ) gene. These cells expressed both TNPO3-wt and a mutated formwith additional 15aa in the C terminus (TNPO3-mut). The interaction between TNPO3 and CPSF6 has been involved in HIV-1 decapsidation and transport of viral DNA to the nucleus, as well as in gene splicing. Our group described previously at HIV-1 pre-integration level was impaired in CD4+ T cells from LGMD1F patients. Now we have immortalized B lymphocytes from LGMD1F patients with the Epstein-Barr virus in order to characterize the mechanism of action of TNPO3-mut to thwart HIV-1 replication. Methods: The interaction between TNPO3-wt and TNPO3-mut was analyzed in Jurkat E6-1 cells by co-transfection of tagged proteins along with pNL4-3_Renilla infectious clone. The level of expression of CPSF6 in LGMD1F cells and the role of TNPO3-mut in the viral splicing was analysed in B cells immortalized with Epstein-Barr virus from four patients with LGMD1F and two healthy relative controls. B cells were infected with a pseudotyped VSV-DEnv NL4-3_LUC virus. Results: 1) Co-transfection of pNL4-3_Renilla in the presence of increasing concentrations of tagged TNPO3-mut proved that this formwas interfering with HIV-1 replication in Jurkat E6-1 cells, likely due to the hijacking of TNPO3-wt, as was observed by immunoprecipitation. 2) Analysis by immunoblotting and immunofluorescence showed that LGMD1F cells had higher levels of the splicing proteins CPSF6 and SC35 in the nucleus, although the viral splicing was not affected. Conclusions: HIV-1 replication was thwarted in LGMD1F cells due to the hijack of TNPO3-wt by TNPO3-mut, avoiding the translocation of HIV-1 DNA to the nucleus. As the entry of CPSF6 in the nucleus depends on TNPO3, the presence of TNPO3-mut was somehow inducing the accumulation of high levels of this splicing protein in the nucleus of LGMD1F cells, although the viral splicing was not affected in the immortalized LGMD1F B cells. More experiments would be necessary to determine whether TNPO3-mut is interfering with viral decapsidation through the inhibition of CPSF6 activity. 197 Longitudinal Epigenome-Wide Association Study of Pre- and Post-HIV –I nfected Subjects Sophie Limou 1 ; GeorgeW. Nelson 1 ; Elizabeth Binns-Roemer 1 ; Jia Li 1 ; Fathi Elloumi 1 ; James J. Goedert 3 ; Cheryl A.Winkler 1 1 Frederick National Laboratory Operated by Leidos Biomedical Research, Inc, for the National Cancer Institute, Frederick, MD, US; 2 Frederick National Laboratory Operated by Leidos Biomedical Research, Inc, for the National Cancer Institute, Frederick, MD, US; 3 National Cancer Institute (NCI), Bethesda, MD, US Background: For nearly 25 years, extensive genetic and genomic association studies have revealed essential host factors for HIV control and disease progression, which notably led to the development of a new class of antiretroviral inhibitors ( CCR5- n 32 association and CCR5 antagonists). Overall, the identified associations account for ~20% of the phenotypic variance suggesting that other factors are yet to be discovered. Epigenetic mechanisms are key regulators of gene expression that are not coded by DNA primary sequence and can impact complex diseases. Here, we evaluated for the first time whether HIV-1 infection modifies the host epigenome DNA methylation patterns. Methods: We recruited 19 untreated HIV-infected individuals from the DC Gay cohort with longitudinal follow-up and PBMC samples available from pre-infection, early post- infection (<12 months), post-infection during clinical latency and post-infection at or near the inflection point. Following DNA and RNA extraction from the 80 PBMC samples, DNA was bisulfite-converted and genotyped with the Illumina Infinium HumanMethylation450 arrays, covering over 485,000 methylation sites across the genome. After normalizing the data, we compared the DNA methylation profiles of pre-infection vs. post-infection samples adjusting for batch effect, age, stage of infection and cell composition. WEDNESDAY, FEBRUARY 25, 2015 Session P-A2 Poster Session Poster Hall

Poster Abstracts

201

CROI 2015