CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

192 HIV and SIV Inhibition by RNA-Associated Early Stage Antiviral Factor (REAF) Aine McKnight Queen Mary University of London, London, United Kingdom Background: The interaction of viruses with their human host is a constant war. The discovery of novel anti-viral restriction factors illuminates unknown aspects of innate sensing and immunity. Methods: An siRNA screen of ~20,000 human genes was used to uncover those involved in inhibition of HIV replication. We identified RNA-associated Early-stage Anti-viral Factor (REAF) as an inhibitor of HIV replication Results: 114 genes were identified to be potentially involved in intrinsic resistance. Focusing on the most potent factors led us to REAF. REAF (previously RPRD2) was annotated in the human genome but with no known function. We observed more than 50 fold rescue of HIV-1 infection following knockdown of REAF by specific siRNA. Quantitative PCR was used to measure the effect of REAF knockdown on two steps in the replication cycle – production of reverse transcripts and integration of viral cDNA. Both steps were strongly enhanced. Conversely, when REAF is over expressed in target cells fewer reverse transcripts are produced. Human REAF can also inhibit HIV-2 and simian immunodeficiency virus (SIV) infection. REAF interacts (either directly or indirectly) with HIV RNA or RNA:DNA intermediates during reverse transcription. Also, during the process of reverse transcription REAF protein is degraded, within one hour of infection, in a proteosomal dependent manner. Furthermore, REAF can inhibit HIV replication via different routes of entry into cells. Its potency is, however, highly dependent on the pathway of entry used and we show it is the lentiviral restriction factor 2 (Lv-2) 1,2 . Conclusions: We propose that REAF is part of an anti-viral surveillance system destroying incoming retroviruses. This novel mechanism could apply to invasion of cells by any intracellular pathogen. 1 Marchant, D., Neil, S. J., Aubin, K., Schmitz, C. & McKnight, A. An envelope-determined, pH-independent endocytic route of viral entry determines the susceptibility of human immunodeficiency virus type 1 (HIV-1) and HIV-2 to Lv2 restriction. J Virol 79 , 9410-9418, (2005). 2 Schmitz, C., Marchant, D., Neil, S. J., Aubin, K., Reuter, S., Dittmar, M. T. & McKnight, A. Lv2, a novel postentry restriction, is mediated by both capsid and envelope. J Virol 78 , 2006-2016, (2004). 193 Translational Control of APOBEC3G/F Restriction Factors by the HIV-1 Vif Protein Camille Libre 1 ; Santiago X. Guerrero 2 ; Julien Batisse 1 ; Roland Marquet 1 ; Jean-Christophe Paillart 1 1 IBMC CNRS, Strasbourg, France; 2 Centre for Genomic Regulation, Barcelona, Spain Background: The human immunodeficiency virus type 1 (HIV-1) requires the concerted contribution of many cellular factors to achieve efficient replication. Similarly, mammalian cells express a set of proteins called restriction factors to suppress viral replication. Among these factors, the family of APOBEC3 (Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 or A3) proteins and in particular A3G and A3F are the most efficient against HIV-1. They belong to a large family of cytidine deaminases that catalyze the deamination of cytidines to uridines in single stranded DNA substrate during HIV-1 retrotranscription and is lethal for the virus. The antiviral activity of A3G/F is counteracted by HIV-1 Vif (Viral infectivity factor) protein. Vif significantly reduces their expression in cell and their incorporation into viral particles by 1) recruiting an E3 ubiquitin ligase complex to induce their degradation by the proteasome, and 2) regulating their translation. Up to now, the mechanisms by which Vif regulates the translation of A3G/F are not known. Methods: To address the role of Vif in the regulation of A3G/F translation, we tested the importance of the unstranslated regions (UTRs) of A3G/F mRNA in the translational inhibition. HEK 293T cells were transfected with wild-type and mutated constructions of A3G and A3F mRNAs (∆UTRs, ∆5’UTR, ∆3’UTR and ∆SL) in presence or absence of Vif. These experiments were also performed with a proteasome inhibitor (ALLN) in order to distinguish the proteasomal degradation pathway from the translational inhibition. Results: Although the translation of wild-type A3G/F mRNA is significantly reduced by Vif, we showed that the suppression of their 5’UTRs does not allow these mRNAs to be regulated by Vif anymore, suggesting that the 5’UTR is important for the translation repression. Next, we showed that the two distal stem-loops in the 5’UTR of A3G mRNA are crucial for the translational inhibition. Finally, we observed a strong correlation between the level of A3G/F protein translation in cell, their incorporation into viral particles, and the infectivity of released virions. Conclusions: Experiments are in progress to identify with precision the mechanisms of A3G/F translational regulation and determine Vif domains involved in this process. Regulating the translation of A3G/F could thus be considered as a new target to restore a functional expression of A3G and viral restriction. 194 Evidence for Lentivirus-Driven Evolution of APOBEC3C CristinaWittkopp ; Michael Emerman Fred Hutchinson Cancer Research Center, Seattle, WA, US Background: The Human APOBEC3 (A3) locus is a family of seven antiviral proteins on chromosome 22. Unlike A3D, A3F, A3G, and A3H, human A3C has little to no activity against HIV-1. However, we have previously found that for A3D and A3H, the homolog from other primates more potently blocks lentiviruses than the human version of these genes. Therefore, we hypothesized that A3C may potently restrict lentiviruses in non-human primates. To test this, we cloned A3Cs from several species and assayed for lentivirus restriction, and also performed evolutionary analyses to look for evidence of a virus-host arms race involving A3C. Methods: We obtained A3C sequences from 12 primate species, and used the PAML software suite to perfom evolutionary analyses . Five of the A3C genes were cloned into expression vectors and their antiviral activity and sensitivity to Vif were functionally characterized. Antiviral activity was assessed by infectivity assays performed with HIV-1 and SIVagm in the absence of the viral antagonist, Vif. Sensitivity to Vif antagonismwas assessed using infectivity assays with HIV-1 proviruses encoding Vifs from diverse lentiviruses. Cells were co-transfected with a provirus and each A3C or a vector control, and virions were harvested and used for infection. Virion and A3C protein expression were analyzed by Elisa and Western blot. Results: A3Cs were functionally assayed for antiviral activity, and we found that in striking contrast to human A3C, several non-human primate A3Cs potently block HIV-1 and SIVagm (Vif deleted). In support of A3C being an active restriction factor, we found that A3C is evolving under positive selection. Futhermore, we identified rapidly evolving residues within A3:Vif binding domains, suggesting that activity against lentiviruses may drive the evolution of primate A3Cs. Preliminary evidence indicates that primate A3Cs have differential sensitivity to Vif antagonism. Conclusions: Our functional and evolutionary data suggest that A3C is in genetic conflict with lentiviruses. The potent anti-lentiviral activity of several primate A3Cs, as well as the rapid evolution of residues important for Vif binding, indicate that A3C may play an important role in protecting primates from lentiviruses, but that human A3C has lost this activity. Thus, humans have lost the activity of an APOBEC3 antiviral gene that is present in other primates. We speculate that this lack of activity could impact our susceptibility to cross-species viral transmissions.

Poster Abstracts

200

CROI 2015