CROI 2017 Abstract e-Book

Abstract eBook

Poster and Themed Discussion Abstracts

responsible for the effect of CsA on HIV-1 infection and Mx2 sensitivity. Some, but not all of these nucleoporins also interact with the viral CA. Finally, we demonstrate that the N-terminal domain of Mx2 attached to a heterologous scaffold that does not localize to the nuclear pore complex is still able to restrict HIV-1 infection, albeit with altered requirements for nucleoporins. Conclusion: Our data highlight the complexity of the interaction between HIV-1, the nuclear pore complex, and other cellular proteins that interact with the viral capsid, and reveal heterogeneity in nucleocytoplasmic trafficking that influences viral infection and susceptibility to an innate immune effector. 161 UBP43 (USP18) ABROGATES SAMHD1-MEDIATED RESTRICTION OF HIV-1 Edmund Osei-Kuffour 1 , Kerstin Schott 2 , Dieter Häussinger 1 , Philipp A Lang 1 , Karl S. Lang 3 , Renate König 4 , Carsten Münk 1 1 Heinrich Heine Univ Hosp, Düsseldorf, Germany, 2 Paul Ehrlich Institut, Fed Inst for Vaccines and BioMeds, Düsseldorf, Germany, 3 Univ Hosp, Essen, Düsseldorf, Germany, 4 Paul Ehrlich Institut, Langen, Germany Background: The innate immune system is the first line of defense against pathogens including HIV-1. Host restriction factors are mediating the innate immune responses including the dNTPase, SAMHD1, which is efficiently antagonized by the HIV-2/SIV viral protein VPX. SAMHD1 is a protein that strongly contributes to the HIV-1 resistance of myeloid cells and of resting CD4+ T-cells. SAMHD1’s dNTPase and antiviral functions in non-cycling cells are thought to be negatively regulated by phosphorylation at residue T592 by cyclin A2/CDKs. The specific phosphatase that renders SAMHD1 active and the mechanism of SAMHD1 mediated restriction in the non-cycling cells is not totally clear. Intrigued by the recent model of enforced viral replication in murine USP18 expressing macrophages (Honke et al. Nature Immunology 2011), we asked whether human USP18 ISG15 isopeptidase would be a factor influencing HIV-1 replication. Methods: THP-1 macrophage-like cell lines were generated that stably expressed USP18 or active site mutants C64A and C64S. Undifferentiated cycling and PMA-differentiated THP-1 cells were infected by luciferase reporter viruses based on HIV-1 and HIV-2 (+/- vpx). The viral transduction was monitored by their luciferase activity in cell lysates. Immunoblots analyzed the cellular expression of USP18, SAMHD1 and phospho-SAMHD1. The interaction of SAMHD1 with USP18 was analyzed by pull-down assays and also visualized by confocal microscopy. Furthermore, the effect of USP18 on the cell cycle was analyzed by flow cytometry after fluorescent staining of the cellular DNA with propidium iodide. Results: PMA-differentiated THP-1 cells are resistant to HIV-1 infection due to the expression of unphosphorylated SAMHD1. This restriction is ablated when USP18 is expressed. We found that the expression of USP18 in differentiated THP-1 cells increases HIV-1 infection by 16-fold and HIV-2Δvpx by 7-fold. USP18 directly bound to SAMHD1 in the cell nucleus, which induced phosphorylation of SAMHD1. Surprisingly, the presence or absence of USP18 did not influence the interaction of SAMHD1 and its kinases CDK1/2. However, USP18 interacted with S-phase Kinase Associated Protein 2 (SKP2) and retained cylin A in differentiated THP-1.USP18 cells. These activities of USP18 were independent of its ISG15 isopeptidase activity. Conclusion: This report provides first evidence of direct involvement of USP18 in SAMHD1 restriction of lentiviruses. 162 LNCRNA DISCOVERY IN THE HIV REPLICATION CYCLE ADDS A NEW LAYER IN HIV-HOST INTERPLAY Wim Trypsteen 1 , Pejman Mohammadi 2 , Clarissa Van Hecke 1 , Pieter Mestdagh 1 , Steve Lefever 1 , Pieter De Bleser 3 , Jo Vandesompele 1 , Angela Ciuffi 2 , Ward De Spiegelaere 1 , Linos Vandekerckhove 1 1 Ghent Univ, Ghent, Belgium, 2 Univ of Lausanne, Lausanne, Switzerland, 3 Inflammation Rsr Cntr, Flanders Inst of BioTech, Ghent, Belgium Background: Studying the effects of HIV infection on the host transcriptome has typically focused on protein-coding genes. However, recent advances in the field of RNA sequencing revealed that long non-coding RNAs (lncRNAs) add an extensive additional layer to the cell’s molecular network and are crucial for normal cellular function. lncRNAs exert the ability to control a wide range of (post-)transcriptional processes and offer unique possibilities for pathogens like HIV to hijack the cellular machinery and reshape gene expression in their favor. Therefore, lncRNA discovery can result in new insights into the HIV-host interplay. Methods: We performed transcriptome profiling throughout a characterized primary HIV infection in vitro to investigate lncRNA expression at the different HIV replication cycle processes (reverse transcription, integration and particle production). Subsequently, guilt-by-association, transcription factor and co-expression analysis were performed to infer biological roles for the lncRNAs identified in the HIV-host interplay. Results: Throughout the HIV replication cycle we identified 387 lncRNAs that were differentially expressed with the majority observed at the viral integration phase. Many of these lncRNAs (173) were suggested to play a role in mechanisms at the heart of HIV-host interplay that rely on proteasomal and ubiquitination pathways (113), apoptosis inhibition (12), BRCA1/2 DNA damage responses and ATR cell cycle regulation (12). Through transcription factor binding analysis, we found that lncRNAs display a distinct transcriptional regulation profile (ao. TAF1/3/7, CHD1 and ATF3) as compared to protein coding mRNAs (ao. KLF4, SUZ12 and SOX2), suggesting that mRNAs and lncRNAs are independently modulated during HIV replication. In addition, we identified five differentially expressed lncRNA-mRNA pairs with mRNA involvement in HIV pathogenesis with possible cis regulatory lncRNAs that control nearby mRNA expression and function (ao. lnc-HES5-1 and TNFRSF14). Conclusion: Altogether, the present study demonstrates that lncRNAs add a new dimension to the HIV-host interplay and exhibit an independent transcriptionally regulated response. These identified lncRNAs are involved in viral and antiviral response pathways and should be further investigated as they may represent possible biomarkers or targets for controlling HIV replication.

Poster and Themed Discussion Abstracts

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CROI 2017