CROI 2016 Abstract eBook

Abstract Listing

Poster Abstracts

206 Novel Mutant HIV-1 Strains With Greater Resistance to Cynomolgus Macaque TRIMCyp Tahmina Sultana 1 ; Emi E. Nakayama 1 ; Satoshi E.Tobita 2 ;Yohei Seki 3 ; Akatsuki Saito 4 ; Masako Nomaguchi 5 ; Akio Adachi 5 ; Hirofumi Akari 4 ;Tatsuo E. Shioda 1 1 Rsr Inst for Microbial Diseases, Osaka Univ, Suita, Japan; 2 Osaka Univ, Suita, Japan; 3 Kyoto Univ, Kyoto, Japan; 4 Inst for Virus Rsr, Kyoto Univ, Kyoto, Japan; 5 Inst of Hlth BioScis, The Univ of Tokushima Grad Sch, Tokushima, Japan Background: An antiretroviral factor, TRIM5α, produced by Old World monkeys strongly suppresses HIV-1 replication. A fusion protein comprising cynomolgus macaque (CM) TRIM5 and cyclophlin A (CM TRIMCyp) also potently suppresses HIV-1 replication. However, CM TRIMCyp fails to suppress a mutant HIV-1 that encodes a mutant capsid protein containing a SIVmac239-derived loop between α-helices 4 and 5 (L4/5) though it costs replicative capability. There are seven amino acid differences between HIV-1 L4/5 and SIVmac239 L4/5. Here, we investigated the minimum numbers of amino acid substitutions that would allow HIV-1 to evade CM TRIMCyp-mediated suppression. Methods: We used PCR-based randommutagenesis to construct a library of HIV-1 variants that contained mutations in L4/5. We then applied a functional screen to isolate resistant viruses; specifically, we recovered replication-competent viruses from CD4+MT4 cells that expressed high levels of CM TRIMCyp. Results: We generated novel mutant HIV-1 strains that were resistant to the CM TRIMCyp. CM TRIMCyp-resistant viruses were obtained after three rounds of selection in MT4 cells that expressed CM TRIMCyp. Viruses isolated under CM TRIMCyp-selection pressure contained four amino acid substitutions in L4/5 of a capsid protein that is H87R, A88G, P90D, and P93A. These four amino acid substitutions were then confirmed to be sufficient to confer CM TRIMCyp resistance to HIV-1 with efficient replicative capability. We also obtained novel CM TRIM5α -resistant HIV-1 strains that were generated via a similar selection method, which required six rounds of selection and rescue cycles. Sequence analysis of CA region of provirus in MT4 cells expressing CM TRIM5α revealed V86A and G116E mutations. V86AG116E mutation conferred partial resistance to CM TRIM5α without substantial fitness cost. Conclusions: Four amino acid substitutions in L4/5 of HIV-1 capsid protein are enough to confer resistance against CM TRIMCyp, while another two amino acid substitutions are required for partial resistance against CM TRIM5α. These results suggested that randommutagenesis of live viruses followed by the selection with restriction factors would be useful in understanding detailed molecular interaction between virus and host. 207 The G1/S Specific Cyclin D2 Acts As a Viral Restriction Factor in Primary Macrophages Ester Ballana 1 ; Roger Badia 1 ; Maria Pujantell 1 ; Eva Riveira-Muñoz 1 ; José A. Esté 1 ; Bonaventura Clotet 2 1 IrsiCaixa Inst for AIDS Rsr, Badalona, Spain; 2 Lluita Contra la SIDA Fndn, Germans Trias i Pujol Univ Hosp, Barcelona, Spain Background: Macrophages are a heterogenic cell population with properties strongly influenced by stimuli present during their differentiation frommonocytes. Distinct differentiation stimuli lead to differential cell activation and proliferation, differential expression of proteins controlling cell cycle progression and differential capacity to support HIV-1 replication. Cell cycle control plays also a fundamental role in SAMHD1-mediated virus restriction, as it is regulated through phosphorylation by cyclin-dependent kinases (CDK). Here, we describe an HIV-1 novel restriction pathway determined by the differential expression of the G1/S-specific cyclin D2. Methods: Primary monocytes were transfected with siRNA and differentiated into monocyte-derived macrophages (MDM). Monocyte differentiation consisted in 4-day incubation with M-CSF or GM-CSF. Susceptibility to HIV-1 infection was examined by flow cytometry after infection of a VSV-pseudotyped NL4-3 GFP-expressing virus. Total viral DNA formation was quantified by qPCR in MDM infected with full-replicative R5 HIV-1 strain BaL. Co-immunoprecipitation (CoIP) was performed in lysates from 293T cells overexpressing cyclin D2-HA or Flag-p21 fusion proteins and were incubated with anti-FLAG or anti-HA antibodies covalently attached to agarose. Protein expression and phosphorylation and CoIP were analyzed by immunoblotting. Results: Differential gene expression profile of cell cycle proteins was observed in GM-CSF vs. M-CSF MDM, being the most relevant the upregulation in GM-CSF MDM of all D-type cyclins (D1, 2-fold, p=0.0006; D2, 40-fold, p=0.0045; and D3, 3-fold p=0.0002, respectively) and the CDK inhibitor p21 (4-fold, p=0.038). Efficient and specific siRNA-mediated downregulation of cyclin D2 led to a significant increase of HIV-1 replication and total viral DNA formation only in GM-CSF MDM (3-fold increase; p=0.0037), but not in M-CSF MDM. CyclinD2 knockdown was linked to an increased phosphorylation of SAMHD1, without additional differences in the expression profile of cell cycle proteins. Characterization of the restriction mechanism by CoIP and additional siRNA experiments led to the identification of a protein complex formed by cyclin D2 and p21 as the responsible of a novel viral restriction mechanism. Conclusions: The G1/S specific cyclin D2 acts as a restriction factor for HIV-1 in primary macrophages. These results further demonstrate the importance of cell cycle control in viral replication. 208 Characterizing Dynamics of Proteo-Transcriptomic Response to HIV-1 Infection Monica Golumbeanu 1 ; Pejman Mohammadi 2 ; Sébastien Desfarges 3 ; Céline Hernandez 4 ; Manfredo Quadroni 5 ; AmalioTelenti 6 ; Niko Beerenwinkel 1 ; Angela Ciuffi 3 1 ETH Zürich, Basel, Switzerland; 2 New York Genome Cntr, New York, NY, USA; 3 Inst of Microbiology, Lausanne Univ Hosp and Univ of Lausanne, Lausanne, Switzerland; 4 Inst de Biologie de l’École Normale Supérieure, Paris, France; 5 Univ of Lausanne, Lausanne, Switzerland; 6 Univ of California San Diego, San Diego, CA, USA Background: During the 24-hour-long replication cycle, HIV-1 enters the host cell, integrates its genome, and utilizes the host cellular machinery in order to produce new virions. T cells respond to infection by transcriptional and proteomic changes. Here, we have conducted genome-wide measurements on transcriptomic, proteomic, and phosphoproteomic levels, in uninfected and infected CD4+ T cells, in order to define the dynamic, integrated proteo-transcriptomic response of the cell to infection. Methods: SupT1 cells were either mock-treated or infected with a VSV-G pseudotyped HIV-based vector (NL4-3ΔEnv/GFP) at a high multiplicity of infection to reach universal infection. Cells were collected at 6h intervals during 24h. Both mock and HIV-infected samples were subjected to transcriptome analysis with mRNA-Seq, and proteome and phosphoproteome analysis using SILAC. An integrative data analysis based on hierarchical clustering was used to identify infection-specific proteome and transcriptome temporal patterns. Results: We evaluated time-series transcript and protein levels in both HIV-infected and mock samples for 3285 genes and detected phosphorylation events in 524 of them. The 3285 genes were further stratified based on their combined proteo-transcriptomic HIV/mock temporal progression. We isolated gene clusters enriched in canonical pathways, including nuclear import and cell cycle, as well as HIV co-factors, supporting different stages of HIV-1 replication. We observed specific post-transcriptional and post-translational regulation after infection. A total of 307 transcriptionally invariable genes in HIV/mock, enriched in HIV-1 life cycle and host interaction factors, expressed differential behavior at proteome or phosphoproteome level. Of 279 differentially expressed proteins in HIV/mock, 16.13% (n=45) showed differential expression also at the transcriptome level. A clear separation between genes being progressively up or down-regulated over 24h was observed, as well as a consistent 6-hour delay in the protein response compared to the transcriptome. Among 93 differentially phosphorylated genes in HIV/mock, 21.5% (n=20) showed differential transcriptome or proteome expression. Conclusions: Integrated time-series proteo-transcriptomic analyses of HIV-1 and mock-infected T-cells define host response to HIV-1 infection. This study exposes novel regulation mechanisms of virus-host interaction through an integrated temporal investigation of different omics measurements. 209LB Kinesin-1 and Nup358 Cooperatively Mediate the Nuclear Import of HIV During Infection Edward Campbell ; Adarsh Dharan; SarahTalley Loyola Univ Chicago, Maywood, IL, USA Background: Fusion of viral and target cell membrane releases the viral conical capsid in to the cytoplasm of target cells which houses the viral RNA and accessory proteins for viral reverse transcription and integration in to the host cell nuclei. The viral capsid undergoes a gradual dissociation of the viral capsid core, a process called uncoating allowing subsequent nuclear import of the viral genome. Nup358, a component of the nuclear pore complex has been shown to be important for HIV-1 nuclear import and infectivity.

Poster Abstracts

80

CROI 2016