CROI 2016 Abstract eBook

Abstract Listing

Poster Abstracts

Results: Vif derivatives that do not bind to CBFβ preferentially bind to MDM2, and over-expression of CBFβ disrupted the interaction between MDM2 and Vif. Knockdown of CBFβ reduced steady-state level of Vif in MDM2 -proficient cells, but not in MDM2 -null cells. Kinetic studies revealed that Vif E88A/W89A, which is deficient in CBFβ binding, degraded faster than wild-type Vif in MDM2 -proficient cells, but not in MDM2 -null cells, suggesting that Vif stabilization by CBFβ is mainly caused by impairing MDM2-mediated degradation. We identified Vif R93E as a Vif variant that does not bind to MDM2, and the virus with this substitution was more resistant to A3G than parental virus. Arginine residue at position 93 is located on the surface of the crystal structure of Vif complex, and not fully covered by, but very close to CBFβ, supporting our hypothesis. Combinatory substitution of Vif residues required for binding CBFβ and MDM2 showed comparable steady-state levels to that of wild-type Vif, but partial counteraction to A3G, suggesting other essential roles for CBFβ than Vif augmentation. Conclusions: Vif augmentation by CBFβ is mainly due to protection fromMDM2-mediated degradation.R93 of Vif is involved in MDM2 binding, and R93E-horboring virus is more resistant to A3G. Stabilization of Vif is not the only function of CBFβ for supporting Vif-mediated counteraction to A3 proteins. 203 MARCH8 Restricts HIV-1 Infection by Reducing Envelope Incorporation Into Virions Kenzo Tokunaga 1 ;TakuyaTada 1 ;Yanzhao Zhang 1 ;Takayoshi Koyama 1 ; MinoruTobiume 1 ;YasukoTsunetsugu-Yokota 1 ; ShojiYamaoka 2 ; Hideaki Fujita 3 1 Natl Inst of Infectious Diseases, Tokyo, Japan; 2 Tokyo Med and Dental Univ, Tokyo, Japan; 3 Nagasaki Intl Univ, Nagasaki, Japan Background: Membrane-associated RING-CH (MARCH) 8 is a member of a recently discovered MARCH family of RING-finger E3 ubiquitin ligases, 11 members of which have been identified. MARCH8 downregulates multiple host transmembrane proteins, such as MHC-II, CD86, IL-1 receptor accessory protein, TRAIL receptor 1, and transferrin receptor. However, its physiological roles remain largely unknown. Here, we identify MARCH8 as a novel antiviral factor. Methods: An envelope (Env)-deficient HIV-1 luciferase reporter construct was cotransfected with wild-type and mutant MARCH8 expression plasmids, together with a plasmid expressing vesicular stomatitis G (VSV-G) or HIV-1 Env, into 293T cells. The resulting viruses were used for infectivity/entry assays and ultracentrifugation analyses, and producer cells were subjected to flow cytometry, immunofluorescence or immunoblotting analyses. Interaction between MARCH8 and HIV-1 Env was analyzed by co-immunoprecipitation. MARCH8 knockdown/knockout experiments in macrophages were performed using lentiviral shRNA and CRISPR/Cas9 systems. Results: The ectopic expression of MARCH8 in virus-producer cells, but not in target cells, impaired viral infectivity. MARCH8 substantially reduced viral entry by blocking the incorporation of HIV-1 Env into virus particles. This was due to MARCH8-mediated Env downregulation from the cell surface likely through an interaction. The inhibitory effect of MARCH8 on VSV-G was similar but even more marked, implying a broad-spectrum inhibition of enveloped viruses. Notably, the endogenous expression of MARCH8 was especially high in monocyte-derived macrophages and dendritic cells. HIV-1 infectivity and multiple replication were strongly enhanced by MARCH8 depletion in macrophages. Conclusions: Our findings indicate that MARCH8 is highly expressed in terminally differentiated myeloid cells and is a powerful antiviral protein that targets viral envelope glycoproteins by reducing their virion incorporation. 204 Sirtuins Modulate HIV Integration and Replication: New Cellular Anti-HIV Targets Michelino Di Rosa 1 ; Ilaria Gnemmi 2 ; Beatrice Riva 2 ; Ubaldina Galli 2 ; Armando Genazzani 2 ; Pier Luigi Canonico 2 ; Marilia R. Pinzone 1 ; Fabrizio Condorelli 2 ; Giuseppe Nunnari 1 1 Univ of Catania, Catania, Italy; 2 Sch of Pharm, Univ of Piemonte Orientale A. Avogadro, Novara, Italy Background: Highly active antiretroviral therapy (HAART) has increased the survival of HIV-infected individuals, although it cannot eradicate the virus. At the molecular level, HIV life cycle critically relies not only on the action of viral proteins, but also on host genes. In particular, integration of the proviral DNA into the host genome is due to the interaction between proviral DNA complex, integrase, and broken-DNA fixing proteins, provided by the host cell, in a process called “post-integration repair”. We investigated the potential impact of those enzymes that catalyse the removal of acetyl groups from chromatin proteins on HIV integration. In particular, we studied the sirtuin class of proteins, since this family of NAD + -dependent deacetylases is recruited to the sites of DNA damage to establish functional interactions with “repairing factors”, such as Ku70 or the ATM/Nsb1 complex. Methods: To quantitatively assess the integration of viral DNA into the host genome, we challenged HeLa cells with an HIV-based, replication-defective, lentivirus that carries the GFP reporter gene. By infecting HeLa cells in a range of 0.1-0.2 MOI, we were able to measure “integrational” events by flow cytometry, counting and sorting cells characterized by a brighter green fluorescence, enabled by the integration of the GFP gene into the host genome. Alu- gag integrated DNA was measured to confirm the results. Sirtuins inhibitors were identified and tested from a library of commercially available and newly synthesized compounds. Results: Two molecules, B2 and compound-2, were capable to inhibit HIV integration as efficiently as the integrase inhibitor raltegravir with a good concentration/toxicity profile. Importantly, flow cytometry data were validated by the detection and quantification of the Alu - gag sequences, via nested PCR. To further show the involvement of sirtuins in the molecular steps leading to viral integration, treatments with resveratrol, which activates the whole family of de-acetylases by increasing intracellular NAD + levels, enhanced HIV integration. Conclusions: Taken together, our observations provide the first evidence that sirtuins are involved in the HIV integration process, hence they may be considered as a new potential class of targets for HIV therapy. Importantly, this paves the way to the development of drugs able to inhibit HIV life cycle on the cellular side in order to avoid resistance to therapies. 205 SAMHD1 Phosphorylation Affects dNTPase Activity and HIV-1 Replication Capacity Roger Badia 1 ; Eva Riveira-Muñoz 1 ; Maria Pujantell 1 ; JavierTorres-Torronteras 2 ; Bonaventura Clotet 3 ; Luis Menéndez-Arias 4 ; Ramón Martí 2 ; Ester Ballana 1 ; José A. Esté 1 1 IrsiCaixa Inst for AIDS Rsr, Badalona, Spain; 2 Vall d`Hebron Inst de Recerca, Barcelona, Spain; 3 Lluita Contra la SIDA Fndn, Germans Trias i Pujol Univ Hosp, Barcelona, Spain; 4 Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas UAM), Madrid, Spain Background: SAMHD1 is a triphosphohydrolase that restricts HIV-1 by limiting the intracellular dNTP pool required for reverse transcription. Lose of SAMHD1 antiviral activity in cycling cells has been associated to CDK-dependent phosphorylation at residue T592, without affecting its tryphosphohydrolase activity. In addition, an endonuclease function of SAMHD1 has been postulated as the mode of anti-HIV-1 activity. A thorough evaluation of SAMHD1 function depending on its phosphorylation state is required. Methods: HIV-1 replication capacity was assessed in CD4+MT4 and TZM cell lines. Knock-out (KO) of SAMHD1 was achieved though CRISPR-Cas9 genome-editing of SAMHD1 TZM cells. SAMHD1 phosphorylation sites were predicted in silico and phosphorylation defective mutants were constructed by site-directed mutagenesis. Functional evaluation of SAMHD1 mutants was performed by transient expression in SAMHD1 KO cells. HIV-1 replication capacity in the presence of AZT or control drugs was evaluated by b-Gal staining at 72h post-infection. HIV-2 Vpx expression was achieved by cell transduction with viral like particles containing Vpx. SAMHD1 expression and phosphorylation was analyzed by Western blot. Results: SAMHD1-induced degradation with HIV-2 Vpx affects the dNTP pool and HIV-1 replication capacity, measured through its sensitivity to AZT. Similarly, SAMHD1 KO cells showed increased replicative capacity in the presence of AZT. Re-expression of wild type SAMHD1 reduced virus replication in the presence of AZT but sensitivity to a non- nucleoside inhibitor (nevirapine) or the integrase inhibitor raltegravir was not affected. Five phosphorylation sites were: 4 located at the N-terminus (S18, T21, T25, S33) and T592 at the C-terminus. Phosphorylation defective mutants were constructed and expressed in KO cells. Single point mutations at the N-terminal region of SAMHD1 did not significantly affect the overall phosphorylation. However, the combination of three mutations (S18A, T21A, T25A) or mutation T592A significantly prevented SAMHD1 phosphorylation and decreased HIV-1 replication in the presence of AZT, indicating that a change in dNTP levels affected HIV-1 replication. Conclusions: Phosphorylation of the N-terminal region as well as residue T592 affects SAMHD1 function affects virus restriction capacity. Thus, inactivation of SAMHD1 by phosphorylation affects HIV-1 replication through modifications in the dNTP pool, suggesting that the SAMHD1 tryphosphohydrolase activity is responsible for HIV-1 restriction.

Poster Abstracts

79

CROI 2016