CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

Results: We found that the majority of residues involved in binding DCAF1 were conserved in essentially all SAMHD1-degrading Vpx and Vpr. We identified highly conserved amino acids flanking the regions of SIVmac Vpx involved in binding SAMHD1. These conserved motifs served as breakpoints to create chimeric proteins between Vpr and Vpx from SIVs infecting macaque, red-capped mangabey, and African green monkeys. We were then able to retarget Vpx and Vpr from different lentiviruses to degrade heterologous SAMHD1. Depending on the lineage, the specificity of Vpx/r in degrading SAMHD1 maps to one or two regions in the viral protein. Conclusions: The structure of Vpx and Vpr proteins and their interaction with the host ubiquitin ligase is widely conserved, despite high levels of sequence variation across lineages. Given the evolutionary constriction in maintaining this ubiquitin ligase binding, similar regions of Vpr/x are used to target SAMHD1, allowing the mapping of sites that govern SAMHD1 antagonism in species-specific interactions. 204 Differentiation Stimuli Strongly Impact the Ability of Macrophages to Support HIV-1 Replication Due to SAMHD1 Restriction Ester Ballana 1 ; Roger Badia 1 ; Eva Riveira-Muñoz 1 ; Alba Ruíz 1 ; JavierTorres-Torronteras 2 ; Bonaventura Clotet 1 ; Eduardo Pauls 1 ; Ramon Marti 2 ; José Esté 1 HIV Pathogenesis Research Group 1 IrsiCaixa Institute for AIDS Research, Badalona, Spain; 2 Institut de Recerca Hospital Universitari Vall d’Hebron, Barcelona, Spain Background: Macrophages are a highly heterogenic cell population with cellular properties strongly influenced by the factors present during differentiation. Monocytes are refractory to HIV infection and only become susceptible to infection after differentiation, due to deactivation of the restriction factor SAMHD1 by phosphorylation. We have shown that SAMHD1 deactivation is controlled by cyclin-dependent kinases (CDK), which in turn, control cell activation and proliferation. Here, we used human primary monocyte-derived macrophages (MDMs) differentiated under different conditions to evaluate cell activation and proliferation, differential gene and protein expression patterns and susceptibility to HIV-1 infection. Methods: CD14+monocytes were differentiated with M-CSF or GM-CSF to generate M-CSF (M-MDM) or GM-CSF-induced MDM (GM-MDM). Expression of cell surface antigens and cell activation and proliferation markers was evaluated by flow cytometry. Susceptibility to HIV-1 infection was examined by flow cytometry after infection with a VSV-pseudotyped NL4-3 GFP-expressing virus in the presence or not of drugs targeting HIV or inhibiting cell activation or proliferation. SAMHD1 restriction was bypassed by VLP-containing Vpx. Gene expression was assessed by quantitative PCR and protein expression and phosphorylation by immunoblotting. dNTP levels were determined using a polymerase-based method. Results: MDMs displayed different morphological characteristics dependent on the differentiation conditions, but no significant differences in cell surface antigens expression were observed. M-MDM were roughly 10-fold more susceptible to HIV-1 infection than GM-MDM, which correlated with higher dNTP levels in M-MDM compared to GM-MDM (from 3 to 80 fold difference depending on the nucleotide, being dCTP the lowest and dTTP the highest). Although no differences were found in SAMHD1 expression, a change in SAMHD1 activation was observed, as a consequence of different cell activation. Degradation of SAMHD1 by Vpx increased HIV replication in both cases, minimizing the original differences in susceptibility. Accordingly, SAMHD1 degradation led to an increase in dNTP intracellular levels under both conditions. Conclusions: Differentiation strongly impacts the ability of MDMs to support HIV-1 replication, mainly due to SAMHD1 function determined by cell activation differences. These resultst suggest that the choice of macrophage model is important for evaluation of virus-cell interactions during HIV infection. 205 Essential Role of Cyclin D3 in dNTP Pool Control and HIV-1 Replication in Macrophages Ester Ballana 1 ; Alba Ruíz 1 ; Eduardo Pauls 1 ; JavierTorres-Torronteras 2 ; Roger Badia 1 ; Eva Riveira-Muñoz 1 ; Bonaventura Clotet 1 ; Ramon Marti 2 ; José Esté 1 HIV Pathogenesis Research Group 1 IrsiCaixa Institute for AIDS Research, Badalona, Spain; 2 Institut de Recerca Hospital Universitari Vall d’Hebron, Badalona, Spain Background: Cyclins control the activation of cyclin-dependent kinases (CDK), which in turn, control cell proliferation. We have shown that the virus restriction factor SAMHD1 is controlled by CDK6, a CDK controlling early G0 to G1 transition of the cell cycle. However, the cyclin controlling the process of SAMHD1 function has not been clearly identified in primary cells. Methods: Monocytes were isolated and transfected with siRNA against different cyclins controlling cell cycle. Transfected monocytes were differentiated in macrophages using M-CSF. RNA interference was assessed by quantitative PCR and Western blot. Primary monocyte-derived macrophages (MDM) were infected with a VSV-pseudotyped NL4-3 GFP-expressing virus or full-replicative R5 HIV-1 strain BaL. Total viral DNA formation was quantified by qPCR and HIV replication measured by flow cytometry. SAMHD1 phosphorylation and protein expression were analyzed by immunoblotting. dNTP intracellular levels in siRNA-treated MDMwere determined using a polymerase-based method. Results: RNAi of eight distinct cyclins led to efficient and specific downregulation (up to 90% efficacy) of the corresponding cyclin as compared to mock-transfected cells or cells transfected with a non-targeting siRNA in MDM. Downregulation of the CDK6-associated cyclin D3 showed the strongest inhibitory effect (p=0.0002) of HIV-1 replication and total viral DNA formation in acutely infected MDM. Cyclins D1 and E2 (associated to CDK6 and CDK2, respectively) had a lower inhibitory effect. Cyclin A2, B1, B2 and D2 did not block HIV-1 replication. Alternative siRNA sequences targeting cyclin D3 confirmed the effect of cyclin D3 in HIV-1 infection and total viral DNA formation. Cyclin D3 downregulation led to a significant (p<0.01) reduction of SAMHD1 phosphorylation (at residue T592), CDK2 activation as measured by phosphorylation of T160 and decreased intracellular dNTP levels. The effect of cyclin D3 RNA interference was lost after degradation of SAMHD1 by HIV-2 Vpx. Conclusions: Knockdown of cyclin D3 has a major impact in SAMHD1 phosphorylation, dNTP levels and HIV-1 reverse transcription and replication. Cyclin D3 is the catalytic partner of CDK6. Thus, our results indicate a fundamental role of the CDK6-cyclin D3 complex in SAMHD1-mediated virus restriction during GO to G1 transition. Agents targeting cell proliferation may prevent new rounds of infection and hypothetically, might prevent the proliferation of persistently infected cells. 206 Low SAMHD1 Expression Renders Activated and Proliferated CD4 + Susceptible to HIV-1 Nabila Seddiki 1 ; Nicolas Ruffin 1 ;Vedran Brezar 1 ; Diana Ayinde 2 ; Julian Schulze zurWiesch 3 ; Jan van Lunzen 3 ; Olivier Schwartz 2 ; Jean-Daniel Lelièvre 1 ; Jacques Banchereau 1 ;Yves Lévy 1 1 Inserm U955 Eq16/UPEC, Créteil, France; 2 Institut Pasteur, Paris, France; 3 University Medical Center, Hamburg-Eppendorf and Heinrich-Pette-Institute, Hamburg, Germany Background: HIV-1 replication depends on the state of cell activation and division. It is established that SAMHD1 restricts HIV-1 infection of resting CD4 + T cells. The modulation of SAMHD1 expression during T-cell activation and proliferation however remains unclear, as well as a role for SAMHD1 during HIV-1 pathogenesis. Methods: SAMHD1 expression was assessed in CD4 + T cells ex vivo , after their activation and in vitro HIV-1 infection. We performed phenotype analyzes and functional studies using CD4 + T cells from peripheral blood and lymph nodes from cohorts of HIV-1 infected subjects under anti-retroviral treatment or not, and controls. Results: We show that SAMHD1 expression decreased during CD4 + T cell proliferation in association with an increased susceptibility to in vitro HIV-1 infection. Additionally, circulating memory CD4 + T cells are enriched in cells with low levels of SAMHD1. These SAMHD1 low cells are highly differentiated, exhibit a large proportion of Ki67 + cycling cells and are enriched in Th17 cells. Importantly, memory SAMHD1 low cells were depleted from peripheral blood of HIV-infected individuals. We also found that lymph node follicular helper T (Tfh) cells lacked the expression of SAMHD1, which was accompanied by a higher susceptibility to HIV-1 infection in vitro .

Poster Abstracts

204

CROI 2015