CROI 2016 Abstract eBook

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Poster Abstracts

POSTER ABSTRACTS

182 Identification of MDM2/HDM2 as a Positive Regulator of HIV-1 in Human Macrophages

Yann Breton ; Alexandre Deshiere; Michel Ouellet; Michel J.Tremblay Univ Laval - Cntr de Recherche du CHU de Québec, Québec, QC, Canada

Background: Macrophages play an important role in the establishment and propagation of HIV-1 infection. Upon exposure to HIV-1, only a small proportion of macrophages are infected whereas most remain uninfected. To shed light on this issue, transcriptomic and proteomic analyses were performed to compare the infected and bystander populations and determine the molecular basis of HIV-1 permissiveness in macrophages. Precise mapping of the interactions between HIV-1 and host signaling pathways identified several new potential regulators of HIV-1 replication in macrophages. One of these was the human ortholog of murine double minute 2 (mdm2), HDM2, an E3 ubiquitin ligase regulating the turn-over of various proteins, including p53. Methods: We developed an experimental model based on the identification and isolation of infected monocyte-derived macrophages (MDMs) within a heterogeneous population. MDMs are exposed to a competent R5 HIV-1 reporter virus expressing all viral genes before immuno-magnetic sorting. Transcriptomic and proteomic analyses led to the selection of 50 genes that were all tested for their functional role on HIV-1 replication by siRNA screen. MDMs were reverse transfected with 2 different siRNA sequences per target and infected with GFP- or luciferase-encoding HIV-1. Results: Although silencing of some specific targets resulted in an increase in viral replication, only the knockdown of MDM2/HDM2 induced a 2-fold decrease of HIV-1 expression. This reduction in viral replication was confirmed by flow cytometry and p24 ELISA immunoassay. Viability assays further demonstrated that no apparent toxicity was associated with MDM2/HDM2 silencing at 72h. Efficiency of siRNA-mediated silencing was assessed by qRT-PCR and western blot analysis and revealed that no change in the mRNA level of MDM2/HDM2 could be observed at 72h post-transfection. Earlier time points were thus tested and showed an efficient silencing of MDM2/HDM2 only at 24h post-transfection. Conclusions: Our results indicate that the resistance to HIV-1 associated with MDM2/HDM2 silencing is maintained in MDMs even if MDM2/HDM2 mRNA level is restored, thus suggesting that this protein might be indirectly involved in HIV-1 infection. Identification of viral cofactors regulated by MDM2/HDM2 will bring a new understanding of signaling events controlling HIV-1 replication in macrophages. 183 HIV-1 Induces p21-Mediated Cellular Senescence in Human Primary Macrophages Eva Riveira-Muñoz 1 ; Roger Badia 1 ; Maria Pujantell 1 ; Bonaventura Clotet 2 ; Ester Ballana 1 ; José A. Esté 1 1 IrsiCaixa Inst for AIDS Rsr, Badalona, Spain; 2 Lluita Contra la SIDA Fndn, Germans Trias i Pujol Univ Hosp, Barcelona, Spain Background: Cellular senescence (CS) represents a state of permanent cell cycle arrest in response to a variety of stressors in which cells irreversibly stop dividing but remain metabolically active. This state is maintained by either or both the p53/p21 and p16INK4a/pRb tumor suppressive pathways that cross-regulate each other. HIV-1 infection is a known external inducer of cell cycle arrest and cellular senescence and although is associated with clinical symptoms of accelerated aging, it has been difficult to detect an accelerated aging effect at a molecular level. Methods: Monocytes purified from healthy blood donors were differentiated to macrophages (MDM) with M-CSF and infected with a full-replicative R5 HIV-1 strain BaL. Senescent cells were identified by staining of SA- β-gal and visualization under optical microscope. mRNA relative levels of the interest genes were measured by qPCR and protein expression was assessed by Immunoblot. Cell cycle profile and proliferation status was investigated by flow cytometry. siRNA interference against the Rb gene was performed in isolated monocytes and the differentiated MDM were infected with VSV-pseudotyped NL4-3 GFP-expressing virus and a R5 HIV-1 strain BaL. Total viral DNA formation was quantified by qPCR and HIV replication measured by flow cytometry. Results: Monocyte differentiation into macrophages with M-CSF led to cell proliferation and susceptibility to HIV-1 infection that, in turn, induced cell cycle arrest at G2-M. Established HIV-1 infection activated STAT1 phosphorylation, transcription of interferon-stimulated genes and production of β-galactosidase, a marker of senescent cells. In addition, there was an increased expression of p21 and subsequent inactivation of cyclin-CDK2 activity leading to a hypo-phosphorylated active retinoblastoma protein (pRb) and deactivation of E2F1-dependent transcription. Additionally, HIV infection led to downregulation of the ribonucleotide reductase subunit R2 (RNR2) and reactivation of the HIV-1 restriction factor SAMHD1. pRb knockdown in primary MDM does not block HIV-1 viral DNA formation and HIV-1 replication Conclusions: HIV-1 induces a state of cellular senescence in primary macrophages. In addition, characterization of the cell-signaling pathway involved in the establishment of the HIV-1 induced senescence phenotype showed that it is driven by p21-dependent CDK inactivation that leads to pRb hypo-phosphorylation and SAMHD1 reactivation. 184 HIV-1 Attached to Monocytes, but Not Lymphocytes, Transmits Infection to Human Tissue Victor Barreto-de-Souza ; ChristopheVanpouille; Leonid Margolis Eunice Kennedy Shriver NICHD, Bethesda, MD, USA Background: Semen of HIV-1 infected men contains free virus as well as infected cells. It remains a matter of debate whether cell-associated or cell-free virus in the semen is predominantly transmitted in the course of HIV-1 sexual transmission. Seminal infected cells produce virus that upon release will essentially become free. However HIV can be also adsorbed on seminal cells, such as seminal monocytes and lymphocytes. Here, we focused on this cell-adsorbed virus and investigated whether it could contribute to HV sexual transmission from an infected man to his sexual partner. Methods: We developed a protocol to adsorb HIV on the cell surface and investigated whether this adsorbed virus can induce productive infection of human tissue ex vivo . Briefly, elutriated blood human lymphocytes or monocytes were gamma-irradiated (25 Gy) and then exposed to free R5 HIV-1 BaL for 2h at 4 o C to prevent virus internalization. To evaluate adsorbed HIV-1, cells were treated with trypsin, washed and lysed for p24 quantification. Results: We demonstrated that, under our protocol, ≥ 95% of virions were located on the cell surface (cell-associated p24: 0.92 ng/ml before and 0.043 ng/ml after trypsin treatment; n=3). We compared the ability of cell-adsorbed virus (on lymphocytes or monocytes) and cell-free virus to infect human tissues ex vivo . Towards this goal, we prepared free viral suspension with p24 concentration equal to that of cell-adsorbed virus and inoculated matched tissues with both viral preparations. HIV-1 adsorbed on Lymphocytes did not transfer infection to tissue ex vivo even after 15 days of culture (n=5). Using TZM-bl cells, we showed that lymphocyte-associated viruses were not transferred to target cells either. In contrast, HIV-1-adsorbed on monocytes were able to induce productive infection in tissue ex vivo similarly to that of free virus (p=0.41), as assessed by flow cytometry and by measuring p24 in cultural media (n=8). When 25% of human semen was added to monocyte-adsorbed HIV, we observed that viral replication in tissue was increased by 4. Conclusions: Our results suggest that HV-1 adsorbed on the surface of monocytes, but not on lymphocytes, can initiate a productive infection in human tissues with the same efficiency as free HIV, and that semen facilitates free HIV-1 infection probably by increasing virus transfer to target cells.

Poster Abstracts

72

CROI 2016