CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

also up-regulated CD169 and HIV-1 fusion in treated MDMs was enhanced (2-fold) in a CD169-dependent manner. Interestingly, CD169 enhanced virus replication in MDMs even in the presence of IFN α (>2-fold higher compared to MDMs pretreated with α CD169 blocking antibody). Finally, LNs from SHIV-infected macaques showing signatures of immune activation contained more CD169 + cells than those of uninfected animals and, intriguingly, a large proportion of p27 gag+ cells were also CD169 + . Conclusions: These studies suggest that HIV-1 has exploited CD169 to attenuate IFN-I-induced antiviral state in myeloid cells. 189 Interferon-Induced Transmembrane Proteins (IFITMs) Antagonize Postintegration Replication of HIV but Are Overcome by Viral Membrane Accessory Proteins Wingyiu Lee ; Ifrah Omar; Richard D. Sloan Queen Mary University of London, London, United Kingdom Background: Interferon response triggered by acute HIV infection modulates the expression of genes in both infected and non-infected cells to control viral replication and virus spread. To date, a number of genes have been identified as host antiviral factors that interfere with various steps of the HIV replication cycle. Members of the interferon-induced transmembrane proteins (IFITMs) are induced in human macrophages and CD4 + T cells upon upon exposure to interferons. While IFITMs have been shown to inhibit entry of HIV-1, the effects of IFITMs on other stages of the viral replication cycle are not known. Thus, we investigated the effects of IFITMs expression on the post-entry steps of the HIV replication cycle and how post-translational modification of IFITMs modulates these effects. Methods: In transfection assays, wild-type or accessory gene-deleted HIV proviral DNA and expression vectors for IFITM1-3 were co-transfected into adherent HEK293T cells. In infection assays, HIV-1 viruses were made from transfected 293T cells and subsequently used to infect SupT1 cells that express human IFITMs upon exposure to doxycycline. Virus released from transfected or infected cells was quantified by p24 ELISA. Expression of viral proteins, IFITMs and other antiviral proteins were evaluated by immunoblotting or intracellular staining and flow cytometry. Quantitative PCR was used to measure the amount of total, multiply-spliced and singly-spliced viral RNA. Immunofluorescence and confocal microscopy were used to visualize IFITMs and viral components in cells. Results: We show that expression of human IFITMs reduces the quantity of released HIV-1 viral particles produced in cells compared to vector controls. In the absence of the HIV accessory proteins Nef and Vpu, IFITM-mediated antagonism of HIV is more potent (20-30 fold). The reduction of viral output is caused by a decrease in synthesis of viral proteins independent of transcriptional control. While mutation of cysteine residues in IFITM1-2 that perturbs their palmitoylation and concomitant plasma membrane retention led to a reduction in all classes of viral RNA transcripts. This results in highly reduced HIV particle output by 10 to 100-fold compared to the wild-type IFITM1. Conclusions: Our studies identify a novel role for IFITMs in controlling HIV replication at the post-transcriptional level and show that it is possible to engineer highly active anti-HIV IFITM variants by modulation of their palmitoylation status. 190 HSV-1-Induced Enhancement of HIV-1 Replication Is Dependent on Decrease in IFITM3 Levels Viviane Andrade 1 ; Milene Miranda 2 ; Mario Stevenson 1 ;Thiago M. Souza 2 1 University of Miami, Miami, FL, US; 2 Oswaldo Cruz Foundation, Rio de Janeiro, Brazil Background: The IFITM3 restriction factor has been described as a protein that impairs HIV-1 replication upon IFN induction. Since herpesvirus infections are extremely common in HIV-1-infected individuals and some HSV-1 proteins inhibit the IFN cascade, we analyzed whether HSV-1 could enhance HIV-1 replication by diminishing the levels of IFITM3. Methods: Monocyte-derived macrophages were obtained from healthy donors and cells were infected with HSV-1 AR-29 (0.1 or 1 MOI) or co-infected with HIV-1 Ba-L (10 ng/mL p24 antigen) during 2 or 14 days, respectively. To access HIV-1 quantification, we performed luciferase assay with infected TZM-Bl from supernatants of infected-macrophages. The expression of IFITM3 was analyzed by immunoblotting with an anti-rabbit antibody against IFITM3. Knockdown of HSV-1 proteins was performed with siRNA against Us11 and VHS transcripts and further qPCR analysis to confirm knock down. Results: Upon HIV-1-HSV co-infection, we observed a 3- 7 fold increase in HIV-1 production. At an HSV-1 MOI of 1,we observed a reduction in IFITM3 levels by 48 h post-infection. We analyzed changes in IFITM3 levels during the first 24 h of HSV-1 infection. Changes in IFITM3 occurred primarily at the late stages of HSV-1 infection. Treatment with ACV or IFN-2 α prevented reduction of IFITM3, indicating that viral replication and ability to overcome IFN signaling are critical for IFITM3 modulation. To confirm that HSV-1 proteins produced at the late phase of replication are indirectly reducing IFITM3 levels by inhibiting the IFN cascade, Us11 and VHS was silenced. This was sufficient to prevent HSV-1- induced IFITM3 reduction. siRNA-mediated silencing of US11 and VHS also consistently prevented HSV-1-mediated enhancement of HIV-1 production. Conclusions: Increased HIV-1 replication observed during HIV-1/HSV-1 co-infection is due to the production of Us11 and VHS by HSV-1. Upon inhibiting the IFN cascade, IFTM3 levels are reduced which contributes to an increased replication of HIV-1 in co-infected macrophages. Since co-infections are common in HIV-1 infected-individuals, our findings Background: Host restriction factors are proteins hampering virus replication, and share common features including positive selection, viral counteraction, interferon-inducible expression and differential expression among HIV+ patients. APOL6 was identified in a screen aiming at identifying novel HIV-1 candidate host restriction factors. Another member of the APOL family, APOL1, has been previously described for its protective role against the parasite Trypanosoma brucei and more recently as a new HIV restriction factor (Taylor et al., 2013). The aim of the study was to characterize the activity of APOL6. Methods: We evaluated the ability of APOL6 primate orthologues, chimeras and mutants to inhibit the GFP expression from an HIV-1 based genomic vector in co-transfection experiments. We also assessed the impact of APOL6 on transduction of reporter viruses. Analysis was carried out using flow cytometry Results: APOL6-mediated restriction was validated in a co-transfection assay with an HIV-1 LTR-EF1-GFP, showing up to 10-fold reduced GFP expression in APOL6-expressing cells compared to control cells. Species-specific restriction of APOL6 primate orthologs co-transfected with HIV-1 LTR-EF1-GFP revealed a higher GFP inhibition mediated by hominids and new world monkey APOL6 compared to old world monkeys APOL6. A similar APOL6-mediated inhibition was observed when APOL6 was co-transfected with alternate GFP expression vectors. In contrast, APOL6 was not able to restrict HIV-based vector transduction nor adenovirus or LCMV. Furthermore, APOL1 and APOL3 constructs were also tested in co-transfection and transduction experiments and followed the same tendency as APOL6. Through co-transfection analyses using human and rhesus APOL6, respectively displaying high and low inhibition ability, we identified a specific APOL6 domain and residue mediating APOL6 effects. Conclusions: All together, these data suggest that APOL6-mediated activity is not virus-specific, but rather displayed a broad action against various promoter constructs. This points out to a specific APOL6 mechanism, potentially acting at the level of nucleic acid (DNA or RNA) sensing and/or degradation. APOL6 domain and residue responsible for the activity was identified. The mechanism used by APOL6 is very likely to be shared by other members of the family. We are currently identifying APOL6 cellular interactants by Mass spectrometry to elucidate the mechanism of APOL6-mediated restriction. provide additional for exacerbation of HIV-1 replication in HSV-1/HIV-1 co-infected individuals. 191 Characterization of the Activity of an Innate Immunity Protein, the Apolipoprotein L6 Nitisha Pyndiah 1 ; Angela Ciuffi 1 ; AmalioTelenti 2 1 Institute of Microbiology, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland; 2 J Craig Venter Institute, San Diego, CA, US

Poster Abstracts

199

CROI 2015