CROI 2020 Abstract eBook

Abstract eBook

Poster Abstracts

Conclusion: Loop 1 of human A3C restriction factor was identified as a novel protein domain that binds DNA and thereby drastically gains in antiviral activity against HIV-1. 177LB IFITM3 REDUCES RETROVIRAL ENV FUNCTION AND IS COUNTERACTED BY GLYCOGAG Yadvinder S. Ahi 1 , Diborah Yimer 1 , Guoli Shi 1 , Saliha Majdoul 1 , Kazi Rahman 1 , Alan Rein 1 , Alex A. Compton 1 1 National Cancer Institute, Frederick, MD, USA Background: The interferon-induced transmembrane (IFITM) proteins are known for inhibiting the entry of a wide array of viruses into host cells. Furthermore, when IFITM3 is present in virus-producing cells, it reduces the fusion potential of HIV-1 virions, but the mechanism is poorly understood. Methods: To describe the antiviral mechanism of IFITM3 and to discover modes of viral evasion, we took advantage of a murine leukemia virus (MLV)-based pseudotyping system. By controlling IFITM3 and envelope (Env) levels in virus- producing cells, we found that IFITM3 potently inhibits MLV infectivity when Env levels are limiting. Results: Loss of infectivity was associated with defective proteolytic processing of Env and lysosomal degradation of the Env precursor. Ecotropic and xenotropic variants of MLV Env, as well as HIV-1 Env and vesicular stomatitis virus glycoprotein (VSV-G), are sensitive to IFITM3, whereas Ebola glycoprotein is resistant, suggesting that IFITM3 selectively inactivates certain viral glycoproteins. Furthermore, endogenous IFITM3 in human and murine cells negatively regulates MLV Env abundance. However, the negative impact of IFITM3 on virion infectivity is greater than its impact on Env incorporation into virions, suggesting that IFITM3 also impairs Env function. Finally, we demonstrate that the presence of glycosylated Gag (glycoGag), the only accessory protein encoded by MLV, confers resistance to the IFITM-mediated loss of infectivity. GlycoGag has previously been shown to counteract another antiviral transmembrane proteins known as SERINC. Importantly, glycoGag rescues virus infectivity in the presence of IFITM3 without enhancing Env incorporation, indicating that glycoGag counteracts the cryptic function of IFITM3 which acts on Env function. This represents the first description of a viral auxiliary protein displaying the capacity to antagonize or enable viral evasion of IFITM3. Conclusion: Overall, we demonstrate that IFITM3: (i) impairs virion infectivity by decreasing Env quantity and Env function, (ii) glycoGag confers virions with resistance to IFITM3 and, (iii) the antiviral activities of IFITM3 and SERINC3/5 may be linked. We are now testing whether the antiviral function of IFITM3 is maintained in SERINC5 knockout cells and whether other retroviral accessory proteins, such as HIV-1 Nef and EIAV S2, also exhibit the capacity to counteract IFITM3. 178 SEQUENCE CHANGES CAUSING REV ACTIVITY DIFFERENCES IN HIV-1 PRIMARY ISOLATES Patrick E. Jackson 1 , Jordan Holsey 1 , Godfrey Dzhivhuho 1 , David Rekosh 1 , Marie- Louise Hammarskjold 1 1 University of Virginia, Charlottesville, VA, USA Background: The HIV-1 Rev-Rev Response Element (RRE) regulatory axis is required for the nucleocytoplasmic export of intron-containing viral mRNAs, an essential step in viral replication. A viral protein, Rev, binds to the RRE, an RNA structure found on incompletely spliced viral mRNAs, multimerizes, and recruits cellular factors to export the transcript. We previously described two Revs from primary isolates which display markedly different levels of activity. The high-activity, 9-G, and low-activity, 8-G, Revs differ by a total of 29 amino acids spanning across all domains including the bipartite oligomerization domain (OD), arginine rich motif (ARM), and nuclear export site (NES) (see Figure). Here, we define key residues causing differential activity. Methods: Chimeric Revs were generated by exchanging regions between 8-G and 9-G sequences. Rev activity was determined using a recently described assay. Two constructs were created: an HIV vector modified to produce two fluorescent proteins in a Rev-dependent or Rev-independent fashion, and a murine stem cell virus vector producing different Revs and a third fluorescent marker. Both constructs were packaged and used to co-transduce lymphoid cells. Rev activity was determined by measuring relative intensity of the fluorescent markers. Results: 9-G Rev displayed about 4-fold greater activity than 8-G Rev (p=0.001). Chimeric Revs created by exchanging the turn or link, the c-terminus,

replication upon infection in vitro (n=3; p=0.0084). Similarly, CH223191 significantly down regulated IL-22, IL-17A, IL-10, production (p<0.0001); increased wild type HIV replication (p=0.0016), as well as HIV-DNA integration/ transcription upon single-round infection with HIV-VSVG pseudotyped viruses (n=5; p=0.001); and increased >2-fold HIV reactivation in VOA (n=7). At the opposite, FICZ significantly increased IL-22 and IL-10 production and inhibited viral replication in vitro and reactivation in VOA. Conclusion: Our results identify the AhR as a novel negative regulator of HIV replication in Th17/Th22-polarized cells thus raising the interest in testing natural/synthetic AhR agonists/antagonists for HIV remission/cure strategies. 175 ANTI-HIV ACTIVITIES OF THE 12 INTERFERON-ALPHA SUBTYPES Aexandra Tauzin 1 , Armando Espinosa 1 , Olivia Blake 1 , Fabrizio Mammano 1 1 INSERM, Paris, France Background: The human genome encodes for 12 different interferon (IFN)- alpha subtypes, which share a common receptor on target cells, and trigger similar signaling cascades through Jak-STAT pathways. Several studies have collectively shown that this apparent redundancy may be justified by specific properties of the different IFN subtypes. Accordingly, the sets of genes induced by different IFN subtypes do not completely overlap, and different viruses, including HIV, are differently sensitive to individual subtypes. Methods: We have measured the inhibition of HIV replication by the 12 IFN- alpha subtypes in primary T-lymphocytes and in a T-cell line using a multiple cycle replication assay. We have then measured the efficacy of inhibition on specific steps of the HIV replication cycle, including viral entry, reverse transcription, integration and budding. In parallel, we have measured the impact of IFN-alpha subtypes on cell proliferation, whose modification could indirectly participate in the overall antiviral effect. Results: Working with primary T-lymphocytes and a T-cell line, we have first confirmed the differential potencies of the 12 IFN-alpha subtypes on HIV replication. The order of potency was similar in the two experimental settings, suggesting the induction of similar sets of antiviral genes. Using dedicated assays, we found that some subtypes act more potently on the early steps of HIV replication, while others target more efficiently the late steps. Conclusion : Our findings support the notion that different genes with anti-HIV potential are induced by the different IFN subtypes. They allow to identify those characterized by potent direct antiviral effect with minimal perturbation of cellular proliferation. Our study also prompts the search for new anti-HIV factors, targeting specific steps of virus replication. 176 LOOP 1 OF HUMAN APOBEC3C REGULATES THE ANTIVIRAL ACTIVITY AGAINST HIV-1 Ananda A. Jaguva Vasudevan 1 , Zeli Zhang 1 , Kannan Balakrishnan 2 , Christoph Gertzen 2 , Ignacio G. Bravo 3 , Gerald G. Schumann 4 , Dieter Häussinger 1 , Holger Gohlke 2 , Carsten Münk 1 1 Heinrich Heine University Hospital, Düsseldorf, Germany, 2 Heinrich Heine University Düsseldorf, Düsseldorf, Germany, 3 IRD, Montpellier, France, 4Paul Ehrlich Institut, Langen, Germany Background: The APOBEC3 (A3) family of single stranded DNA deaminases defends hosts from Human immunodeficiency virus (HIV)-1 lacking viral infectivity factor (vif) (HIV-1Δvif). A3 catalyzes the dC to dU deamination in the viral DNA/genome, causing hypermutation that abrogates the virus. Human APOBEC3C (hA3C) is known as a strong restriction factor of Vif-deficient Simian immunodeficiency virus (SIVΔvif), but exhibits a weak inhibition against HIV-1Δvif. The reason for this specificity of A3C’s antiviral function remains unknown. Methods: Experiments were performed in cell culture using virus infections, expression of APOBEC3 proteins, biochemistry to study protein DNA interaction and enzyme activity, which were complemented with structural protein modelling and gene evolution studies. Results: We report that residues in loop 1 of A3C govern their anti-HIV-1 activity to the level compared to that of A3G. We identified that exchanging WE to RK in loop 1 in A3C drastically enhances A3C’s deamination activity. Molecular modeling and EMSA experiments demonstrated that A3C.WE-RK interacts with ssDNA substrate stronger than that of wild-type, which consecutively facilitates catalytic function. As the RK residues are naturally presenting in A3F at the equivalent position, we swapped themwith WE and found a marginal decrease in HIV-1Δvif inhibition. The gain-of-function A3C variant also exhibited stronger LINE-1 restriction capacity but RK-WE exchange did not crucially disarm A3F.

Poster Abstracts

59

CROI 2020