CROI 2018 Abstract eBook

Abstract eBook

Poster Abstracts

variables (sex; age; risk factor; stage; baseline viro-immunological data) was explored. Results: Patients were mainly males (85%), MSM (54%), median age 35 yrs (range 27-44), 41%with a recent HIV infection, AIDS in 15%, 67% infected with subtype B, and 82%with R5 variants. V2 data are shown in Table 1. No association was found between patients’ features and V2 length, PNGs or NC. LDV was the most common V2 179-181 tripeptide (41.2%), followed by LDI (24.5%); Asp 180 was highly conserved in both B and non-B strains (99%). 59.8% and 40.2% of B vs non-B sequences (p≤0.001) had a Leu 179 . Val 181 was detected in 62% and 38% of B/non-B (p=0.04) while Ile 181 in 73% vs 27% of B/non-B subtypes (p=0.034), respectively. No correlation between CRT and V2 179-181 variants, or presence of LDV vs LDI motif was observed. Patients with L-strains had more frequently a recent infection (65% vs 37% in non-L, p=0.01); no other association between patient features and 179L/non-L variants was found. Conversely, patients with Val 181 were younger (median age 32.3 vs 37.8, p=0.004), mainly MSM (63% vs 41%, p≤0.001), with higher CD4 (p=0.04), and an earlier stage at diagnosis (C3 1% vs 10%, p≤0.001) than patients with other variants. A multivariate analysis confirmed correlation with risk factor and stage. Conclusion: Our results show a certain variability in V2 structure, including the α4β7 binding tripeptide, described as highly conserved. Due to potential therapeutic implications, further studies confirming our findings of a possible association of V2 features with viro-immunological characteristics are warranted.

the infectivity of virions produced by these types of cells we used single round infection assays of TZM-bl cells. Results: Using the iFRET construct, we discovered that up to 4% of virions remain immature and out of the ones that complete maturation, only 10% have a sealed core (in 293T cells). The infectivity of virions produced by Jurkat cells, which HIV-1 naturally targets, was up to ten-fold higher than that of virions produced by 293T cells, possibly also due to a higher number of mature, closed core, infectious virions released by Jurkat cells. We determined that Jurkat cells do indeed produce up to 2.2 times more sealed core virions than 293T cells (p<0.05). Conclusion: Our single virion imaging system based on FRET can be used to distinguish and quantify immature and open/closed core mature HIV-1 populations in different cell type settings. Jurkat cells produce a significantly higher number of sealed core virions compared to 293T cells. Our results suggest that cell line dependent factors may alter the core formation during virus maturation. 186 NEF UPREGULATES FASL EARLY WHILE MHCs ARE DOWNREGULATED LATE IN HIV-INFECTED CELLS Rodrigo Matus-Nicodemos , Daniel Douek, Richard A. Koup NIAID, Bethesda, MD, USA Background: HIV-1 Nef is a multifunctional early expressed protein. It selectively downregulates MHC molecules to prevent recognition by HIV-specific CD8 T cells. Nef also upregulates Fas ligand which leads to apoptosis of CD4 T cells and CD8 T cells. The temporal dynamics of these functions throughout the HIV replicative cycle is not known. To determine when these functions occur we developed a replicative cycle reporter system and measured these functions in HIV-infected cells. Methods: A GFP IRES NEF reporter construct was inserted into the Nef ORF of the NL4-3 (X4-tropic) and ADA-AD8 (R5-tropic) replication-competent molecular clones. A separate GFP reporter virus without Nef was also generated. For infection, PHA/anti-CD28-activated primary CD4 T cells were incubated with reporter virus for 3 hours, washed, and further incubated for 15, 24 and 48 hours. At the indicated time point we performed surface staining for HLA-A02 or HLA-B07, then intracellular staining for FasL and Gag p24. We also stained for the downregulation of CD4, T cell activation by CD69 expression, and cell viability by co-staining for Annexin V and Vivid. Our experimental control was primary CD4 T cells that went through the experiment without virus infection. Results: At 15, 24, and 48 hours after infection we detected two HIV-infected cell populations: GFP+p24-, expressing only Nef, and GFP+p24+, expressing Nef and Gag. CD4 was partially downregulated in the GFP+p24- population and completely downregulated in the GFP+p24+ population. FasL upregulation was detected at 15hr in both populations and remained high throughout the replicative cycle. Downregulation of MHC molecules was only observed in the GFP+p24+ population at the 48hr time point were HIV-induced cell death was observed. HIV-induced cell death was partially blocked by the caspase inhibitor, Z-VAD-FMK. We observed a significant increase in MHC expression in the both populations at 15 and 24hr. Finally, we also observed a significant increase in MHC expression in cells that up-regulated CD69. Conclusion: Our results show that FasL is upregulated early in HIV-infected cells by Nef while downregulation of MHC molecules occurs late. These data suggest that MHC molecules presenting virus peptides in HIV-infected cells could be expressed for most of the virus replicative cycle and recognized by CD8 T cells. Therefore, strategies targeting early Nef functions, such as FasL upregulation, in HIV-infected cells could be more detrimental to the virus than late Nef functions. 187 VISUALISATION OF THE HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 CDNA BY CLICK CHEMISTRY Flore De Wit, Frauke Christ , Sambasivarao Pillalamarri, Akkaledevi Venkatesham, Arthur Van Aerschot, Zeger Debyser Katholieke University Leuven, Leuven, Belgium Background: One of the key steps in HIV-1 replication is the reverse transcription of viral RNA into a double-stranded copy DNA (cDNA). This cDNA is subsequently transported in the form of a pre-integration complex (PIC) to the nucleus where it is finally integrated into the host chromosome by the viral integrase (IN). Although much is known about the biochemistry and inhibition of reverse transcription, many questions remain about its timing and location within the cellular environment.

Poster Abstracts

185 HIV-1 CORE MORPHOLOGY ASSESSMENT USING SINGLE VIRION IMAGING BASED ON FRET Anamaria D. Sarca 1 , Hirofumi Fukuda 1 , Luca Sardo 2 , Jessica Smith 3 , Kotaro Shirakawa 1 , Kazuki Horikawa 4 , Taisuke Izumi 1 , Akifumi Takaori-Kondo 1 1 Kyoto University, Kyoto, Japan, 2 University of the Sciences, Philadelphia, PA, USA, 3 United States Army Medical Research Institute of Infectious Diseases, Frederick, MD, USA, 4 University of Tokushima, Tokushima, Japan Background: There is an increasing importance to accurately characterize the morphology of HIV-1 virions, as it is closely related to their behavior as infectious particles. HIV-1 maturation, via Gag processing, leads to the formation of either closed (sealed) or open (unsealed) core virions. However, a microscopy method that allows the distinction of these HIV cores at single-virion level is not available. Thus, we developed the single virion visualization technology based on the principle of fluorescence resonance energy transfer (FRET). Methods: Based on the infectious and replication-competent interdomain green fluorescence protein (iGFP) HIV-1 designed by Hubner et al (J Virol. 2007), we created the iFRET construct in which GFP is replaced with the cyan and yellow fluorescent protein pair (CFP and YFP) between the Gag MA-CA domains, with cleavage sequences between them and the adjacent domains. As control, we used iFRET∆Pro, the protease deficient construct. FRET occurs between CFP and YFP in immature (uncleaved Gag) virions. Post Gag cleavage, these fluorescent proteins are dispersed inside and outside of the core. Stripping the viral membrane with detergent leads to complete loss of signal for some virions, indicating the presence of core sealing defects. To assess the quality of mature virion production by different cell types, we used the human T lymphocyte cell line (Jurkat cells) and the human embryonic kidney cell line, 293T cells. To check

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CROI 2018