CROI 2017 Abstract e-Book

Abstract eBook

Poster and Themed Discussion Abstracts

POSTER ABSTRACTS

154 PHENOTYPING AND SORTING OF INDIVIDUAL FREE INFECTIOUS HIV PARTICLES BY FLOW CYTOMETRY Michał Bonar , John Tilton Case Western Reserve Univ, Cleveland, OH, USA

Background: Flow cytometry has revolutionized our understanding of cell biology and immunology by revealing previously unappreciated phenotypic and functional heterogeneity of cell subsets. In contrast, our understanding of viral heterogeneity – and the role it may play in transmission, immune responses, and vaccination – is not nearly as well defined. Recent advancements in flow cytometry allow for nanoparticle analysis using a strong fluorescent signal. Here, we utilized fluorescently labeled viral particles to develop techniques to measure viral heterogeneity and perform functional analysis of viral populations purified by FACS. Methods: We compared a standard flow cytometer and a cytometer designed for sub-micron particles for their ability to detect unlabeled or fluorescently labeled viral particles. In addition to labeling structural proteins, we adapted an MS2 RNA loop system to assess viral genome incorporation. Viral particles containing different fluorescent structural and RNA markers were sorted by FACS and analyzed for their ability to infect CD4+ T cells. Specificity of detection and purity of sorts were confirmed by confocal microscopy and standard bulk biochemical techniques. Results: Fluorescently labeled – but not unlabeled – single viral particles could be identified and distinguished from exosomes on both cytometers. Viral genome incorporation was successfully monitored by colocalization of the fluorescently labeled structural and MS2-tagged proteins with agreement between flow cytometry and microscopy techniques. Viral particles could be sorted by genome incorporation and retained the ability to infect target cells. Conclusion: Starting with fluorescently labeled viruses, we have developed a ‘toolkit’ to explore the composition and infectivity of individual live viral particles with unprecedented detail. We are currently extending this technique to unlabeled HIV using fluorescently labeled neutralizing and non-neutralizing antibodies against Env proteins. We anticipate that viral heterogeneity will reveal critical insights into viral transmission and vaccine design. 155 SINGLE VIRUS IMAGING OF HIV-1 WITH FLUORESCENTLY LABELED CA Zeger Debyser, Irena Zurnic, Lieve Dirix, Veerle Lemmens, Veerle Lemmens, Susana Rocha, Johan Hofkens, Jelle Hendrix, Frauke Christ Katholieke Univ Leuven, Leuven, Belgium Background: HIV-1 capsid uncoating is a crucial step in HIV-1 replication. It is tightly regulated in a spatiotemporal fashion. Both accelerated and delayed uncoating hinder productive infection, and require a complex interplay between reverse transcription, uncoating and nuclear import. Although different available in vitro and in vivo uncoating assays already revealed valuable information, a consensus uncoating model is lacking. To date, uncoating is studied mostly indirectly in a heterogeneous pool of viruses during asynchronous infection, lacking the ability to discern the transient stages of uncoating. Therefore, conflicting results on the dynamics of this process have been obtained. Recently, single virus studies with imunolabelled CA suggested the presence of CA in the nucleus. Resolving the dynamics of capsid uncoating during infection awaits the development of a method to image functional virus containing labeled CA. Methods: We fluorescently labeled CA within the molecular clone at different selected positions, and evaluated the production efficiency and single round infectivity of these labeled viruses. We generated dual labeled VSV-G pseudotyped particles containing CA-eGFP and Vpr-transincorporated IN-mCherry. Using confocal microscopy, we evaluated the potential to study HIV-1 uncoating at a single virus level. Hereto we investigated the cellular distribution of CA and IN at a single virus level in fixed HeLaP4 cells. Validation of the model was provided through the use of inhibitors blocking HIV replication at discrete steps. Results: A specific (1:10) mixture of labeled and unlabeled CA was required to produce viral particles containing both labeled CA and IN yet maintaining infectivity in single round experiments. After entry, colocalisation of CA and IN was observed. When reaching the nuclear membrane, CA containing complexes accumulated in the perinuclear area and were depleted for IN. Using both eGFP-labeled CA and immunocytochemistry, we confirmed the presence of CA in the nucleus, although the role and nature of this nuclear CA remains unclear. Still, complexes with a high CA-eGFP content were mainly found in the cytoplasm and not in the nucleus. Most CA complexes in the nucleus did not contain IN. Conclusion: Directly labeled CA allows single virus imaging of uncoating and provides novel insights in the cellular distribution of CA. In agreement with others we detect CA in the nucleus, but not associated with IN. Ongoing research will prove the significance of nuclear CA. 156 CHARACTERIZING CELLULAR FACTORS INVOLVED IN HIV-1 GAG TRAFFICKING TO ASSEMBLY SITES Rachel Van Duyne , Philip R. Tedbury, Eric O. Freed NCI, Frederick, MD, USA Background: In the late stages of the viral replication cycle, the Gag precursor, Pr55Gag, is synthesized in the cytoplasm, cotranslationally myristylated, and recruited to the plasma membrane (PM) through binding to the phospholipid PI(4,5)P2. Though the mechanisms of virus release are well studied, the trajectory by which Gag traffics remains one of the least understood aspects of HIV-1 replication. We hypothesize that HIV-1 Gag requires a network of cooperative endosomal and secretory pathway cofactors to localize to sites of virus assembly. We previously determined that overexpression of the Golgi-localized γ-ear containing Arf-binding (GGA) proteins reduces HIV-1 particle production by impairing Gag trafficking to the membrane through disruption of Arf (ADP-ribosylation factor) protein activity. We therefore aim to characterize the role of the Arf family of vesicular trafficking proteins in the context of HIV-1 assembly and release. Methods: We screened dominant negative (DN) mutants of the Arf family members to determine the effect that functional disruption of these proteins has HIV-1 release. Results: Expression of Arf1DN potently inhibits not only HIV-1 release efficiency, but also processing of the viral envelope (Env) precursor gp160, and subsequently, particle infectivity. Interestingly, Arf3DN does not confer the same phenotype, despite greater than 96% sequence identity with Arf1. We demonstrate that Arf1DN inhibits virus release independent of matrix (MA), Env, and Nef as well as of mutants defective in budding, maturation, and membrane targeting. This virus release defect is not due to the mis-targeting of Gag to intracellular sites of assembly or the loss of Gag membrane binding, but rather due to the accumulation of Gag at intracellular membranes away from the PM. The retention of Gag membrane binding function, but the loss of assembly may be due to a defect in Gag oligomerization. Treatment of cells with Arf inhibitors also results in a defect in Env processing and virus release. Conclusion: This study suggests that disruption of Arf1 function severely affects the early steps of HIV-1 Gag trafficking. The accumulation of Gag at internal membranes upon disruption of Arf1 suggests Gag may interact with other endosomal/secretory membranes and vesicles prior to association with the PM. The characterization of Arf1DN-mediated re-localization of Gag to specific membranes will assist in the elucidation of the steps in the HIV-1 Gag trafficking pathway.

Poster and Themed Discussion Abstracts

61

CROI 2017