CROI 2020 Abstract eBook

Abstract eBook

Poster Abstracts

183 THE HIV ANTISENSE PROTEIN ASP IS A TRANSMEMBRANE PROTEIN OF THE VIRAL ENVELOPE Zahra Gholizadeh 1 , Yvonne Affram 1 , Juan C. Zapata 1 , Hongshuo Song 1 , Rui Li 1 , Maria Iglesias-Ussel 1 , Krishanu Ray 1 , Olga Latinovic 1 1 University of Maryland, Baltimore, MD, USA Background: The negative strand of the HIV-1 genome encodes a highly hydrophobic antisense protein (ASP) with no known homologs. Humoral and cellular immune responses against ASP show that it is expressed in vivo, but its role remains unknown. We studied ASP expression in chronically infected myeloid and lymphoid cell lines, its impact on viral replication, and ASP sequence evolution during natural infection. Methods: Flow cytometry was performed on Millipore Guava flow and analyzed with FlowJo. Confocal microscopy was performed with Zeiss LSM 800 and analyzed with Zen Blue. Fluorescence Correlation Spectroscopy (FCS) was performed with ISS Q2 confocal microscope and ISS VistaVision. Longitudinal sequences were downloaded from the Los Alamos HIV database and were aligned using GeneCutter. Results: Using a monoclonal antibody (324.6) against an epitope mapping between two transmembrane domains of ASP, we detected ASP in the nuclei of all infected cell lines. Confocal microscopy showed a polarized nuclear distribution of ASP, and accumulation in areas containing actively transcribed chromatin. PMA treatment caused translocation of ASP to the cytoplasm and cell membrane. Cell surface detection of ASP without membrane permeabilization shows extracellular exposure of the 324.6 epitope. We found that ASP and gp120 co-localize on the membrane of PMA-treated cells (Manders overlap coefficient 76%), suggesting that ASP might be incorporated in the membrane of budding virions. Indeed, 324.6 captured HIV-1 particles with efficiency similar to anti-gp120 VRC01. Also, FCS showed that 324.6 binds single virions in solution with ~30% efficiency. Altogether, these two assays demonstrate the presence of ASP on the surface of HIV-1 virions. ASP-knockout HIV-1 particles displayed a ~50% reduction in replication rate compared to wildtype virus. Longitudinal sequence analysis shows that during natural infection viruses with intact ASP preserve the ORF, and viruses with early stop codons in ASP undergo deletion or recombination events that restore the ORF. Conclusion: ASP is a transmembrane protein found on the surface of productively infected cells, and on the envelope of mature HIV-1 virions. Knocking out ASP expression reduced viral replication. Preservation or restoration of functional ASP ORF during natural infection indicates that ASP may provide a selective advantage to HIV-1. Jennifer Currenti 1 , Abha Chopra 2 , Mina John 3 , Shay Leary 2 , Elizabeth McKinnon 2 , Eric Alves 1 , Mark Pilkinton 4 , Ramesh Ram 2 , Becker Law 1 , Francine Noel 5 , Simon Mallal 4 , Joseph Conrad 4 , Spyros Kalams 4 , Silvana Gaudieri 1 1 University of Western Australia, Crawley, Australia, 2 Murdoch University, Murdoch, Australia, 3 Royal Perth Hospital, Perth, Australia, 4 Vanderbilt University, Nashville, TN, USA, 5 GHESKIO, Port-au-Prince, Haiti Background: Human immunodeficiency virus (HIV) can adapt to an individual’s T cell immune response via genetic mutations that affect antigen recognition and impact disease outcome. In vaccine design, it is vital to understand this complex host-viral interaction including the mechanisms that underpin viral adaptations that subvert/alter the immune response. In this study, we assign the putative replicative cost and immune benefit of specific HIV adaptations in the unique setting of vertical HIV transmission. Single cell transcriptomics of antigen-specific T cells was also utilised to further delineate the dynamics of specific adaptations that may reflect a novel mechanism of adaptation. These results could be used to inform vaccine designs and cure strategies to combat the issue of immune adaptation. Methods: Specifically, we utilised a deep sequencing approach to determine the HIV quasispecies in 26 mother/child transmission pairs where the potential for founder viruses to be pre-adapted is high. The resultant sequences and previously determined viral adaptations for specific host genotypes were used to generate adaptation scores for the transmitted virus. We used intra-cellular cytokine staining to assess specific antigen-specific T cell immune responses and single cell technologies to compare T cell receptor (TCR) repertoire and transcriptome data for a specific HIV epitope in which adaptation is associated with continued immune recognition. Results: We showed that the dynamics of HIV adaptations following transmission provides insight into the in vivo replicative cost associated with 184 HIV ADAPTATION FOLLOWING VERTICAL TRANSMISSION

biological phenotype of virus variants such as per-particle infectivity, response to neutralizing antibody (nAb), Maraviroc (MVC) and Interferon alpha (IFN-a). Results: Based on the genotypic and phenotypic analysis, we identified 10 TF viruses from 8 infants. TF viruses were characterized by shorter V1V2 regions, reduced number of potential N-linked glycosylation sites and higher infectivity titer as compared to the non-transmitted (NT) variants. The sensitivity of the TF variants to MVC and a standard panel of nAbs (VRC01, PG09, PG16, and PGT121) were found to be much lower in TF than NT variants. IFN-a resistance by NT viruses were comparatively lower throughout the experiment when compared to TF viruses. Unexpectedly the productivity (amount of p24) of the NT viruses was found to be higher than TF viruses with the influence of IFN-a and MVC. Conclusion: Despite small sample size, the study identified precise molecular and biological phenotype of the TF viral strains and its evolutionary dynamics on transmission-associated bottlenecks. In general, these studies improve our knowledge about the very diverse and highly adaptable nature of the TF virus, and hopefully takes us a few steps closer to addressing the challenges that come in the way of developing an effective vaccine or cure against HIV-1 infection.

Poster Abstracts

182 NEW HIV-1 CAPSID LABELING SYSTEM DOES NOT SUPPORT UNCOATING DURING NUCLEAR IMPORT Chenglei Li 1 , Ryan C. Burdick 1 , Mohamed Husen Munshi 1 , Ferri Soheilian 2 , Wei-Shau Hu 1 , Vinay K. Pathak 1 1 National Cancer Institute, Frederick, MD, USA, 2 Leidos Biomedical Research, Inc, Frederick, MD, USA Background: HIV-1 uncoating (capsid core disassembly) is a prerequisite for viral DNA integration into the host genome and a promising target for antiviral therapy. However, the timing and cellular location of uncoating remain elusive, in part because current methods are unable to directly and accurately measure the amount of capsid protein (CA) loss from the infectious viral complexes. Bulk measurement of CA loss by biochemical methods or imaging of viral complexes may not reflect the behavior of infectious viral complexes, since only a small fraction of the viral complexes in the cell lead to infection. Quantification of CA by immunostaining with anti-CA antibody or live-cell imaging of viral complexes labeled with fluorescently-tagged cyclophilin A (CypA) is confounded by loss of epitope accessibility to the antibody or loss of interactions to CypA. Methods: We developed a method to directly label CA with green fluorescent protein (GFP) in infectious viral complexes, determined virus infectivity in HeLa and CEM-SS cells, characterized GFP-CA core incorporation and stability by sucrose gradient fractionation and Western blot, and quantified the core- associated CA during nuclear import using live-cell imaging. Results: The GFP-CA labeling method is highly efficient and results in >96% of the virions being fluorescently labeled. Importantly, the GFP-CA labeling resulted in only a ~2-fold loss of virus infectivity in HeLa and CEM-SS T cells, indicating that GFP-CA-labeled viral complexes are infectious. Sucrose-gradient fractionation of virions indicated that GFP-CA was incorporated into viral cores and did not affect the core stability. Moreover, analysis of infected HeLa cells indicated that GFP-CA-labeled cores can efficiently associate with the nuclear envelope and enter the nucleus. We analyzed the amount of GFP-CA associated with viral cores docked at the nuclear envelope just before and after their translocation into the nucleus. No significant loss of GFP-CA was observed in the nuclear viral complexes compared to those at the nuclear envelope, indicating that uncoating does not occur during nuclear import. Conclusion: These studies provide a new robust method for quantification of CA associated with viral complexes and will facilitate studies of HIV-1 post-entry events. Our results do not support the model that viral core uncoating occurs during nuclear import.

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CROI 2020