CROI 2024 Abstract eBook

Abstract eBook

Oral Abstracts

Methods: To study HIV-1 transcription start site usage, we established a next-generation sequencing (NGS)- based 5' rapid amplification of cDNA end (5' RACE) method. The accuracy and reproducibility of this assay were verified using in vitro-transcribed RNAs as templates and by determining the 5' context of HIV-1 unspliced RNA in multiple biological replicates of infected cells and virions. Using samples with characterized HIV-1 transcripts, we further determined the copy number of HIV-1 unspliced RNA required to obtain accurate measurement. Results: To study HIV-1 transcription start site usage in vivo, we used NGS-based 5' RACE to analyze RNA samples isolated from PBMCs of infected individuals. In most samples, there are several HIV-1 unspliced RNA species with varied 5' ends, indicating that multiple transcription start sites are used. Furthermore, 3G RNA is often the most abundant RNA species in PBMC samples. We have also examined the HIV-1 RNA species in corresponding plasma samples. Intriguingly, the distribution of HIV-1 unspliced RNA species in the plasma is distinct from that of the PBMCs: 1G RNA is the most abundant RNA species in the patient plasma samples, consistent with preferential packaging of 1G RNAs into virions. Conclusion: Our preliminary results indicate that in vivo, HIV-1 uses heterogeneous transcription start sites to generate multiple unspliced RNA species. Furthermore, these 99.9% identical HIV-1 unspliced RNA species differ functionally. The 1G RNA is preferentially selected as virion genome to transfer genetic information to the progeny. Investigation of the Functional Role of DDX42 in HIV-1 Viral RNA Splicing Xiao Lei 1 , Ann Emery 2 , Peng Zhang 1 , Arjun Kanjarpane 3 , Ronald Swanstrom 2 , Paul D. Bieniasz 1 1 The Rockefeller University, New York, NY, USA, 2 University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, 3 University of Maryland, Baltimore, MD, USA Background: Understanding the mechanism of HIV-1 viral RNA splicing could lead to the discovery of therapeutic potential drugs that target HIV-1 viral splicing pathways. HIV-1 splicing is regulated by cis-acting regulatory sequences and trans-acting splicing factors. We aim to identify host splicing factors that play roles in HIV-1 splicing through high-throughput assays. Methods: We generated a stable HEK-293T cell line harboring an HIV-1 splicing reporter which contains GFP in Gag position and RFP in Nef position in the viral genome. This reporter indicates the expression of unspliced viral RNAs and completely spliced viral RNAs through the expression of GFP and RFP respectively. We introduced genome-wide CRISPR knockout sgRNA library into the splicing reporter cell line and sorted cells with significantly higher or lower GFP/RFP expression through fluorescence-activated cell sorting. We analyzed enriched sgRNA guides in each sorted cell populations to identify genes whose disruption might affect HIV-1 viral RNA splicing. Results: We identified an RNA helicase DDX42, whose disruption caused an increase in completely spliced viral RNA species and a decrease in incompletely spliced viral RNA species. HIV-1 viral RNA splicing assays based on next generation sequencing show that the usage of specific HIV-1 viral RNA splice acceptors is affected in DDX42 knockout cells. For example, the usage of viral splicing acceptor A3 is increased significantly upon DDX42 knockout, which is also reflected in the increase of Tat protein expression in DDX42 knockout cell clones in western blot analysis. RNAseq analysis also reveals a role for DDX42 in the regulation of alternative splicing for a subset of host genes. Conclusion: DDX42 plays a role in the regulation of HIV-1 viral RNA splicing through up- and down- regulation of the usage of specific viral RNA splicing acceptors. Structural Basis of Translation Inhibition by MERS-CoV Nsp1 Reveals a Conserved Mechanism for β-CoVs Michael Vetick , Swapnil Devarkar, Shravani Balaji, Ivan Lomakin, Yong Xiong Yale University, New Haven, CT, USA Background: Since 2002, three deadly human betacoronaviruses (β-CoVs) have emerged: SARS-CoV, MERS, and SARS-CoV-2, however our therapeutic arsenal remains inadequate to restrict current and novel β-CoVs. All β-CoVs encode non-structural protein 1 (Nsp1), an essential pathogenicity factor that potently inhibits host gene expression. Three distinct mechanisms are proposed for Nsp1: two cytosolic involving the ribosome and one nuclear stopping mRNA export. Also, across β-CoVs Nsp1 has very low amino acid conservation. Previous literature suggests MERS Nsp1 function is restricted to the nucleus and does not bind 40S ribosome, contrasting SARS-CoV-2 Nsp1. However, due to conserved

structure prediction, we hypothesized that Nsp1 function is highly conserved across β-CoVs. Methods: Nsp1's effects on translation were evaluated in-vitro. HeLa cytoplasmic extracts were incubated with an exogenous luciferase reporter mRNA and recombinant Nsp1. Nsp1-40S ribosome binding was evaluated via fluorescence polarization. Fluorescein was conjugated to Nsp1 and incubated against a range of 40S ribosomes. A high-resolution structure of MERS Nsp1 binding the 40S ribosome was obtained by single particle cryo-EM of in vitro assembled complexes. Results: Contrary to previous reports, MERS Nsp1 robustly inhibits the translation of luciferase mRNA in- vitro. Compared to MERS Nsp1, SARS-CoV-2 Nsp1 inhibits translation at the ribosomal level more efficiently. However, fluorescence polarization showed that MERS and SARS-CoV-2 Nsp1 bind the 40S ribosome with very similar Kd's, 40nM and 27nM respectively. We resolved a 2.6-Å structure of MERS Nsp1 binding the 40S ribosome. The CTD of MERS Nsp1 adopts a helix-turn-helix motif binding in the mRNA entry channel of the 40S subunit interacting with the 18S rRNA and ribosomal proteins uS3 and uS5. Mutating this binding interface completely ablates Nsp1 function in vitro. Conclusion: We show divergent Nsp1 proteins exhibit a remarkably conserved mechanism by targeting the 40S ribosome to restrict host gene expression. Since SARS-CoV-2 and MERS Nsp1 have similar Kd's for the 40S ribosome, we speculate the increased potency of SARS-CoV-2 Nsp1 is due to effects beyond initial binding to the 40S ribosomal subunit. Diversified therapeutics targeting multiple stages of the viral life cycle will be critical for containing β-CoV outbreaks. We present that Nsp1 of β-CoVs is an essential pathogenicity factor and an attractive target for therapeutic intervention which can broadly restrict β-CoVs.

Oral Abstracts

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“Traitor”-Virus-Guided Discovery of Novel Antiviral Factors Caterina Prelli Bozzo 1 , Alexandre Laliberté 1 , Aurora De Luna 1 , Chiara Pastorio 1 , Meta Volcic 1 , Alexander Graf 2 , Stefan Krebs 2 , Helmut Blum 2 , Konstantin M. Sparrer 1 , Frank Kirchhoff 1 1 Ulm University Medical Center, Ulm, Germany, 2 University of Munich, Munich, Germany Background: Cellular innate defense mechanisms govern the outcome of pathogen exposure. Studies on HIV-1 identified a variety of restriction factors (RFs) that may inhibit different viral pathogens at various steps of their replication cycle. They also revealed, however, that RFs playing roles in HIV-1 transmission and/or are targeted by the viral accessory Vif, Vpr, Vpu and Nef protein remain to be discovered. Thus, improved screens are urgently needed for a better understanding of host-pathogen interplay and antiviral defense mechanisms. Methods: To develop effective, sensitive and versatile screens for novel RFs, we combined the CRISPR/Cas9 technology with the adaptive capacity of replication-competent HIV-1. In brief, we generated libraries of infectious molecular clones (IMCs) of HIV-1 equipped with single guide RNAs (sgRNAs) that target >500 potential restriction factors. Passaging of the library in Cas9 expressing CD4+ T cell lines allows enrichment of HIV-1 variants expressing sgRNAs associated with increased replication fitness. Enrichment of the sgRNAs thus identifies anti-viral genes. Results: Passage of NL4-3 and CH077 based HIV-1 gRNA constructs in CEM-M7 and SupT1 cells stably expressing Cas9 revealed that sgRNAs against GRN, CIITA,

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CROI 2024