The Cell Biology of HIV-1 Genome Trafficking

/ ABSTRACT There are more than 38 million people still living with HIV-1 infection worldwide, and more than 1.1 million in the United States alone. HIV-1 acute infection and latency reversal are both dependent on the expression of full- length unspliced viral RNAs (US-vRNAs) that serve dual roles in the cytoplasm as either (1) viral mRNAs encoding Gag and Gag-Pol proteins that drive virus particle assembly or (2) viral RNA genome substrates bound by Gag/Gag-Pol for packaging into virions. This project combines live cell and super-resolution imaging, biochemical assays, and other research tools to address key foundational issues in HIV-1 US-vRNA subcellular trafficking with the goal of exposing new virus and cell biology of potential relevance to novel antiviral strategies. Aim 1 is to determine why HIV-1 evolved to exploit a highly unconventional RNA export machinery regulated by Exportin-1 (XPO1, also known as CRM1) for US-vRNA nucleocytoplasmic transport, and how cooperativity- based RNA-protein interactions regulated by the viral Rev protein serve to activate this pathway. Our preliminary data and prior studies support a model wherein Rev multimerization on the US-vRNA’s Rev response element (RRE) forms a multi-NES complex needed to recruit at least two XPO1 proteins for activation of US-vRNA nuclear export. Moreover, live cell imaging has indicated that US-vRNA transcripts first build up in the nucleus prior to exhibiting “burst-like” nuclear export kinetics when virion production is initiated. We will test the hypothesis that burst export is explained HIV-1 US-vRNAs, Rev, and XPO1 interact in a stepwise manner to ultimately link viral transcription to nuclear pores, thereby upregulating late-stage delivery of US-vRNAs to the cytoplasm. To this end, we will carefully dissect Rev and the US-vRNA’s transitional stages over space and time in single cells using a combination of live cell imaging and an innovative “cell expansion” superresolution light microscopy technique. Our goal is to precisely define the subnuclear sites of Rev-US-vRNA and Rev-XPO1 interactions and understand the effects of prescribed perturbations to these essential processes, including a profound cell-specific block to HIV-1 US-vRNA nuclear export observed in mice and other rodents. For Aim 2, we will extend these studies to the cytoplasm, again using live cell and super-resolution imaging in combination with RNA capture proteomics to test the hypotheses that Rev-dependent nuclear export licenses viral RNAs for (1) enhanced stability in the cytoplasm and (2) preferred delivery to sites of genome packaging into virions. These experiments will take advantage of newly described mutant forms of HIV-1 that allow us to, for the first time, study US-vRNAs that are specifically programmed for either translation or packaging fates. We will also exploit these comparative conditions to identify new host proteins/pathways specifically associated with US-vRNA translation vs. packaging. Collectively, these studies will provide new mechanistic insights into the viral and cellular machines that drive HIV-1 genome trafficking and advance the development of new technologies for studying HIV-host interactions. Project Number: 2R01AI110221-11A1 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Nathan Sherer | Institution: UNIVERSITY OF WISCONSIN-MADISON, MADISON, WI | Award Amount: $376,863 | Activity Code: R01 | Study Section: Special Emphasis Panel[ZRG1 IIDA-K (02)] View on NIH RePORTER: https://reporter.nih.gov/project-details/2R01AI11022111A1