Tuesday, January 28, 2025
1/28/2025
Abstract Virtually every step of HIV-1 replication as well as numerous cellular antiviral defense mechanisms are regulated by viral and cellular RNA-binding proteins (RBPs) that recognize distinct sequence or structural features on viral RNAs. One such interaction takes place between the HIV-1 major structural protein, Gag, and the viral genomic RNA (gRNA). Through its nucleocapsid (NC) domain, Gag selects two copies of an unspliced positive strand gRNA from a pool of cellular and spliced viral mRNAs in excess and orchestrates key steps of virion assembly. How gRNAs are selected for packaging, why only a dimer is packaged in a single virus particle and whether NC-gRNA interactions guide other RBPs that target the same gRNA remain poorly understood. Previous CLIP studies demonstrated that Gag predominantly binds to guanosine-rich sequences in the cytosol, but its binding preference shifts towards sequences with adenosine-rich nucleotide composition (including binding sites on cellular mRNAs) at the plasma membrane where virions assemble. Here we propose to formally test whether adenosine-richness of the HIV-1 gRNA facilitates its selective packaging through complementary genetic and biochemical approaches (Aim 1). According to a widely accepted model, recruitment of a dimeric gRNA to the plasma membrane nucleates virion assembly, resulting in progressive recruitment of Gag molecules from the cytosol. As the late arriving pool of Gag molecules at the nucleation site needs to be devoid of RNAs, we hypothesize that affinity of Gag towards cognate RNAs is a key parameter that underpins dimeric gRNA packaging. In Aim 2, we propose to disrupt this balance by generating Gag chimeras with altered affinity/avidity towards target RNAs and determine the impact on dimeric gRNA trafficking to the PM, gRNA packaging and virus particle assembly. In Aim 3, using the Gag chimeric viruses as tools, we propose to target key NC functions and determine the NC- and gRNA-dependence of other viral and host RBPs that are critical regulators of HIV-1 replication and host defenses. Specifically, we propose to examine whether members of the antiviral APOBEC3 protein family infiltrate into virions through binding to the viral gRNA or other virion- incorporated host RNAs, such as 7SL. As part of this aim, Gag chimeras that can package gRNA independent of NC will be used to test the dependence of integrase-gRNA binding, an event critical for proper virion maturation, on NC and NC-gRNA interactions. Understanding the interplay between multiple RBPs that target the same gRNA is highly relevant to our fundamental understanding of HIV-1 replication and virus-host interactions. Currently, no antiretrovirals target gRNA packaging and assembly despite these steps presenting a clear vulnerability for HIV-1. Our preliminary studies demonstrate that Gag-gRNA interactions are finely tuned and can be targeted. Proposed work is thus significant as it may guide the development of NC- or gRNA-targeted drugs that abrogate gRNA packaging, virion assembly and infectivity.
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