RNA binding and packaging by retroviral Gag proteins

Ohio State University, Columbus OH

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Abstract

Summary HIV-1 packages two copies of unspliced viral RNA as a dimer into newly budding virions. The unspliced viral RNA also serves as an mRNA template for translation of viral proteins. Recent studies showed that the fate of the viral RNA (genome vs. mRNA) is determined at the level of transcription. Host RNA polymerase II uses heterogeneous transcription start sites (TSSs) to generate major transcripts that differ in only two guanosines at the 5ʹ end (referred to here as 1G and 3G RNA). Remarkably, this two-nucleotide (nt) difference is sufficient to alter the structure of the 5ʹ-untranslated region (UTR) and generate two pools of RNA with distinct functions; 1G RNA is selectively packaged by the viral Gag protein and functions as the genome, whereas 3G RNA is preferentially translated by the ribosome. The presence of both RNA species is needed for optimal viral replication and fitness, but gaps remain in our understanding of the mechanism by which different RNA structures dictate viral and host factor interactions and thereby impact function. Our long-term goal is to understand how TSS choice influences HIV-1 RNA interactions and function. We previously showed that the stability of the “polyA” hairpin in the 5′UTR is a key regulator of the conformational equilibria, resulting in exposed (1G) versus sequestered (3G) dimerization and Gag binding sites. Tuning polyA hairpin stability allowed us to alter the conformational landscape and packaging selectivity. HIV variants that only express 1G or 3G major transcripts are in hand and will serve as key tools that will enable us to address exciting open questions. The proposed aims will test our major hypothesis that the different RNA structures formed due to TSS heterogeneity significantly impact viral and host factor interactions, RNA 5ʹ-modification, and ribosome loading and scanning. We will: (1) identify and characterize the HIV-1 1G and 3G interactomes in vitro and in cells; (2) determine how HIV-1 TSS choice affects RNA 5ʹ-end modification and dynamics; and (3) determine how HIV-1 TSS choice affects translation in vitro and in cells. The results will help guide the design of novel therapeutic agents that target the 5′UTR and interfere with its essential conformational plasticity.