Poster abstracts
Poster number 103 submitted by Michael Muccio
Transcriptional start site usage in the HIV-1 genome dictates dimerization and selective packaging by modulating polyA stem stability
Michael R. Muccio (Ohio State University, Department of Chemistry and Biochemistry, Centers for RNA Biology and Retrovirus Research, Columbus, OH), Joseph G. Kanlong (Ohio State University, Department of Chemistry and Biochemistry, Centers for RNA Biology and Retrovirus Research, Columbus, OH), Jonathan P. Kitzrow, Olga Nikolaitchik (NCI, DRP, Viral Recombination Section, Frederick, MD), Vinay K. Pathak (NCI, DRP, Viral Mutation Section, Frederick, MD), Saiful Islam, Zetao Cheng, Akhil Chameettachall, Wei-Shau Hu (NCI, DRP, Viral Recombination Section, Frederick, MD), Karin Musier-Forsyth (Ohio State University, Department of Chemistry and Biochemistry, Centers for RNA Biology and Retrovirus Research, Columbus, OH)
Abstract:
Human immunodeficiency virus type 1 (HIV-1) is a retrovirus that encodes a 9.2 kb RNA genome. The HIV-1 unspliced (US) RNA serves as both the genomic RNA (gRNA) and as an mRNA template for translation of Gag and Gag-Pol polyproteins. The 5' untranslated region (UTR) of HIV-1 RNA is a major regulatory element and contains distinct structural motifs. These include the 5' terminal transactivation response element (TAR) and polyA hairpins, followed by three stem loops (SL1-SL3) that contain binding sites for Gag and a palindromic loop known as the dimerization initiation signal (DIS), which serves as the primary site for gRNA dimerization initiation. Recently, HIV-1 transcription start site (TSS) heterogeneity has been shown to produce major HIV-1 RNA transcripts with three guanosines (3G) or one guanosine (1G) at the 5' end. 3G RNAs are the major species of US RNA in the cytoplasm, whereas 1G RNAs are selectively packaged into new viral particles. 1G RNAs adopt a major conformation that stabilizes the polyA hairpin, exposing the DIS and Gag binding sites crucial for genome packaging, while 3G RNAs have an unstable polyA stem loop and adopt a conformation that sequesters these elements and favors mRNA translation. Thus, differences in the stability of the polyA hairpin appear to modulate the function of HIV-1 US RNA. Here, we performed UV thermal melting studies to investigate the stability of the polyA hairpins in various contexts. We first showed that the polyA hairpin was significantly less stable in the 3G HIV-1 TAR-PolyA RNA relative to the 1G construct. Viral packaging assays performed using a TSS mutant that produces RNAs with either a single 5' adenosine (1A) or a 5' GGA, showed that neither of the RNAs were selectively packaged; Tm studies showed that the differences in polyA hairpin stability were also eliminated. A compensatory mutant was identified that rescued selective 1A packaging and Tm studies showed that the polyA hairpin stability differences were also restored. We also performed native gel electrophoresis assays and RNA structure probing to observe how these mutants influenced the global 5’ UTR structure. Overall, these studies confirm a remarkable correlation between packaging selectively and differential polyA hairpin stability to inform new RNA-targeted therapeutic strategies.
Keywords: HIV-1, RNA Structure, Viral Packaging