Poster abstracts
Poster number 25 submitted by Joe Kanlong
Transcription Start Site Mutations Disrupt HIV-1 Genomic RNA Packaging via RNA Structure-guided Regulatory Mechanisms
Joseph G. Kanlong (Department of Chemistry and Biochemistry, Center for RNA Biology, Center for Retrovirus Research, The Ohio State University, Columbus, OH), Jonathan P. Kitzrow, Olga Nikolaitchik, Saiful Islam (Viral Recombination Section, HIV DRP, NCI, Frederick, MD), Michael R. Muccio (Department of Chemistry and Biochemistry, Center for Retrovirus Research, Center for RNA Biology, The Ohio State University, Columbus OH), Vinay K. Pathak (Viral Mutation Section, HIV DRP, NCI, Frederick, MD), Zetao Cheng, Akhil Chameettachall, Wei-Shau Hu (Viral Recombination Section, HIV DRP, NCI, Frederick, MD), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for Retrovirus Research, Center for RNA Biology, The Ohio State University, Columbus OH)
Abstract:
During HIV-1 replication, two copies of a 9.2 kb viral RNA are packaged into nascent virions as a dimer. This unspliced viral RNA (US HIV RNA) functions as both the genome (gRNA) and as an mRNA that encodes for the polyprotein Gag, which plays a major role in gRNA packaging. Recent work has demonstrated that the fate of US HIV RNA is regulated at the level of transcription. Heterogeneous transcription start site (TSS) usage by RNA pol II produces US HIV RNA that contains one (1G), two, or three (3G) 5ʹ guanosines and a 5ʹ cap. Whereas 3G RNAs are the major transcripts found in the cytoplasm and function as mRNAs, 1G RNAs are selectively packaged as the viral genome. The 5ʹ UTRs of the 1G and 3G RNAs adopt distinct conformational ensembles that expose or sequester structural elements crucial for gRNA packaging. The conformational switch is dictated by differences in the stability of a hairpin element termed PolyA. To investigate how transcription initiation and RNA fate are regulated by nucleotides in the HIV-1 TSS, we designed a set of mutants that targeted the tract of three consecutive Gs. We analyzed both cellular and virion RNAs produced from mutant proviruses. While G->T/C mutations inhibited transcription from the mutated residue, G->A mutations supported robust transcription initiation. We found that a GGG->GGA (G3A) mutation produced transcripts from both the first (5ʹ-GGA) and third (5ʹ-A) positions; however, neither of the mutant RNAs were selectively packaged into virions. Selective packaging of 5ʹ-A RNAs was rescued by a single downstream compensatory mutation. To investigate the effects of these mutations on PolyA hairpin stability and global 5ʹ UTR structure in vitro, we performed UV thermal melting studies and RNA structure probing studies using SHAPE-MaP. We observed a correlation between PolyA hairpin stability and packaging selectivity. The G3A mutation results in a destabilized polyA hairpin, which shifts the 5ʹ UTR into a non-packageable conformation; this effect is reversed with the compensatory mutation. Structural differences in the 5ʹUTR of TSS mutants are similarly correlated with packaging selectivity. These results demonstrate how the identity of the HIV-1 TSS is critical in regulating transcription to produce RNAs with distinct 5ʹ UTR conformations that direct HIV-1 RNA fate.
Keywords: HIV-1, Transcription, RNA structure