Poster abstracts
Poster number 34 submitted by Wuxun Lu
N6-methyladenosine of HIV-1 RNA regulates viral replication and gene expression
Wuxun Lu (Center for Retrovirus Research; Department of Veterinary Biosciences, The Ohio State University), Nagaraja Tirumuru (Center for Retrovirus Research; Department of Veterinary Biosciences, The Ohio State University), Pratibha Chowdary Koneru (Center for Retrovirus Research; College of Pharmacy, The Ohio State University), Chang Liu (Department of Chemistry, Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, The University of Chicago), Mamuka Kvaratskhelia (Center for Retrovirus Research; College of Pharmacy, The Ohio State University), Chuan He (Department of Chemistry, Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, The University of Chicago)
Abstract:
The internal N6-methyladenosine (m6A) modification of cellular RNA controls post-transcriptional gene expression. The dynamic addition, removal, and recognition of m6A in cellular RNAs are coordinately regulated by three groups of host proteins, including methyltransferases (writers), demethylases (erasers), and m6A-binding proteins (readers). The YTH domain family proteins (YTHDF1-3, readers) bind to m6A-modified cellular RNA and modulate RNA metabolism and processing. Three recent studies highlighted the importance of m6A modification of HIV-1 RNA in regulating viral replication and gene expression (Lichinchi, Nat. Microbiol. 2016; Kennedy, Cell Host Microbe. 2016; Tirumuru, eLife, 2016). We reported that overexpression of YTHDF1-3 proteins in cells inhibits HIV-1 infection, mainly by decreasing HIV-1 reverse transcription, while knockdown of endogenous YTHDF1-3 in primary CD4+ T-cells increases HIV-1 infection (Tirumuru, eLife, 2016). Here, we aim to better understand the mechanisms by which YTHDF1-3 inhibit HIV-1 infection. We hypothesize that the m6A modification of HIV-1 RNA may affect its structure and/or binding to YTHDF1-3 proteins, thereby regulating HIV-1 reverse transcription and viral replication. Using an AlphaScreen-based binding assay, we determined that recombinant YTHDF1-3 proteins strongly bound to an m6A-modified HIV-1 RNA fragment compared to an unmodified RNA counterpart. We generated three HIV-1 mutants that could impair m6A modification in the 5’ leader sequence of HIV-1 RNA. We investigated the effect of these mutations on HIV-1 replication and viral protein expression in cells. Our results indicate an important role of m6A modification of HIV-1 RNA in viral replication and gene expression.
Keywords: m6A modification, HIV-1 replication