Poster abstracts
Poster number 61 submitted by Nagaraja Tirumuru
Functional significance of the demethylase ALKBH1 in HIV-1 infection and gene expression
NAGARAJA TIRUMURU (Center for Retrovirus Research; Department of Veterinary Biosciences; Center for RNA Biology, The Ohio State University, Columbus, Ohio 43221, USA), Chuan He (Department of Chemistry, Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics; Howard Hughes Medical Institute, The University of Chicago, Chicago,Illinois 60637, USA.), Li Wu (Center for Retrovirus Research; Department of Veterinary Biosciences; Center for RNA Biology, The Ohio State University, Columbus, Ohio 43221, USA)
Abstract:
Modification of cellular RNA controls the expression of different genes in organisms, ranging from humans to bacteria. Such modifications are regulated by a diverse group of demethylases including the ALKBH (AlkB homolog) family proteins, which has 9 members including ALKBH1-8 and fat mass and obesity-associated protein (FTO). ALKBH1 has six distinct enzymatic activities, including demethylation of m3C in DNA, demethylation of methylated lysine in histone H2A, demethylation of m6A in DNA, double hydroxylation of m5C in mitochondrial tRNA, demethylation of m1A in tRNA, and lyase cleavage of apurinic/ apyrimidinic sites in DNA. We have reported that the HIV-1 genome RNA contains m6A modification and that ALKBH5 and FTO negatively regulate HIV-1 protein expression in virus-producing cells (Tirumuru et al, eLife, 2016). However, the role of ALKBH1 in HIV-1 infection is unknown. Since HIV-1 replication requires human tRNAs in its lifecycle, we investigated whether ALKBH1 plays a role in HIV-1 infection and viral gene expression. We hypothesize that ALKBH1 may affect HIV-1 infection by regulating proviral DNA transcription, viral protein synthesis and virus production. We knocked-down ALKBH1 expression in target cells with specific siRNA, and infected the cells with single-cycle reporter HIV-1. We found that knockdown of ALKBH1 reduced HIV-1 Gag protein synthesis, while overexpression of ALKBH1 increased HIV-1 Gag protein synthesis in virus-producing cells. Moreover, compared to control cells, ALKBH1 knockdown or overexpression either reduced or increased cell proliferation, respectively. To examine the role of ALKBH1 in HIV-1 production, we transfected an HIV-1 proviral DNA plasmid into ALKBH1-overexpressing cells and observed reduced HIV-1 protein synthesis compared to control cells. Our results suggest that ALKBH1 plays an important role in cell proliferation and translation of HIV-1 proteins.
Keywords: ALKBH1, HIV-1, Demethylase