Poster abstracts
Poster number 2 submitted by Jenna Marie Antonucci
SAMHD1 suppresses HIV-1 gene expression and reactivation of viral latency in CD4+ T-cells
Jenna M. Antonucci (Center for Retrovirus Research, Center for RNA Biology, Department of Veterinary Biosciences, Department of Microbiology), Alice A. Duchon (Center for Retrovirus Research, Center for RNA Biology, Department of Chemistry and Biochemistry ), Olga Buzovetsky (Department of Molecular Biophysics and Biochemistry, Yale University), Yong Xiong (Department of Molecular Biophysics and Biochemistry, Yale University), Karin Musier-Forsyth (Center for Retrovirus Research, Center for RNA Biology, Department of Chemistry and Biochemistry ), Dr Li Wu (Center for Retrovirus Research, Center for RNA Biology, Department of Veterinary Biosciences)
Abstract:
The cellular dNTP hydrolase SAMHD1 restricts HIV-1 replication in non-dividing cells by degrading intracellular dNTPs to a level that limits efficient viral reverse transcription. Recombinant SAMHD1 binds HIV-1 DNA and RNA fragments in vitro, but the functional significance of the binding remains unclear. SAMHD1 is highly expressed in cell types that contribute to HIV-1 latent reservoirs, such as resting CD4+ T cells and myeloid cells. Transcriptional suppression of proviral DNA gene expression contributes to HIV-1 latency. However, it is unknown whether SAMHD1 regulates HIV-1 proviral gene expression in latently infected cells. Here, we investigate the effect of SAMHD1 on HIV-1 gene expression and the underlying mechanisms. We found that overexpression of SAMHD1 in HEK293T cells suppressed HIV-1 LTR promoter-driven luciferase expression in a dose-dependent manner at the level of transcription. We hypothesize that SAMHD1 may bind to the HIV-1 LTR to transcriptionally suppress viral gene expression in latently infected CD4+ T-cells. To study the effect of SAMHD1 on gene expression of integrated HIV-1 proviral DNA, we utilized the HIV-1 latently infected J-Lat cell line. We expressed exogenous SAMHD1 in J-Lat cells and observed that SAMHD1 reduced reactivation of HIV-1 gene expression. Additionally, a chromatin immunoprecipitation assay followed by qPCR revealed that SAMHD1 binds preferentially to the LTR in J-Lat cells, though it also binds to other HIV-1 gene sequences such as gag, rev, and vpr. We further investigated the in vitro binding affinity of recombinant SAMHD1 to single-stranded HIV-1 LTR and gag DNA fragments by fluorescence anisotropy. Binding assays performed over a range of salt concentrations are consistent with more specific binding of SAMHD1 to the LTR-derived DNA fragment relative to the gag fragment. Our data suggest that SAMHD1-mediated suppression of HIV-1 gene expression likely contributes to viral latency in CD4+ T-cells.
Keywords: HIV-1, SAMHD1, Latency