Following cell entry, the RNA genome of HIV-1 is reverse transcribed into double-stranded DNA that ultimately integrates into the host-cell genome to establish the provirus. These early phases of infection are notably vulnerable to suppression by a collection of cellular antiviral effectors, called restriction or resistance factors. The host antiviral protein APOBEC3G (A3G) antagonizes the early steps of HIV-1 infection through the combined effects of inhibiting viral cDNA production and cytidine-to-uridine-driven hypermutation of this cDNA. In seeking to address the underlying molecular mechanism for inhibited cDNA synthesis, we developed a deep sequencing strategy to characterize nascent reverse transcription products and their precise 3′-termini in HIV-1 infected T cells. Our results demonstrate site- and sequence-independent interference with reverse transcription, which requires the specific interaction of A3G with reverse transcriptase itself. This approach also established, contrary to current ideas, that cellular uracil base excision repair (UBER) enzymes target and cleave A3G-edited uridine-containing viral cDNA. Together, these findings yield further insights into the regulatory interplay between reverse transcriptase, A3G and cellular DNA repair machinery, and identify the suppression of HIV-1 reverse transcriptase by a directly interacting host protein as a new cell-mediated antiviral mechanism.
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The authors acknowledge support (and debate) from members of the Malim laboratory, the insights of J. Ule and R. Oakey on optimization of the sequencing protocol, and M. Emerman and S. Hughes for the provision of reagents. The authors thank M. Arno at the King’s College London Genomic Centre and D. Hughes at the University College London (UCL) Institute for Neurology Next Generation Sequencing Facility for help with MiSeq sequencing runs. The work was supported by the UK Medical Research Council (G1000196 and MR/M001199/1 to M.M. and MR/K015664/1 to M.P.), the Wellcome Trust (106223/Z/14/Z to M.M.), the European Commission’s Seventh Framework Programme (FP7/2007-2013) under grant agreement no. PIIF-GA-2012-329679 (to D.P.), King’s alumni community sponsored King’s Undergraduate Research Fellowships (to R.D.L.), King’s School of Medicine Summer Studentship Award (to J.C.) and the Department of Health via a National Institutes for Health Research Comprehensive Biomedical Research Center award to Guy’s and St. Thomas’ NHS Foundation Trust in partnership with King’s College London and King’s College Hospital NHS Foundation Trust (guysbrc-2012-1).
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