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Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and ...

King's College London

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.

Andrew Sobala

Department of Infectious Diseases,
School of Immunology & Microbial Sciences,
Department of Infectious Diseases, School of Immunology & Microbial Sciences

King's College London

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

Andrew Sobala

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King's College London

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

Department of Infectious Diseases,
School of Immunology & Microbial Sciences,
Department of Infectious Diseases, School of Immunology & Microbial Sciences