title: Frequent incorporation of ribonucleotides during HIV-1 reverse transcription and their attenuated repair in macrophages.
abstract: Macrophages are well known long-lived reservoirs of HIV-1. Unlike activated CD4(+) T cells, this nondividing HIV-1 target cell type contains a very low level of the deoxynucleoside triphosphates (dNTPs) required for proviral DNA synthesis whereas the ribonucleoside triphosphate (rNTP) levels remain in the millimolar range, resulting in an extremely low dNTP/rNTP ratio. Biochemical simulations demonstrate that HIV-1 reverse transcriptase (RT) efficiently incorporates ribonucleoside monophosphates (rNMPs) during DNA synthesis at this ratio, predicting frequent rNMP incorporation by the virus specifically in macrophages. Indeed, HIV-1 RT incorporates rNMPs at a remarkable rate of 1/146 nucleotides during macrophage infection. This greatly exceeds known rates for cellular replicative polymerases. In contrast, little or no rNMP incorporation is detected in CD4(+) T cells. Repair of these rNMP lesions is also substantially delayed in macrophages compared with CD4(+) T cells. Single rNMPs embedded in a DNA template are known to induce cellular DNA polymerase pausing, which mechanistically contributes to mutation synthesis. Indeed, we also observed that embedded rNMPs in a dsDNA template also induce HIV-1 RT DNA synthesis pausing. Moreover, unrepaired rNMPs incorporated into the provirus during HIV-1 reverse transcription would be generally mutagenic as was shown in Saccharomyces cerevisiae. Most importantly, the frequent incorporation of rNMPs makes them an ideal candidate for development of a new class of HIV RT inhibitors.
authors.affiliation: Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York 14642, USA.
authors.forename: Edward M
authors.initials: EM
authors.lastname: Kennedy
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Amie
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bambara
authors.forename: Baek
authors.initials: B
authors.lastname: Kim
mesh_terms: D001483:Base Sequence; D015496:CD4-Positive T-Lymphocytes / Q000821:virology*; D004260:DNA Repair; D004261:DNA Replication; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D019169:Jurkat Cells; D007700:Kinetics; D008264:Macrophages / Q000166:cytology / Q000821:virology*; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000235:genetics; D048348:Reverse Transcription / Q000502:physiology*; D016914:Ribonuclease H / Q000378:metabolism; D012265:Ribonucleotides / Q000235:genetics; D013997:Time Factors
chemical_list: D009711:Nucleotides; D012265:Ribonucleotides; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; C091139:ribonuclease HII; D016914:Ribonuclease H