Frequent Incorporation of Ribonucleotides during HIV-1 Reverse Transcription and Their Attenuated Repair in Macrophages
2012; Elsevier BV; Volume: 287; Issue: 17 Linguagem: Inglês
10.1074/jbc.m112.348482
ISSN1083-351X
AutoresEdward M. Kennedy, Sarah M. Amie, Robert A. Bambara, Baek Kim,
Tópico(s)Cytomegalovirus and herpesvirus research
ResumoMacrophages 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. 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. IntroductionLentiviruses such as human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and simian immunodeficiency viruses uniquely replicate in both nondividing and dividing cells, whereas other retroviruses such as oncoretroviruses replicate exclusively in the latter (1Lewis P. Hensel M. Emerman M. Human immunodeficiency virus infection of cells arrested in the cell cycle.EMBO J. 1992; 11: 3053-3058Crossref PubMed Scopus (394) Google Scholar, 2Lewis P.F. Emerman M. Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus.J. Virol. 1994; 68: 510-516Crossref PubMed Google Scholar). Specifically, HIV-1 primarily infects activated CD4+ T cells and to a lesser extent, monocyte-derived macrophages. HIV-1 infection of terminally differentiated/nondividing cells such as macrophages and microglia directly impact viral pathogenesis. These cells are long-lived upon infection (3Chugh P. Fan S. Planelles V. Maggirwar S.B. Dewhurst S. Kim B. Infection of human immunodeficiency virus and intracellular viral Tat protein exert a pro-survival effect in a human microglial cell line.J. Mol. Biol. 2007; 366: 67-81Crossref PubMed Scopus (46) Google Scholar), in contrast to activated CD4+ T cells (4Stewart S.A. Poon B. Jowett J.B. Chen I.S. Human immunodeficiency virus type 1 Vpr induces apoptosis following cell cycle arrest.J. Virol. 1997; 71: 5579-5592Crossref PubMed Google Scholar), and produce both virus and proinflammatory factors known to compound HIV-1-associated neurocognitive disorder (5Achim C.L. Heyes M.P. Wiley C.A. Quantitation of human immunodeficiency virus, immune activation factors, and quinolinic acid in AIDS brains.J. Clin. Invest. 1993; 91: 2769-2775Crossref PubMed Scopus (182) Google Scholar, 6Gendelman H.E. Zheng J. Coulter C.L. Ghorpade A. Che M. Thylin M. Rubocki R. Persidsky Y. Hahn F. Reinhard Jr., J. Swindells S. Suppression of inflammatory neurotoxins by highly active antiretroviral therapy in human immunodeficiency virus-associated dementia.J. Infect Dis. 1998; 178: 1000-1007Crossref PubMed Scopus (153) Google Scholar, 7Price R.W. Brew B. Sidtis J. Rosenblum M. Scheck A.C. Cleary P. The brain in AIDS: central nervous system HIV-1 infection and AIDS dementia complex.Science. 1988; 239: 586-592Crossref PubMed Scopus (1081) Google Scholar). Macrophages also serve as a cellular reservoir of HIV-1 within the host and are compartmentalized in several tissues, especially in the brain, where elimination is challenging but essential for virus eradication (8Bagasra O. Bachman S.E. Jew L. Tawadros R. Cater J. Boden G. Ryan I. Pomerantz R.J. Increased human immunodeficiency virus type 1 replication in human peripheral blood mononuclear cells induced by ethanol: potential immunopathogenic mechanisms.J. Infect. Dis. 1996; 173: 550-558Crossref PubMed Scopus (76) Google Scholar, 9Schrager L.K. D'Souza M.P. Cellular and anatomical reservoirs of HIV-1 in patients receiving potent antiretroviral combination therapy.JAMA. 1998; 280: 67-71Crossref PubMed Scopus (285) Google Scholar, 10Schnell G. Joseph S. Spudich S. Price R.W. Swanstrom R. HIV-1 replication in the central nervous system occurs in two distinct cell types.PLoS Pathog. 2011; 7: e1002286Crossref PubMed Scopus (168) Google Scholar).Activated CD4+ T cells and macrophages differ enormously in cell cycle status and thus expression of ribonucleotide reductase R2 subunit, which determines the levels of the principal substrates, 2′-deoxynucleoside triphosphates (dNTPs), 3The abbreviations used are: dNTP2′-deoxynucleoside triphosphateFEN1flap endonuclease 1qRT-PCRquantitative real time PCRrNMPribonucleoside monophosphaterNTPribonucleoside triphosphate2LTRtwo long terminal repeatsRNase Hαribonuclease Hα. required for viral DNA synthesis mediated by HIV-1 reverse transcriptase (RT) (11Björklund S. Skog S. Tribukait B. Thelander L. S-phase-specific expression of mammalian ribonucleotide reductase R1 and R2 subunit mRNAs.Biochemistry. 1990; 29: 5452-5458Crossref PubMed Scopus (157) Google Scholar). Moreover, a substantial challenge that lentiviruses encounter during infection of macrophages is the poor cellular dNTP availability (12Diamond T.L. Roshal M. Jamburuthugoda V.K. Reynolds H.M. Merriam A.R. Lee K.Y. Balakrishnan M. Bambara R.A. Planelles V. Dewhurst S. Kim B. Macrophage tropism of HIV-1 depends on efficient cellular dNTP utilization by reverse transcriptase.J. Biol. Chem. 2004; 279: 51545-51553Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 13Jamburuthugoda V.K. Guo D. Wedekind J.E. Kim B. Kinetic evidence for interaction of human immunodeficiency virus type 1 reverse transcriptase with the 3′-OH of the incoming dTTP substrate.Biochemistry. 2005; 44: 10635-10643Crossref PubMed Scopus (17) Google Scholar). This low intracellular dNTP concentration causes substrate binding to RT to be the rate-limiting step in proviral DNA synthesis in human primary macrophages (20–40 nm dNTP concentration), which harbor ∼50–200 times lower dNTPs than activated CD4+ T cells (1–5 μm dNTP concentration) (12Diamond T.L. Roshal M. Jamburuthugoda V.K. Reynolds H.M. Merriam A.R. Lee K.Y. Balakrishnan M. Bambara R.A. Planelles V. Dewhurst S. Kim B. Macrophage tropism of HIV-1 depends on efficient cellular dNTP utilization by reverse transcriptase.J. Biol. Chem. 2004; 279: 51545-51553Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 14Kennedy E.M. Gavegnano C. Nguyen L. Slater R. Lucas A. Fromentin E. Schinazi R.F. Kim B. Ribonucleoside triphosphates as substrate of human immunodeficiency virus type 1 reverse transcriptase in human macrophages.J. Biol. Chem. 2010; 285: 39380-39391Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar).It is increasingly apparent that rNTPs, which serve as the substrates of cellular RNA polymerases, cellular kinases, and are metabolic energy carriers, can be incorporated into DNA by cellular DNA polymerases during chromosomal DNA replication (15Nick McElhinny S.A. Kumar D. Clark A.B. Watt D.L. Watts B.E. Lundström E.B. Johansson E. Chabes A. Kunkel T.A. Genome instability due to ribonucleotide incorporation into DNA.Nat. Chem. Biol. 2010; 6: 774-781Crossref PubMed Scopus (287) Google Scholar, 16Nick McElhinny S.A. Watts B.E. Kumar D. Watt D.L. Lundström E.B. Burgers P.M. Johansson E. Chabes A. Kunkel T.A. Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 4949-4954Crossref PubMed Scopus (299) Google Scholar). These events are normally infrequent because DNA polymerases, including HIV-1 RT, rigorously discriminate against nucleotides containing a 2′ OH such as rNTPs (17Astatke M. Ng K. Grindley N.D. Joyce C.M. A single side chain prevents Escherichia coli DNA polymerase I (Klenow fragment) from incorporating ribonucleotides.Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 3402-3407Crossref PubMed Scopus (181) Google Scholar, 18Bonnin A. Lázaro J.M. Blanco L. Salas M. A single tyrosine prevents insertion of ribonucleotides in the eukaryotic-type phi29 DNA polymerase.J. Mol. Biol. 1999; 290: 241-251Crossref PubMed Scopus (74) Google Scholar, 19Cases-Gonzalez C.E. Gutierrez-Rivas M. Ménendez-Arias L. Coupling ribose selection to fidelity of DNA synthesis: the role of Tyr-115 of human immunodeficiency virus type 1 reverse transcriptase.J. Biol. Chem. 2000; 275: 19759-19767Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 20Gao G. Orlova M. Georgiadis M.M. Hendrickson W.A. Goff S.P. Conferring RNA polymerase activity to a DNA polymerase: a single residue in reverse transcriptase controls substrate selection.Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 407-411Crossref PubMed Scopus (162) Google Scholar). However, high cellular concentrations of rNTPs (millimolar range) relative to dNTPs (nanomolar range) can promote rNMP incorporation during DNA replication. Therefore, when HIV-1 infects macrophages, HIV-1 RT faces the substrate specificity challenge during proviral DNA synthesis in the presence of limited dNTPs, which kinetically promotes rNMP incorporation. Here, we demonstrate that HIV-1 RT frequently incorporates rNMPs during proviral DNA synthesis in macrophages and that their capacity for repair of the resulting rNMP lesions is greatly reduced relative to activated CD4+ T cells.DISCUSSIONThere is considerable evidence that cellular DNA polymerases do not always properly use their canonical dNTP substrates, such that in the context of an entire genome, rNMPs are commonly incorporated (15Nick McElhinny S.A. Kumar D. Clark A.B. Watt D.L. Watts B.E. Lundström E.B. Johansson E. Chabes A. Kunkel T.A. Genome instability due to ribonucleotide incorporation into DNA.Nat. Chem. Biol. 2010; 6: 774-781Crossref PubMed Scopus (287) Google Scholar, 16Nick McElhinny S.A. Watts B.E. Kumar D. Watt D.L. Lundström E.B. Burgers P.M. Johansson E. Chabes A. Kunkel T.A. Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 4949-4954Crossref PubMed Scopus (299) Google Scholar). Yeast DNA polymerase ϵ incorporates rNMPs during chromosomal DNA replication at a rate of 1 in 1,250 nucleotides (16Nick McElhinny S.A. Watts B.E. Kumar D. Watt D.L. Lundström E.B. Burgers P.M. Johansson E. Chabes A. Kunkel T.A. Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 4949-4954Crossref PubMed Scopus (299) Google Scholar). Results indicate that HIV-1 incorporates rNMPs during proviral DNA synthesis in macrophages at the rate of 1 in 460 nucleotides. Interestingly, this noncanonical rNMP incorporation rate is 20–30 times higher than the incorrect dNMP incorporation rate of HIV-1 RT (28Preston B.D. Poiesz B.J. Loeb L.A. Fidelity of HIV-1 reverse transcriptase.Science. 1988; 242: 1168-1171Crossref PubMed Scopus (676) Google Scholar). This suggests that the initial rNMP incorporation during HIV-1 proviral DNA synthesis is frequent, and reverse transcription in macrophages results in 130 rNMP molecules incorporated/HIV-1 genome during infection. Frequent rNMP incorporation during proviral DNA synthesis in macrophages was logically predicted by our previous biochemical observation about the larger concentration disparity between dNTPs and rNTPs in macrophages compared with activated CD4+ T cells. Considering that cellular DNA polymerases also frequently incorporate noncanonical rNMPs and that cells are well equipped with a repair system for removing rNMPs embedded in dsDNA, it is a reasonable assumption that rNMPs incorporated during DNA synthesis must be detrimental to the cells. Indeed, defects in RNase H2, which is a key initial repair enzyme that specifically recognizes rNMPs embedded in dsDNA, cause the severe genetic disorder Aicardi-Goutières syndrome (33Crow Y.J. Leitch A. Hayward B.E. Garner A. Parmar R. Griffith E. Ali M. Semple C. Aicardi J. Babul-Hirji R. Baumann C. Baxter P. Bertini E. Chandler K.E. Chitayat D. Cau D. Déry C. Fazzi E. Goizet C. King M.D. Klepper J. Lacombe D. Lanzi G. Lyall H. Martínez-Frías M.L. Mathieu M. McKeown C. Monier A. Oade Y. Quarrell O.W. Rittey C.D. Rogers R.C. Sanchis A. Stephenson J.B. Tacke U. Till M. Tolmie J.L. Tomlin P. Voit T. Weschke B. Woods C.G. Lebon P. Bonthron D.T. Ponting C.P. Jackson A.P. Mutations in genes encoding ribonuclease H2 subunits cause Aicardi-Goutières syndrome and mimic congenital viral brain infection.Nat. Genet. 2006; 38: 910-916Crossref PubMed Scopus (524) Google Scholar).Unlike DNA, RNA is labile to both heat and alkaline hydrolysis (26Lipkin D. Talbert P.T. Cohn M. J. Am. Chem. Soc. 1954; 76: 2871-2872Crossref Scopus (38) Google Scholar). Therefore, after cellular polymerases misincorporate rNMPs in DNA, they are quickly repaired by enzymes of the RNase H2 family found in prokaryota, eukaryota, and archaea. RNase H2 specifically nicks 5′ of an rNMP, or RNA oligomer, in DNA. Our data demonstrate that macrophages express RNase H2, but it appears that the RNase H2 activity in macrophages is lower than that in CD4+ T cells. Interestingly, different forms of the RNase H2 subunit (Fig. 3A and supplemental Fig. S4B) appear to exist in different cell types, supporting the idea that their activities may be differentially regulated. In addition, after RNase H2 cleavage of the 5′ side of embedded rNMPs, the highly coordinated actions of multiple enzymes are required to complete the repair process. One major kinetic block of the rNMP repair process that specifically exists in macrophages is DNA gap repair, which requires cellular dNTPs. Considering the high Km values of host cellular DNA polymerases for dNTPs (micromolar range) and the extremely poor availability of cellular dNTPs in macrophages (20–40 nm), it is a logical assumption that the gap repair process after rNMP removal is kinetically impaired in macrophages (Fig. 4), compared with activated CD4+ T cells.Our observations detailed herein are directly relevant to other recent descriptions of the cellular incorporation and repair of rNMPs in chromosomal DNA. First, recent work with DNA polymerase ϵ revealed (16Nick McElhinny S.A. Watts B.E. Kumar D. Watt D.L. Lundström E.B. Burgers P.M. Johansson E. Chabes A. Kunkel T.A. Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 4949-4954Crossref PubMed Scopus (299) Google Scholar) that the replication of an rNMP-containing DNA template induces pausing of the DNA polymerase, which is one of the known elements that induces mutation synthesis (32Ji J. Hoffmann J.S. Loeb L. Mutagenicity and pausing of HIV reverse transcriptase during HIV plus-strand DNA synthesis.Nucleic Acids Res. 1994; 22: 47-52Crossref PubMed Scopus (41) Google Scholar). In this report, we have clearly demonstrated that HIV-1 RT recapitulates these results (Fig. 5). Second, work addressing the mutagenic impact of rNMPs in DNA has shown that a specific 2-bp deletion is present in the context of elevated ribonucleotide incorporation and in the absence of repair (15Nick McElhinny S.A. Kumar D. Clark A.B. Watt D.L. Watts B.E. Lundström E.B. Johansson E. Chabes A. Kunkel T.A. Genome instability due to ribonucleotide incorporation into DNA.Nat. Chem. Biol. 2010; 6: 774-781Crossref PubMed Scopus (287) Google Scholar). It was further shown in yeast that DNA topoisomerase I, in conjunction with an rNMP, is responsible for this distinct mutational spectrum. Additionally, we have found 45 of the (AT)2 and three (TC)3 topoisomerase I sites described previously (34Kim N. Huang S.N. Williams J.S. Li Y.C. Clark A.B. Cho J.E. Kunkel T.A. Pommier Y. Jinks-Robertson S. Mutagenic processing of ribonucleotides in DNA by yeast topoisomerase I.Science. 2011; 332: 1561-1564Crossref PubMed Scopus (219) Google Scholar) in the NL4–3 HIV-1 genome. Therefore, it is a distinct possibility that mutagenesis impacts HIV-1 replication; however, it is unclear at this time whether topoisomerase I is expressed and active in macrophages. Third, the rNMPs embedded in proviral DNA, more specifically in the minus strand, may also affect host RNA polymerase II (viral transcription). Indeed, the effect of rNMPs on the process of transcription has not been reported, and this issue requires further investigation. Finally, because DNA repair systems change with cell cycle and DNA replication during S phase, most of the DNA repair pathways were extensively studied in dividing cells. The most studied DNA repair system in nondividing cells is transcription-coupled nucleotide excision repair (TC-NER), which is activated by stalled RNA polymerases at DNA damage sites. However, whether TC-NER can recognize the rNMPs embedded in DNA as it does with other well known DNA lesions induced by UV and chemical modifications is still unclear. In summary, the virological impacts of rNMPs embedded in HIV-1 proviral DNA require further investigation.This study reveals that HIV-1 frequently incorporates rNMPs during proviral DNA synthesis in macrophages compared with activated CD4+ T cells. This unexpectedly frequent rNMP incorporation by HIV-1 RT was predicted by the uniquely large concentration disparity between dNTPs and rNTPs in macrophages observed previously (14Kennedy E.M. Gavegnano C. Nguyen L. Slater R. Lucas A. Fromentin E. Schinazi R.F. Kim B. Ribonucleoside triphosphates as substrate of human immunodeficiency virus type 1 reverse transcriptase in human macrophages.J. Biol. Chem. 2010; 285: 39380-39391Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Furthermore, the frequent rNMP incorporation by HIV-1 RT validates 3′ deoxyribonucleosides, termed ribonucleoside RT inhibitors, as a new class of anti-HIV therapeutic agents that specifically target HIV-1 reverse transcription in nondividing macrophages. IntroductionLentiviruses such as human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and simian immunodeficiency viruses uniquely replicate in both nondividing and dividing cells, whereas other retroviruses such as oncoretroviruses replicate exclusively in the latter (1Lewis P. Hensel M. Emerman M. Human immunodeficiency virus infection of cells arrested in the cell cycle.EMBO J. 1992; 11: 3053-3058Crossref PubMed Scopus (394) Google Scholar, 2Lewis P.F. Emerman M. Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus.J. Virol. 1994; 68: 510-516Crossref PubMed Google Scholar). Specifically, HIV-1 primarily infects activated CD4+ T cells and to a lesser extent, monocyte-derived macrophages. HIV-1 infection of terminally differentiated/nondividing cells such as macrophages and microglia directly impact viral pathogenesis. These cells are long-lived upon infection (3Chugh P. Fan S. Planelles V. Maggirwar S.B. Dewhurst S. Kim B. Infection of human immunodeficiency virus and intracellular viral Tat protein exert a pro-survival effect in a human microglial cell line.J. Mol. Biol. 2007; 366: 67-81Crossref PubMed Scopus (46) Google Scholar), in contrast to activated CD4+ T cells (4Stewart S.A. Poon B. Jowett J.B. Chen I.S. Human immunodeficiency virus type 1 Vpr induces apoptosis following cell cycle arrest.J. Virol. 1997; 71: 5579-5592Crossref PubMed Google Scholar), and produce both virus and proinflammatory factors known to compound HIV-1-associated neurocognitive disorder (5Achim C.L. Heyes M.P. Wiley C.A. Quantitation of human immunodeficiency virus, immune activation factors, and quinolinic acid in AIDS brains.J. Clin. Invest. 1993; 91: 2769-2775Crossref PubMed Scopus (182) Google Scholar, 6Gendelman H.E. Zheng J. Coulter C.L. Ghorpade A. Che M. Thylin M. Rubocki R. Persidsky Y. Hahn F. Reinhard Jr., J. Swindells S. Suppression of inflammatory neurotoxins by highly active antiretroviral therapy in human immunodeficiency virus-associated dementia.J. Infect Dis. 1998; 178: 1000-1007Crossref PubMed Scopus (153) Google Scholar, 7Price R.W. Brew B. Sidtis J. Rosenblum M. Scheck A.C. Cleary P. The brain in AIDS: central nervous system HIV-1 infection and AIDS dementia complex.Science. 1988; 239: 586-592Crossref PubMed Scopus (1081) Google Scholar). Macrophages also serve as a cellular reservoir of HIV-1 within the host and are compartmentalized in several tissues, especially in the brain, where elimination is challenging but essential for virus eradication (8Bagasra O. Bachman S.E. Jew L. Tawadros R. Cater J. Boden G. Ryan I. Pomerantz R.J. Increased human immunodeficiency virus type 1 replication in human peripheral blood mononuclear cells induced by ethanol: potential immunopathogenic mechanisms.J. Infect. Dis. 1996; 173: 550-558Crossref PubMed Scopus (76) Google Scholar, 9Schrager L.K. D'Souza M.P. Cellular and anatomical reservoirs of HIV-1 in patients receiving potent antiretroviral combination therapy.JAMA. 1998; 280: 67-71Crossref PubMed Scopus (285) Google Scholar, 10Schnell G. Joseph S. Spudich S. Price R.W. Swanstrom R. HIV-1 replication in the central nervous system occurs in two distinct cell types.PLoS Pathog. 2011; 7: e1002286Crossref PubMed Scopus (168) Google Scholar).Activated CD4+ T cells and macrophages differ enormously in cell cycle status and thus expression of ribonucleotide reductase R2 subunit, which determines the levels of the principal substrates, 2′-deoxynucleoside triphosphates (dNTPs), 3The abbreviations used are: dNTP2′-deoxynucleoside triphosphateFEN1flap endonuclease 1qRT-PCRquantitative real time PCRrNMPribonucleoside monophosphaterNTPribonucleoside triphosphate2LTRtwo long terminal repeatsRNase Hαribonuclease Hα. required for viral DNA synthesis mediated by HIV-1 reverse transcriptase (RT) (11Björklund S. Skog S. Tribukait B. Thelander L. S-phase-specific expression of mammalian ribonucleotide reductase R1 and R2 subunit mRNAs.Biochemistry. 1990; 29: 5452-5458Crossref PubMed Scopus (157) Google Scholar). Moreover, a substantial challenge that lentiviruses encounter during infection of macrophages is the poor cellular dNTP availability (12Diamond T.L. Roshal M. Jamburuthugoda V.K. Reynolds H.M. Merriam A.R. Lee K.Y. Balakrishnan M. Bambara R.A. Planelles V. Dewhurst S. Kim B. Macrophage tropism of HIV-1 depends on efficient cellular dNTP utilization by reverse transcriptase.J. Biol. Chem. 2004; 279: 51545-51553Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 13Jamburuthugoda V.K. Guo D. Wedekind J.E. Kim B. Kinetic evidence for interaction of human immunodeficiency virus type 1 reverse transcriptase with the 3′-OH of the incoming dTTP substrate.Biochemistry. 2005; 44: 10635-10643Crossref PubMed Scopus (17) Google Scholar). This low intracellular dNTP concentration causes substrate binding to RT to be the rate-limiting step in proviral DNA synthesis in human primary macrophages (20–40 nm dNTP concentration), which harbor ∼50–200 times lower dNTPs than activated CD4+ T cells (1–5 μm dNTP concentration) (12Diamond T.L. Roshal M. Jamburuthugoda V.K. Reynolds H.M. Merriam A.R. Lee K.Y. Balakrishnan M. Bambara R.A. Planelles V. Dewhurst S. Kim B. Macrophage tropism of HIV-1 depends on efficient cellular dNTP utilization by reverse transcriptase.J. Biol. Chem. 2004; 279: 51545-51553Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 14Kennedy E.M. Gavegnano C. Nguyen L. Slater R. Lucas A. Fromentin E. Schinazi R.F. Kim B. Ribonucleoside triphosphates as substrate of human immunodeficiency virus type 1 reverse transcriptase in human macrophages.J. Biol. Chem. 2010; 285: 39380-39391Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar).It is increasingly apparent that rNTPs, which serve as the substrates of cellular RNA polymerases, cellular kinases, and are metabolic energy carriers, can be incorporated into DNA by cellular DNA polymerases during chromosomal DNA replication (15Nick McElhinny S.A. Kumar D. Clark A.B. Watt D.L. Watts B.E. Lundström E.B. Johansson E. Chabes A. Kunkel T.A. Genome instability due to ribonucleotide incorporation into DNA.Nat. Chem. Biol. 2010; 6: 774-781Crossref PubMed Scopus (287) Google Scholar, 16Nick McElhinny S.A. Watts B.E. Kumar D. Watt D.L. Lundström E.B. Burgers P.M. Johansson E. Chabes A. Kunkel T.A. Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 4949-4954Crossref PubMed Scopus (299) Google Scholar). These events are normally infrequent because DNA polymerases, including HIV-1 RT, rigorously discriminate against nucleotides containing a 2′ OH such as rNTPs (17Astatke M. Ng K. Grindley N.D. Joyce C.M. A single side chain prevents Escherichia coli DNA polymerase I (Klenow fragment) from incorporating ribonucleotides.Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 3402-3407Crossref PubMed Scopus (181) Google Scholar, 18Bonnin A. Lázaro J.M. Blanco L. Salas M. A single tyrosine prevents insertion of ribonucleotides in the eukaryotic-type phi29 DNA polymerase.J. Mol. Biol. 1999; 290: 241-251Crossref PubMed Scopus (74) Google Scholar, 19Cases-Gonzalez C.E. Gutierrez-Rivas M. Ménendez-Arias L. Coupling ribose selection to fidelity of DNA synthesis: the role of Tyr-115 of human immunodeficiency virus type 1 reverse transcriptase.J. Biol. Chem. 2000; 275: 19759-19767Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 20Gao G. Orlova M. Georgiadis M.M. Hendrickson W.A. Goff S.P. Conferring RNA polymerase activity to a DNA polymerase: a single residue in reverse transcriptase controls substrate selection.Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 407-411Crossref PubMed Scopus (162) Google Scholar). However, high cellular concentrations of rNTPs (millimolar range) relative to dNTPs (nanomolar range) can promote rNMP incorporation during DNA replication. Therefore, when HIV-1 infects macrophages, HIV-1 RT faces the substrate specificity challenge during proviral DNA synthesis in the presence of limited dNTPs, which kinetically promotes rNMP incorporation. Here, we demonstrate that HIV-1 RT frequently incorporates rNMPs during proviral DNA synthesis in macrophages and that their capacity for repair of the resulting rNMP lesions is greatly reduced relative to activated CD4+ T cells.
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