Pyrimidine nucleotide and nucleic acid synthesis in human monocytes and macrophages

Terai, Chihiro; Carson, Dennis A.

doi:10.1016/0014-4827(91)90110-g

Cited by 42 publications

(28 citation statements)

References 35 publications

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“…Primer-templates terminated with nucleotide analogues such as ddAMP or d4TMP that contain smaller 3Ј substituents are also removed efficiently but form DEC at low dNTP concentrations (K ddNTP , 3 to 20 M) (12,22,23,25,26). Therefore, removal of these nucleotide analogues under physiological conditions will depend on the intracellular concentration of dNTPs, which have been reported to range from 0.14 to 5.6 M in resting lymphocytes (10,11,28,33), 2.4 to 26 M in mitogen-stimulated lymphocytes (10,11), and 15 to 170 M in CEM lymphoblasts (28,33). Although AZT resistance mutations confer an increase in removal of most chain terminators and likely confer some resistance to many types of chain terminators in HIV-infected individuals, the impact of these mutations on phenotypic resistance assays is likely underestimated due to very high dNTP levels present in the cells used in these assays.…”

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Effects of Dipeptide Insertions between Codons 69 and 70 of Human Immunodeficiency Virus Type 1 Reverse Transcriptase on Primer Unblocking, Deoxynucleoside Triphosphate Inhibition, and DNA Chain Elongation

Meyer

Lennerstrand

Matsuura

et al. 2003

J Virol

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Finger insertion mutations of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) (T69S mutations followed by various dipeptide insertions) have a multinucleoside resistance phenotype that can be explained by decreased sensitivity to deoxynucleoside triphosphate (dNTP) inhibition of the nucleotidedependent unblocking activity of RT. We show that RTs with SG or AG (but not SS) insertions have three-to fourfold-increased unblocking activity and that all three finger insertion mutations have threefold-decreased sensitivity to dNTP inhibition. The additional presence of M41L and T215Y mutations increased unblocking activity for all three insertions, greatly reduced the sensitivity to dNTP inhibition, and resulted in defects in in vitro DNA chain elongation. The DNA chain elongation defects were partially repaired by additional mutations at positions 210, 211, and 214. These results suggest that structural communication between the regions of RT defined by these mutations plays a role in the multinucleoside resistance phenotype.Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is responsible for replication of the HIV-1 genome and is therefore an important target for antiretroviral therapy. Many inhibitors of HIV-1 RT are nucleoside analogues that are converted to their active triphosphate forms by host cell kinases and are incorporated into the viral genome by HIV-1 RT. Because nucleoside analogues lack a 3Ј-OH group, their incorporation prevents further extension of the DNA chain. HIV-1 RT becomes resistant to nucleoside analogues primarily through two mechanisms (8, 12): (i) increased discrimination against the compounds (9, 13, 35) leading to decreased incorporation and less chain termination and (ii) increased ability to remove chain terminators from blocked DNA chains, allowing DNA synthesis to resume (1,3,19,20,22,26). The removal occurs through excision of the 3Ј-terminal nucleotide by transfer to one of several potential acceptor substrates, including pyrophosphate (PP i ) or ATP, generating the triphosphate form of the chain terminator or a dinucleoside polyphosphate, respectively. Most zidovudine (AZT) resistance mutations, including M41L, D67N, K70R, T215F/Y, and K219Q (29), confer increased removal, compared to wildtype (WT) RT (3,19,22,25,26). The effect of these mutations is greatest when a nucleoside triphosphate, such as ATP, is used as the acceptor substrate, whereas there is little if any effect of AZT resistance mutations on removal when a nucleoside diphosphate or PP i is used as the substrate (3,16,20,22,25).The efficiency of the removal reaction under physiological conditions will depend on the rate of the reaction and the ability of deoxynucleoside triphosphates (dNTPs) to inhibit it. HIV-1 RT bound to a chain-terminated primer-template will still bind the dNTP complementary to the next nucleotide on the template strand (34) to form a stable complex. Since chemical bond formation is prevented due to lack of a 3Ј-OH, this complex has been named a dead-end...

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Effects of Dipeptide Insertions between Codons 69 and 70 of Human Immunodeficiency Virus Type 1 Reverse Transcriptase on Primer Unblocking, Deoxynucleoside Triphosphate Inhibition, and DNA Chain Elongation

Meyer

Lennerstrand

Matsuura

et al. 2003

J Virol

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“…Because primate lentiviruses infect non-dividing macrophage cells that are characterized by low intracellular dNTP pool levels with a low dCTP/dTTP ratio and a high level of dUTP pool (19), they have a non-negligible probability to misincorporate uracil residues into their genome during viral DNA synthesis. Therefore, it is likely that they have evolved to control uracil misincorporation into viral DNA.…”

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Functional Role of HIV-1 Virion-associated Uracil DNA Glycosylase 2 in the Correction of G:U Mispairs to G:C Pairs

Priet

Navarro

Gros

et al. 2003

Journal of Biological Chemistry

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Human monocytes/macrophages are target cells for HIV-1 infection. As other non-dividing cells, they are characterized by low and imbalanced intracellular dNTP pool levels and an excess of dUTP. The replication of HIV-1 in this cellular context favors misincorporation of uracil residues into viral DNA because of the use of dUTP in place of dCTP. We have previously reported that the host uracil DNA glycosylase enzyme UNG2 is packaged into HIV-1 viral particles via a specific association with the integrase domain of the Gag-Pol precursor. In this study, we investigated whether virion-associated UNG2 plays a role similar to that of its cellular counterpart. We show that the L172A mutation of integrase impaired the packaging of UNG2 into viral particles. Using a primer-template DNA substrate containing G:U mispairs, we demonstrate that wild-type viral lysate has the ability to repair G:U mismatched pairs to G:C matched pairs, in contrast to UNG2-deficient viral lysate. Moreover, no correction of G:T mispairs by wildtype HIV-1 viral lysate was observed, which argues for the specificity of the repair process. We also show that UNG2 physically associates with the viral reverse transcriptase enzyme. Altogether our data indicate for the first time that a uracil repair pathway is specifically associated with HIV-1 viral particles. However, the molecular mechanism of this process remains to be characterized further.

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“…ATP is present at millimolar concentrations in lymphocytes (37), making it the most likely acceptor substrate for the removal reaction. Estimates of dNTP concentrations depend on assumptions about cell volume (3,5,11,33,36) and range from 0.14 to 5.6 M in resting lymphocytes (10,11,31,37), 2.4 to 26 M in mitogen-stimulated lymphocytes (10,11), and 15 to 170 M in CEM lymphoblasts (31,37). These values suggest that removal of AZTMP residues can occur in cells at most stages of activation, since the IC 50 for dNTP inhibition is Ͼ100 M, but that removal of d4TMP will be inhibited except in cells that are relatively quiescent.…”

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Differential Removal of Thymidine Nucleotide Analogues from Blocked DNA Chains by Human Immunodeficiency Virus Reverse Transcriptase in the Presence of Physiological Concentrations of 2′-Deoxynucleoside Triphosphates

Meyer¹,

Matsuura²,

Schinazi

et al. 2000

Antimicrob Agents Chemother

103

119

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Removal of 2,3-didehydro-3-deoxythymidine-5-monophosphate (d4TMP) from a blocked DNA chain can occur through transfer of the chain-terminating residue to a nucleotide acceptor by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). ATP-dependent removal of either d4TMP or 3-azido-3-deoxythymidine-5-monophosphate (AZTMP) is increased in AZT resistant HIV-1 RT (containing D67N/K70R/ T215F/K219Q mutations). Removal of d4TMP is strongly inhibited by the next complementary deoxynucleoside triphosphate (50% inhibitory concentration [IC 50 ] of ϳ0.5 M), whereas removal of AZTMP is much less sensitive to this inhibition (IC 50 of >100 M). This could explain the lack of cross-resistance by AZT-resistant HIV-1 to d4T in phenotypic drug susceptibility assays.Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and other retroviral RTs lack 3Ј-5Ј exonuclease activity (2, 30) but can remove 3Ј-terminal chain-terminating residues from blocked DNA chains through a nucleotide-dependent mechanism leading to production of dinucleoside polyphosphates (23, 24) or through pyrophosphorolysis (the reversal of polymerization) (1,4,13,29). We have recently shown (23) that HIV-1 RT containing the 3Ј-azido-3Ј-deoxythymidine (AZT) resistance mutations D67N, K70R, T215F, and K219Q (67/70/215/219 mutant RT) removes AZT-5Ј-monophosphate (AZTMP) from blocked primer-templates through the nucleotide-dependent mechanism more efficiently than does wildtype (WT) RT. The mutant enzyme also removes 2Ј,3Ј-dideoxyadenosine-5Ј-monophosphate (ddAMP) from blocked DNA chains more efficiently than does WT RT. Removal of ddAMP is strongly suppressed by physiological concentrations of deoxynucleoside triphosphates (dNTPs), whereas removal of AZTMP is much less sensitive to this inhibition (23).The chain terminator 2Ј,3Ј-didehydro-3Ј-deoxythymidine-5Ј-triphosphate (d4TTP) is efficiently incorporated into growing DNA chains by HIV-1 RT (39). Resistance to d4T can arise in cell culture through a valine-to-threonine mutation at position 75 (19, 21, 32); however, this mutation is rarely observed in HIV-1 from d4T-treated individuals (6,9,17,22,27,35). Instead, mutations associated with AZT resistance, including M41L, D67N, K70R, L210W, and T215Y/F, are frequently selected (6,8,21,22,27,32,35). The selection of AZT resistance mutations by d4T in the absence of AZT is unexpected, since phenotypic assays show little, if any, cross-resistance between these drugs (20,22). Nonetheless, clinical studies have shown that prior exposure to AZT reduces the efficacy of subsequent treatment with d4T (17), and the presence of AZT resistance mutations is correlated with reduced suppression of viral load in response to d4T-containing therapies (15, 25). These results suggest that the phenotypic assays do not fully reflect the in vivo sensitivity of HIV-1 replication to d4T.In an effort to understand the biochemical basis for the lack of cross-resistance by AZT-resistant mutants to d4T, we have investigated the ability of WT and 67/70/215/219 m...

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Pyrimidine nucleotide and nucleic acid synthesis in human monocytes and macrophages

Cited by 42 publications

References 35 publications

Effects of Dipeptide Insertions between Codons 69 and 70 of Human Immunodeficiency Virus Type 1 Reverse Transcriptase on Primer Unblocking, Deoxynucleoside Triphosphate Inhibition, and DNA Chain Elongation

Effects of Dipeptide Insertions between Codons 69 and 70 of Human Immunodeficiency Virus Type 1 Reverse Transcriptase on Primer Unblocking, Deoxynucleoside Triphosphate Inhibition, and DNA Chain Elongation

Functional Role of HIV-1 Virion-associated Uracil DNA Glycosylase 2 in the Correction of G:U Mispairs to G:C Pairs

Differential Removal of Thymidine Nucleotide Analogues from Blocked DNA Chains by Human Immunodeficiency Virus Reverse Transcriptase in the Presence of Physiological Concentrations of 2′-Deoxynucleoside Triphosphates

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