Reduced dNTP binding affinity of 3TC-resistant M184I HIV-1 reverse transcriptase variants responsible for viral infection failure in macrophage.
ABSTRACT We characterized HIV-1 reverse transcriptase (RT) variants either with or without the (-)-2',3'-deoxy-3'-thiacytidine-resistant M184I mutation isolated from a single HIV-1 infected patient. First, unlike variants with wild-type M184, M184I RT variants displayed significantly reduced DNA polymerase activity at low dNTP concentrations, which is indicative of reduced dNTP binding affinity. Second, the M184I variant displayed a approximately 10- to 13-fold reduction in dNTP binding affinity, compared with the Met-184 variant. However, the k(pol) values of these two RTs were similar. Third, unlike HIV-1 vectors with wild-type RT, the HIV-1 vector harboring M184I RT failed to transduce cell types containing low dNTP concentrations, such as human macrophage, likely due to the reduced DNA polymerization activity of the M184I RT under low cellular dNTP concentration conditions. Finally, we compared the binary complex structures of wild-type and M184I RTs. The Ile mutation at position 184 with a longer and more rigid beta-branched side chain, which was previously known to alter the RT-template interaction, also appears to deform the shape of the dNTP binding pocket. This can restrict ground state dNTP binding and lead to inefficient DNA synthesis particularly at low dNTP concentrations, ultimately contributing to viral replication failure in macrophage and instability in vivo of the M184I mutation.
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ABSTRACT: The fidelity of DNA polymerases is largely attributable to a two-step nucleotide binding mechanism. In the first step, binding contacts are initially made between the template and the incoming dNTP. The selectivity of this ground-state binding is similar in magnitude to the selectivity seen in forming base pairs in solution. In the second step, a change in protein conformation occurs, which leads to rapid incorporation of the dNTP into the growing polymer. This conformational change appears to occur globally in that it is inhibited by mismatches in the dNTP or in any of the three terminal base pairs of the primer/template. The open conformation allows rapid binding of the dNTP from solution, while the closed conformation provides steric checks for the proper Watson-Crick base pair geometry. This conformational change accounts for the extraordinary fidelity of polymerization and also provides selectivity to the exonuclease by inhibiting polymerization over a mismatch in the primer/template. The overall fidelity approaches one error in 10(10) by a combination of selectivity in polymerization (10(5)-10(6)) and in proofreading (10(3)-10(4)). This paradigm provides the theoretical basis for further investigation of the structural basis for fidelity by pointing to the essential elements of the polymerization reaction that need to be examined in order to evaluate active-site-directed mutants of polymerases to test appropriate structure/function relationships.Annual Review of Biochemistry 02/1993; 62:685-713. · 34.32 Impact Factor
Article: Mutations at codon 184 in simian immunodeficiency virus reverse transcriptase confer resistance to the (-) enantiomer of 2',3'-dideoxy-3'-thiacytidine.[show abstract] [hide abstract]
ABSTRACT: Variants of simian immunodeficiency virus (SIV) that display greater than 2,000-fold resistance to the (-) enantiomer of 2',3'-dideoxy-3'-thiacytidine (3TC) were generated through in vitro passage and drug selection. The polymerase regions of several of these resistant viruses were sequenced and were found to share either of two codon alterations at site 184 in reverse transcriptase (ATG to ATA [methionine to isoleucine] and ATG to GTA [methionine to valine]). The biological relevance of these substitutions for 3TC was confirmed by site-directed mutagenesis with the SIVmac239 infectious recombinant clone of SIV.Antimicrobial Agents and Chemotherapy 01/1998; 41(12):2763-5. · 4.84 Impact Factor
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ABSTRACT: Disturbances in neurotransmitter distribution have been observed in cerebral ischemia in the pathophysiologic process of excitotoxicity. The goal of this study was to examine the effect of pressure-induced retinal ischemia on the distribution of the retinal neurotransmitters glutamate and gamma-aminobutyric acid (GABA) within the rat retina. Animals were subjected to increased intraocular pressure of 110 mm Hg for 45 min using an intracameral hydrostatic pressure device. The distribution of glutamate and GABA immunoreactivity (IR) was determined at 0, 2, 4, 8 and 24 hrs after reperfusion by immunogold with silver intensification. Three phases of neurotransmitter immunoreactivity patterns were discernible following retinal ischemia. Immediately following reperfusion (Phase I), a shift of GABA-IR from inner retinal neurons to the Mueller cells and their processes was noted. In contrast, despite marked decreases in neuronal glutamate-IR, a less pronounced shift of glutamate-IR to the Muller cells was simultaneously noted. This shift of neurotransmitter IR to the Mueller cells was transient with the gradual reappearance of IR within the inner retinal neurons noted 2-8 hrs after reperfusion (Phase II). Phase III began at 8 hrs after reperfusion with progressive loss of GABA-IR noted in the inner retina; by 24 hrs, secondary loss of inner retinal glutamate-IR was evident with corresponding dropout and pyknosis of inner retinal neurons apparent. The distribution of glutamate-IR and GABA-IR was significantly altered following retinal ischemia. The alteration noted in Phase I suggested that the regulation of glutamate by Mueller cells was disrupted by this ischemic insult leading to glutamate excitotoxicity, and delayed neuronal cell degeneration as evidenced by the subsequent loss of inner retinal immunoreactivity in Phase III.Current Eye Research 07/1996; 15(6):589-96. · 1.28 Impact Factor