A possible role for cysteine residues in the fidelity of DNA synthesis exhibited by the reverse transcriptases of human immunodeficiency viruses type 1 and type 2

Abstract

HIV reverse transcrlptases (RTs) have few cysteine residues rda t.ive to other RTs and retain their DNA polymerization functions followin8 eheJ~),ieal modification by thiol.speeifie reagents. The functional role or ire cysteines in the fidelity of the DNA-dependent DNA synthesis of HIV RTs has been addressed by chemical modification of the wild-type enzy:.~es in combination with the analysis of an ermymatically active mutant HIV-I RT in which all eysteines were modified to serines. We have observed an increase in Y-terminal mispair extension efficiency exhibited by ehemi,:~.'dly modifi~l HIV-I and HIV-2 RTs. The possible involvement of eysteine residues was further substantiated asia8 the cysteine.free mutant I-IlIV-i RT that displays an increased efficiency of mispair extension. These results provide evidence for a possible role of,~steine residues in the flt"~:lity of DNA synthesis catalyzed by HIV RTs,

Mutational studies of human immunodeficiency virus type 1 reverse transcriptase : the involvement of residues 183 and 184 in the fidelity of DNA synthesis

“…Parallel studies of the Klenow fragment of E. coli DNA polymerase I have revealed that the substitution of Tyr-766 leads to a substantial decrease in fidelity [29]. Reduced fidelity of DNA synthesis has also been observed with HIV-1 RT mutants in which Cys-38 and Cys-280 were replaced by serines [19]. It should be emphasized that all of the above-mentioned mutations result in a decrease in fidelity, while the mutant M184L and Y183F of HIV-1 RT show the opposite --an increase in fidelity.…”

“…The apparent Km and Vm~ steady-state kinetic values for each dNTP were calculated from double-reciprocal plots of initial elongation velocity versus substrate concentrations (not shown) and are summarized in Table 1. As apparent for wild-type RT [16,19], misinsertion of the non-complementary nucleotides and subsequent extension is obtained at substantially higher nucleotide concentrations than complementary insertion of dTTP. The ratios of the insertion efficiencies for the wrong (W) versus right (R) nucleotides provide the frequency of misinsertion (Fins)…”

“…We have already suggested that the low cysteine content in HIV-1 and HIV-2 RTs might be associated with the errorproneness of these enzymes [19]. Two mutants of HIV-1 RT (Tyr-181 ~ lie and Tyr-188 ~ Leu) resistant to non-nucleoside inhibitors, exhibit a 3'-mispair extension frequency that is similar to that of wild-type RT [20].…”

DNA synthesis exhibited by the reverse transcriptase of mouse mammary tumor virus : Processivity and fidelity of misinsertion and mispair extension

“…Clearly, there are subtle structural features in the lentiviral RTs that make them more error prone during DNA synthesis than other RTs. Thus, the low cysteine content in both HIV-1 and HIV-2 RTs has been suggested to be associated with fidelity, since mutants lacking cysteine residues 38 and 280, or wild-type RT enzymes treated with sulfhydryl reagents, have a lower fidelity than that of the untreated wildtype enzymes [31]. It was also suggested that mutations in HIV-1 RT that confer resistance to nucleoside analogs (i.e.…”

The Fidelity of 3′ Misinsertion and Mispair Extension During DNA Synthesis Exhibited by two Drug‐Resistant Mutants of the Reverse Transcriptase of Human Immunodeficiency Virus Type 1 with Leu74→Val and Glu89→Gly

“…We have already suggested that the low cysteine content in HIV-1 and HIV-2 RTs might be associated with the high error proneness of these enzymes [20]. Two other mutants of HIV-1 RT resistant to nonnucleoside inhibitors, i.e.…”