THE FIDELITY OF 3'-MISINSERTION AND MISPAIR EXTENSION DURING DNA-SYNTHESIS EXHIBITED BY 2 DRUG-RESISTANT MUTANTS OF THE REVERSE-TRANSCRIPTASE OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 WITH LEU74-]VAL AND GLU89-]GLY

The relatively low fidelity of DNA synthesis characteristic to the rev erse transcriptases (RTs) of the AIDS-causing viruses, human immunodef iciency viruses types 1 and 2 (HIV-1 and HIV-2, respectively) was impl icated as a dominant factor that contributes to the genetic hypervaria bility of these viruses. The formation of 3'-mispaired DNA and the sub sequent extension of this DNA were shown to be key determinants that l ead to the error proneness of these RTs. As part of our goal to study the structure/function relationship in HIV-1 RT,we have conducted muta tional studies aimed at identifying amino-acid residues involved in af fecting the fidelity of DNA synthesis by the enzyme. We have recently found that two mutants of HIV-1 RT, which show resistance to nucleosid e analog inhibitors ([Leu184]RT and [Phe183]RT), exhibit ia vitro erro r proneness of DNA synthesis lower than that of wild-type enzyme [Bakh anshvili, M., Avidan, O. & Hizi, A. (1996) Mutational studies of human immunodeficiency virus type I reverse transcriptase: the involvement of residues 183 and 184 in the fidelity of DNA synthesist FEES Lett. 3 91, 257-262]. Using both criteria, the current comparative study sugge sts that these two mutant RTs display a substantially enhanced fidelit y of DNA synthesis relative to the wild-type RT counterpart. In the cu rrent study we have analyzed two additional drug-resistant mutants of HIV-1 RT, [Val74]RT and [Gly89]RT, for their in vitro fidelity of DNA synthesis using two parameters of DNA synthesis: 3' mispair formation and elongation of 3'-mismatched DNA. The current comparative study sug gests that these two mutant RTs display a substantially enhanced fidel ity of DNA synthesis relative to the wild-type RT counterpart, using b oth criteria, Analysis of the relative frequencies of misinsertion and mispair extension indicates that the overall error proneness of DNA s ynthesis in HIV-1 RT is wildtype > [Val74]RT > [Gly89]RT mutant. The r esults further support the possible linkage between the capacity of an enzyme to incorporate a nucleoside analog instead of the correct dNTP (leading to drug sensitivity) and the ability to incorporate and exte nd a wrong nucleotide (resulting in mutagenesis). Our results may bear on the potential use of selecting aid maintaining HIV virions with hi gh fidelity and drug-resistant RTs to suppress the subsequent appearan ce of virions resistant to other drugs.