DEOXYNUCLEOSIDE TRIPHOSPHATE CONCENTRATIONS EMPHASIZE THE PROCESSIVITY DEFECT OF LAMIVUDINE-RESISTANT VARIANTS OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REVERSE-TRANSCRIPTASE

The nucleoside drug lamivudine (3TC) triggers the selection of resista nt forms of the human immunodeficiency virus type 1 (HIV-1) reverse tr anscriptase (RT) with a substitution of amino acid 184Met. The 3TC-res istant RT enzymes 184Val and 184Ile exhibit a processivity defect in i s vitro assays that correlates with reduced replication of the corresp onding virus variants in primary cells. However, no replication defect is apparent for these two mutants in the transformed T-cell line SupT 1. One obvious difference between the two cell types is the intracellu lar deoxynucleoside triphosphate (dNTP) level. Primary cells have a mu ch smaller dNTP pool, and this cellular condition may emphasize the pr ocessivity defect of the codon 184 RT variants. Alternatively, cell-sp ecific cofactors that influence the process of reverse transcription m ay exist. Such accessory factors may be packaged into the virion to ex ert an effect on the RT enzyme. To discriminate between these possibil ities we performed additional assays with the wild-type and mutant RT enzymes. The RT proteins were either isolated from virions produced by primary and transformed cell types or expressed as recombinant protei n. We also performed infection assays with cells treated with a drug t hat reduces the intracellular dNTP pool. Furthermore, reverse transcri ption was studied within virus particles in the endogenous assay; whic h allows for the manipulation of the dNTP level. The combined results indicate that the enzymatic defect of the 3TC-resistant HIV-1 variants is stressed at low dNTP concentrations.