EFFECTS OF 3'-DEOXYNUCLEOSIDE 5'-TRIPHOSPHATE CONCENTRATIONS ON CHAINTERMINATION BY NUCLEOSIDE ANALOGS DURING HUMAN-IMMUNODEFICIENCY-VIRUSTYPE-1 REVERSE TRANSCRIPTION OF MINUS-STRAND STRONG-STOP DNA

We have compared the effects of nucleoside analogs in quiescent and ph ytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells ( PBMC) exposed to human immunodeficiency virus type 1 (HIV-1) with thos e of their triphosphorylated derivatives in cell-free HIV-1 reverse tr anscription assays. We observed a substantial decrease in synthesis of early minus-strand proviral DNA products in HIV-l-infected, quiescent PBMC exposed to each of 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideo xyinosine (ddI), and 2',3'-dideoxy-3'-thiacytidine (3TC), in compariso n with nontreated, infected controls. In contrast, no such diminution was observed when PHA-stimulated, HIV-l-infected PBMC were treated wit h the same drugs. This result was attributed to previously reported fi ndings that PHA-stimulated PBMC possessed larger deoxynucleoside triph osphate (dNTP) pools than quiescent cells did. To further investigate this subject, a cell-free HIV-1 reverse transcription reaction involvi ng HIV-1 RNA genomic template, recombinant purified HIV-1 reverse tran scriptase, all four dNTPs and either tRNA(3)(Lys) or a deoxyoligonucle otide as primer was used to monitor chain termi-nation mediated by 2', 3'-dideoxynucleoside triphosphates (ddNTPs) during synthesis of minus- strand strong-stop DNA. Augmented chain termination was observed with decreasing concentrations of both ddNTP and dNTP when the ratio of dNT P to ddNTP was fixed. We also found that both the number and strength of reverse transcription pause sites were increased at low concentrati ons of dNTPs and when a deoxyoligonucleotide primer was used in place of the cognate primer, tRNA(3)(Lys). Preferential incorporation of ddA TP was observed dur-ing reverse transcription opposite a distinct paus e site in a short synthetic RNA template. These results con-firm the n otion that the antiviral activities of ddNTP are dependent on both cel lular dNTP pools and the state of cellular activation, Pausing of HIV- 1 reverse transcriptase during reverse transcription, altered by dNTP concentrations, may be a mechanism that controls the position and exte nt of incorporation of nucleoside analogs.