IDENTIFICATION OF THE AMINO-ACID IN THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REVERSE-TRANSCRIPTASE INVOLVED IN THE PYROPHOSPHATE BINDING OF ANTIVIRAL NUCLEOSIDE TRIPHOSPHATE ANALOGS AND PHOSPHONOFORMATE - IMPLICATIONS FOR MULTIPLE-DRUG RESISTANCE

A recombinant clone of human immunodeficiency virus type 1 reverse tra nscriptase (HIV-1 RT)with reduced sensitivity to 3'-azido-3'-deoxythym idine 5'-triphosphate (AZTTP) and phosphonoformate (PFA), a pyrophosph ate analog, has been obtained from the RNA of HTLV-IIIB infected cells using the polymerase chain reaction. The mutant HIV-1 RT retained pol ymerase activity and was cross-resistant to triphosphate forms of othe r nucleoside analogs including 2',3'-dideoxycytidine 5'-triphosphate, 2',3'-dideoxyadenosine 5'-triphosphate, and 3'-deoxy-2',3'-didehydroth ymidine 5'-triphospate (D4TTP), but remained sensitive to the non-nucl eoside HIV-1 RT inhibitors, such as nevirapine and TIBO R82150. Sequen ce analysis of the mutant HIV-1 RT revealed a single amino acid substi tution (Val--> Ala) at amino acid 90. The substitution of amino acid 9 0 by the closely related amino acids, such its Thr and Gly, also showe d decreased sensitivity to AZTTP, D4TTP, and PFA. All these mutations at amino acid 90 also caused an alteration of K-m for thymidine tripho sphate. These results suggest that Val at this site plays a role in de termining the interaction of the HIV-1 RT enzyme with the pyrophosphat e group of deoxynucleoside triphosphate (dNTP) and that the hydrophobi city of the amino acid at this position was the most important determi nant in the binding of HIV-I RT to dNTP.