HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REVERSE-TRANSCRIPTASE EXPRESSING THE K70E MUTATION EXHIBITS A DECREASE IN SPECIFIC ACTIVITY AND PROCESSIVITY

Adefovir dipivoxil 9-(2-(bispivaloyloxymethyl)phosphonylmethoxyethyl) adenine (bis-POM PMEA)], an oral prodrug of adefovir (PMEA), is curren tly in phase ill clinical testing for the treatment of human immunodef iciency Virus-1 (HIV-1) infection. Previous in vitro experiments have shown that HIV-I recombinant viruses expressing either a K65R or a K70 E mutation in reverse transcriptase (RT) have reduced sensitivity to P MEA and that the K70E mutant also has impaired replication capacity in vitro. Genotypic analyses of samples from patients enrolled in a phas e I/II clinical trial of adefovir dipivoxil demonstrated that the K70E RT mutation developed in two of 29 patients during extended therapy. To further investigate the molecular mechanisms involved in the resist ance to PMEA, we cloned, expressed, and purified HIV-1 RT enzymes carr ying either the K65R or K70E and, for comparison, the M184V mutation. The K<INF>m</INF> values of dNTPs for these mutant enzymes were not si gnificantly altered from wild-type RT. The K<INF>i</INF> values for th e K65R mutant were increased from wild-type by 2-5-fold against a vari ety of inhibitors, whereas the K<INF>i</INF><SUP></SUP> values for the M184V mutant were increased 12-fold specifically for 2',3'-dideoxy-3' -thiacytidine (3TC) triphosphate. The Ki values for the K70E mutant we re increased for PMEA diphosphate and 3TC triphosphate by 2-3-fold. Th ese results are in agreement with antiviral drug susceptibility assay results. The three recombinant enzymes were also evaluated for their s pecific activities and processivities. All mutants were reduced in spe cific activity with respect to wild-type RT. In single-cycle processiv ity studies, the M184V mutant was, as expected, notably impaired. The K70E mutant was also slightly impaired, whereas the K65R mutant was sl ightly more processive than wild-type. These results with recombinant K70E RT are consistent with the reduced in vitro replication capacity of the K70E RT mutant of HIV-1 and further demonstrate that the K70E m utation confers minor PMEA and 3TC resistance to HIV-1.