RT Journal Article
SR Electronic
T1 Human Immunodeficiency Virus Type 1 Reverse Transcriptase Expressing the K70E Mutation Exhibits a Decrease in Specific Activity and Processivity
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 291
OP 297
DO 10.1124/mol.54.2.291
VO 54
IS 2
A1 Michael D. Miller
A1 Patrick D. Lamy
A1 Michael D. Fuller
A1 Andrew S. Mulato
A1 Nicolas A. Margot
A1 Tomas Cihlar
A1 Julie M. Cherrington
YR 1998
UL http://molpharm.aspetjournals.org/content/54/2/291.abstract
AB Adefovir dipivoxil [9-(2-(bispivaloyloxymethyl)phosphonylmethoxyethyl)adenine (bis-POM PMEA)], an oral prodrug of adefovir (PMEA), is currently in phase III clinical testing for the treatment of human immunodeficiency virus-1 (HIV-1) infection. Previous in vitro experiments have shown that HIV-1 recombinant viruses expressing either a K65R or a K70E mutation in reverse transcriptase (RT) have reduced sensitivity to PMEA and that the K70E mutant also has impaired replication capacityin vitro. Genotypic analyses of samples from patients enrolled in a phase 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 resistance to PMEA, we cloned, expressed, and purified HIV-1 RT enzymes carrying either the K65R or K70E and, for comparison, the M184V mutation. TheK m values of dNTPs for these mutant enzymes were not significantly altered from wild-type RT. TheK i values for the K65R mutant were increased from wild-type by 2–5-fold against a variety of inhibitors, whereas the K i values for the M184V mutant were increased 12-fold specifically for 2′,3′-dideoxy-3′-thiacytidine (3TC) triphosphate. TheK i values for the K70E mutant were increased for PMEA diphosphate and 3TC triphosphate by 2–3-fold. These results are in agreement with antiviral drug susceptibility assay results. The three recombinant enzymes were also evaluated for their specific activities and processivities. All mutants were reduced in specific activity with respect to wild-type RT. In single-cycle processivity studies, the M184V mutant was, as expected, notably impaired. The K70E mutant was also slightly impaired, whereas the K65R mutant was slightly 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 mutation confers minor PMEA and 3TC resistance to HIV-1.