In vitro selection and molecular characterization of human immunodeficiency virus type 1 with reduced sensitivity to 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA)

Abstract

9[2-(Phosphonomethoxy)ethyl]adenine (PMEA) is an acyclic nucleotide with potent in vitro activity against human immunodeficiency virus type 1 (HIV-1). The present study was undertaken to determine whether HIV-1 resistance to PMEA could be generated by in vitro selection and if so, to determine which mutations in reverse transcriptase (RT) were responsible. HIV-1 LAI was serially passaged for 10 months in the presence of increasing concentrations of PMEA up to a maximum of 40 μM. After 40 passages, the 50% inhibitory concentration (IC 50) of PMEA had increased almost 7-fold from 4.45 to 30.5 μM. Some cross-resistance to 2′,3′-dideoxycytidine (ddC, zalcitabine), 2′,3′-dideoxyinosine (ddI, didanosine), and 3′-thiacytidine (3TC, lamivudine) was also observed, but no cross-reactive resistance to 3′-azido-3′-thymidine (AZT, zidovudine). Sequencing of the RT encoding region of each of eight pol clones from resistant isolates revealed a Lys-65 → Arg (K65R) substitution. HIV with the K65R mutation inserted by site-directed mutagenesis also had decreased sensitivity to PMEA in H9 cells and a similar cross-resistance profile. Thus, HIV can develop decreased sensitivity to PMEA after long-term in vitro exposure and this change is associated with a K65R substitution. Additional studies will be needed to determine whether a similar mutation in HIV RT develops in patients receiving PMEA or its orally bioavailable prodrug adefovir dipivoxil (bis-POM PMEA).