A HUMAN T-LYMPHOID CELL VARIANT RESISTANT TO THE ACYCLIC NUCLEOSIDE PHOSPHONATE 9-(2-PHOSPHONYLMETHOXYETHYL)ADENINE SHOWS A UNIQUE COMBINATION OF A PHOSPHORYLATION DEFECT AND INCREASED EFFLUX OF THE AGENT

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) is a new antiviral agent wi th activity against herpes viruses and retroviruses, including human i mmunodeficiency virus, but its metabolism and mechanism of action rema in unclear. We have isolated a human T lymphoid cell line (CEMr-1) tha t is resistant to the antiproliferative effects of PMEA. The antiviral effects of PMEA against human immunodeficiency virus-1 infection were also greatly reduced in CEM-r1 cells, compared with the parental cell s. This mutant showed cross-resistance to the related acyclic nucleosi de phosphonates 9-(2-phosphonylmethoxyethyl)diaminopurine and 9-(2-pho sphonylmethoxyethyl)guanine and the lipophilic prodrug 9-(2-phosphonyl methoxyethyl)adenine-(bispom-PMEA), as well as partial resistance to t he purine nucleosides 2-chloredeoxyadenosine, 2-fluro-9-beta-D-arabino sylfuranosyladenine, and adenosine, but did not show resistance to 2'- deoxyadenosine or 9-beta-D-arabinosylfuranosyladenine. We compared the uptake and metabolism of [H-3]PMEA and [H-3]-bispom-PMEA in the mutan t and parental cells. The analysis of radioactive products by high pre ssure liquid chromatography revealed marked alterations in the ability of the mutant cell line to accumulate PMEA and its anabolites, compar ed with the parental cells. Accumulation of PMEA, PMEA monophosphate, and PMEA bisphosphate (major metabolites formed with either PMEA or bi spom-PMEA) decreased by 50, 95, and 97%, respectively. Compared with t he parental cells, the variant cells showed a similar to 7-fold increa se in the rate of efflux of PMEA and a 2-fold decrease in the activity of adenylate kinase. In contrast, other enzymes of nucleotide metabol ism, such as adenosine kinase, deoxycytidine kinase, and 5-phosphoribo syl-1-pyrophosphate synthetase, showed no significant change in the tw o cell lines. Overall, these results suggest that the mutation in this resistant cell line is of a novel type, involving an alteration in th e cellular efflux of PMEA as the major basis for the resistant phenoty pe.