NOVEL MUTATIONS IN REVERSE-TRANSCRIPTASE OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REDUCE SUSCEPTIBILITY TO FOSCARNET IN LABORATORY AND CLINICAL ISOLATES

Foscarnet (phosphonoformic acid) is a pyrophosphate analog that inhibi ts the replication of human immunodeficiency virus type 1 (HIV-1) in v itro and in patients with AIDS. HIV-1 resistance to foscarnet has not been reported despite long-term foscarnet therapy of AIDS patients wit h cytomegalovirus disease. We therefore attempted to select foscarnet- resistant HIV-1 in vitro by serial endpoint passage of virus in 400 mu M foscarnet. After 13 cycles of passage in MT-2 cells, virus exhibiti ng greater than or equal to 8.5-fold foscarnet resistance was isolated . The reverse transcriptase (RT) from resistant virions exhibited a si milar level of foscarnet resistance in enzyme inhibition assays (simil ar to 10-fold resistance). Foscarnet-resistant virus showed increased susceptibility to 3'-azido-3'-deoxythymidine (90-fold) and to the HIV- 1-specific RT inhibitors TIBO R82150 (30-fold) and nevirapine (20-fold ). DNA sequence analysis of RT clones from resistant virus revealed th e coexistence of two mutations in all clones: Gln-161 to Leu (CAA to C TA) and His-208 to Tyr (CAT to TAT). Sequence analysis of six clinical HIV-1 isolates showing reduced susceptibility to foscarnet revealed t he Tyr-208 mutation in two, the Leu-161 mutation in one, and a Trp-88- to-Ser or -Gly mutation in four isolates. Site-specific mutagenesis an d production of mutant recombinant viruses demonstrated that the Leu-1 61, Ser-88, and Tyr-208 mutations reduced HIV-1 susceptibility to fosc arnet 10.5-, 4.3-, and 2.4-fold, respectively, in MT-2 cells. In the c rystal structure of HIV-1 RT, the Gln-161 residue lies in the alpha E helix beneath the putative deoxynucleoside triphosphate (dNTP) binding site. The Gln-161-to-Leu mutation may affect the structure of the dNT P binding site and its affinity for foscarnet. The location of the Trp -88 residue in the beta 5a strand of HIV-1 RT suggests that the Ser-88 mutation affects template-primer binding, as do several mutations tha t affect RT susceptibility to nucleoside analogs.