Raymond F. Schinazi

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 inhibits the replication of human immunodeficiency virus type 1 (HIV-1) in vitro and in patients with AIDS. HIV-1 resistance to foscarnet has not been reported despite long-term foscarnet therapy of AIDS patients with cytomegalovirus disease. We therefore attempted to select foscarnet-resistant HIV-1 in vitro by serial endpoint passage of virus in 400 microM foscarnet. After 13 cycles of passage in MT-2 cells, virus exhibiting > or = 8.5-fold foscarnet resistance was isolated. The reverse transcriptase (RT) from resistant virions exhibited a similar level of foscarnet resistance in enzyme inhibition assays (approximately 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 the coexistence of two mutations in all clones: Gln-161 to Leu (CAA to CTA) and His-208 to Tyr (CAT to TAT). Sequence analysis of six clinical HIV-1 isolates showing reduced susceptibility to foscarnet revealed the 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 and production of mutant recombinant viruses demonstrated that the Leu-161, Ser-88, and Tyr-208 mutations reduced HIV-1 susceptibility to foscarnet 10.5-, 4.3-, and 2.4-fold, respectively, in MT-2 cells. In the crystal 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 dNTP binding site and its affinity for foscarnet. The location of the Trp-88 residue in the Beta5a strand of HIV-1 RT suggest that the Ser-88 mutation affects template-primer binding, as do several mutations that affect RT susceptibility to nucleoside analogs.

The 3'-azido group is not the primary determinant of 3'-azido-3'-deoxythymidine (AZT) responsible for the excision phenotype of AZT-resistant HIV-1

The mechanism of human immunodeficiency virus (HIV) 1 resistance to 3′-azido-3′-deoxythymidine (AZT) involves reverse transcriptase (RT)-catalyzed phosphorolytic excision of the chain-terminating AZT-5′-monophosphate (AZTMP). Primers terminated with AZTMP are generally better substrates for this reaction than those terminated with 2′,3′-dideoxynucleoside-5′-monophosphate (2′,3′-ddNMP) analogs that lack a 3′-azido moiety. This led to the hypothesis that the 3′-azido group is a major structural determinant for maintaining the primer terminus in the appropriate site for phosphorolytic excision of AZTMP by AZT-resistant (AZTR) RT. To test this hypothesis, we evaluated the incorporation, phosphorolytic excision, and antiviral activity of a panel of 3′-azido-2′,3′-ddN including 3′-azido-2′,3′-ddA (AZddA), 3′-azido-2′,3′-ddC (AZddC), 3′-azido-2′,3′-ddG (AZddG), AZT, and 3′-azido-2′,3′-ddU (AZddU). The results indicate that mutations correlated with resistance to AZT (D67N/K70R/T215F/K219Q) confer resistance to the 3′-azidopyrimidine nucleosides (AZddC, AZT, and AZddU) but not to the 3′-azidopurine nucleosides (AZddA and AZddG). The data suggest that the presence of a 3′-azido group on the 3′-terminal nucleotide of the primer does not confer increased phosphorolytic excision by AZTR RT for all 3′-azido-ddNMP analogs. Thus, the 3′-azido group cannot be the only structural determinant important for the enhanced phosphorolytic excision of AZTMP associated with HIV resistance to AZT. Other structural components, such as the base, must play a role in defining the specificity of the excision phenotype arising from AZT resistance mutations.