Selective Pressure of a Quinoxaline Nonnucleoside Inhibitor of Human Immunodeficiency Virus Type 1 (HIV-1) Reverse Transcriptase (RT) on HIV-1 Replication Results in the Emergence of Nucleoside RT-Inhibitor-Specific (RT Leu-74 → Val or Ile and Val-75 → Leu or Ile) HIV-1 mutants

The quinoxaline nonnucleoside RT inhibitor (NNRTI) (S)-4-isopropoxycarbonyl-6-methoxy-3-(methylthio-methyl)-3, 4-dihydroquinoxaline-2(1H)-thione (HBY 097) was used to select for drug-resistant HIV-1 variants in vitro. The viruses first developed mutations affecting the NNRTI-binding pocket, and five of six strains displayed the RT G190→ E substitution, which is characteristic for HIV-1 resistance against quinoxalines. In one variant, a new mutant (G190→ Q) most likely evolved from preexisting G190→ E mutants. The negative charge introduced by the G190→ E substitution was maintained at that site of the pocket by simultaneous selection for V179→ D together with G190→ Q. After continued exposure to the drug, mutations at positions so far known to be specific for resistance against nucleoside RT inhibitors (NRTIs) (L74→ V/I and V75→ L/I) were consistently detected in all cultures. The inhibitory activities of the cellular conversion product of 2′,3′-dideoxyinosine (ddI, didanosine), 2′,3′-dideoxyadenosine (ddA) and of 2′,3′-didehydro-3$ ^{\prime}$-deoxythymidine (d4T, stavudine) against these late-passage viruses were shown to be enhanced with the L74→ V/I RT mutant virus as compared with the wild-type (wt) HIV-1MNisolate. Clonal analysis proved linkage of the codon 74 and codon 75 mutations to the NNRTI-specific mutations in all RT gene fragments. The nonnucleoside- and nucleoside-resistance mutation sites are separated by approximately 35 angstrom. We propose that the two sites ``communicate'' through the template-primer which is situated in the DNA-binding cleft between these two sites. Quinoxalines cause high selective pressure on HIV-1 replication in vitro; however, the implication of these findings for the treatment of HIV-1 infection has yet to be determined.