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The RNase A mismatch cleavage method has been applied to the characterization of natural genetic variation of human immunodeficiency virus (HIV) from different geographical areas. The approach provides a rapid and simple assay for the analysis of differences in closely related viral isolates and allows the establishment of phylogenetic relationships between epidemiologically distinct viruses. Our results show a broad clustering of circulating viruses according to their geographical distribution. We also have analyzed the temporal appearance of mutations associated with the acquisition of resistance to 3'-azido-3'-deoxythymidine (AZT). The results show that mutations in codon 215 of the viral reverse transcriptase can be detected readily by this method in HIV isolates and also directly in peripheral blood from HIV-infected individuals after in vitro amplification of viral sequences with the polymerase chain reaction. The specific recurrence of identical double-nucleotide substitutions in epidemiologically and geographically distant viruses suggests that the restricted amino acid substitutions at this position selected by drug exposure are a critical, rate-limiting step in the acquisition of drug resistance.

proceedings of the national academy of sciences of the united states of america

Characterization of genetic variation and 3'-azido-3'-deoxythymidine- resistance mutations of human immunodeficiency virus by the RNase A mismatch cleavage method.