Alignment of pol gene deduced amino acid sequences of retroviruses of man (HIV-1, II), ape, cow, sheep, mouse, and chicken were made to identify conserved regions, presumably functional domains, of the enzyme reverse transcriptase. Although little overall sequence conservation was found, one short, highly conserved domain was identified and selected for further study. Site-directed mutagensis was employed to alter these conserved amino acid codons and the effects of these changes were measured biologically and biochemically. Moloney murine leukemia virus (MuLV) was used as the model system. Alteration of four of the five individual amino acids resulted in inability of the transfected modified cloned DNA to produce infections virus without use of helper virus. The biochemical effect of these mutations on reverse transcriptase (RT) and the associate RNaseH activity was determined on purified mutant RT expressed in E. coli. One mutation caused a greater than 10 fold decrease in RNase H activity without simultaneously altering RT activity. Other mutant RTs had depressed RT and RNaseH activities.
