Fidelity of DNA synthesis exhibited in vitro by the reverse transcriptase of the lentivirus equine infectious anemia virus

Abstract

The lentivirus equine infectious anemia virus (EIAV) shows high genetic variations. To gain insight into the relative contribution of the reverse transcription process to the EIAV mutation rate, the accuracy of DNA synthesis catalyzed in vitro by the reverse transcriptase (RT) of EIAV was determined. Since the RT of EIAV shows a relatively high sequence homology with other lentiviral RTs, most notable being the RTs of human immunodeficiency viruses (HIVs), type 1 and type 2, it was of interest to study the fidelity of EIAV RT as part of an investigation of the structure-function relationship in lentiviral RTs. Like other RTs, EIAV RT was found to lack a 3'-->5' exonuclease activity. The fidelity of EIAV RT was analyzed by studying two distinct steps that lead to base substitution mutations: nucleotide misinsertions and elongation from 3'-terminal DNA mispairs. Analysis of misincorporation rates opposite the template adenine residue in native phi x174am3 DNA showed that EIAV RT catalyzes nucleotide mismatches with a specificity of A:C >> A:G > A:A. Interestingly, the same order of specificity was also detected during mispair extension with three templates tested (i.e., phi x174am3 DNA, rRNA, and synthetic oligo DNA). The mispair extension efficiency and mispair formation appear to be affected mainly by the increase in apparent Km values, rather than by the change in Vmax values. Furthermore, EIAV RT exhibits similar mispair extension efficiencies with both RNA and DNA templates with identical surrounding sequences. However, dissimilarities were detected in mispair extension frequencies with two DNAs which have different sequences, thus emphasizing the importance of the sequences copied.(ABSTRACT TRUNCATED AT 250 WORDS)