FIDELITY OF DNA-SYNTHESIS EXHIBITED IN-VITRO BY THE REVERSE-TRANSCRIPTASE OF THE LENTIVIRUS EQUINE INFECTIOUS-ANEMIA VIRUS

The lentivirus equine infectious anemia virus (EIAV) shows high geneti c variations. To gain insight into the relative contribution of the re verse 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 seq uence homology with other lentiviral RTs, most notable being the RTs o f 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 R Ts, EIAV RT was found to lack a 3'--> 5' exonuclease activity. The fid elity of EIAV RT was analyzed by studying two distinct steps that lead to base substitution mutations: nucleotide misinsertions and elongati on from 3'-terminal DNA mispairs. Analysis of misincorporation rates o pposite the template adenine residue in native phix174am3 DNA showed t hat EIAV RT catalyzes nucleotide mismatches with a specificity of A:C >> A:G > A:A. Interestingly, the same order of specificity was also de tected during mispair extension with three templates tested (i.e., phi x174am3 DNA, rRNA, and synthetic oligo DNA). The mispair extension eff iciency and mispair formation appear to be affected mainly by the incr ease in apparent K(m) values, rather than by the change in V(max) valu es. Furthermore, EIAV RT exhibits similar mispair extension efficienci es with both RNA and DNA templates with identical surrounding sequence s. However, dissimilarities were detected in mispair extension frequen cies with two DNAs which have different sequences, thus emphasizing th e importance of the sequences copied. The fact that EIAV RT is as erro r-prone as the two HIV RTs further strengthens the correlation between the reduced fidelity of RT and the genomic heterogeneity observed amo ng strains of various lentiviruses. The data also suggest that an impo rtant component of retroviral genetic variability may be attributable to the efficient mismatch extension during the copying of both RNA and DNA templates. In all, as with other RTs studied so far, it is appare nt that the fidelity of DNA synthesis exhibited by EIAV RT is also enz yme-dependent and sequence-related.