Mutations in the SIV env and the M13 lacZa gene generatedin vitro by reverse transcriptases and DNA polymerases

Abstract

To investigate the accuracy of retroviral in vitro DNA replication we have examined with two fidelity assays the reverse transcriptases (RTs) from SIVagm, HIV-1, MoMLV as well for comparison the Klenow fragment from E. coli and DNA polymerase a from calf-thymus. These forward mutation assays measured the loss of bacteriophage M13 lacZa gene function by mutations. In the EnvlacZa assay frameshift mutations occurring during polymerisation of a 176 b long simian immunodeficiency virus (SIV) envelope (env) sequence were phenotypically detected by blue/white-plaque screening. To measure in addition substitutions, a 116 b long M13 lacZa gene DNA template was used as the mutational target (LacZa assay). With the SIVagm env gene DNA template, we observed similar levels of frameshift fidelity for all three RTs. Nevertheless, the SIVagm RT was slightly more accurate than the other RTs and nearly all frameshifts were observed at two homopolymeric runs of its homologous template. Measuring also substitution errors at the lacZa template the mutation frequency of the SIVagm RT increased 2.5 fold and that of the HIV-1 RT was enhanced by a factor of 3.

Nature, Position, and Frequency of Mutations Made in a Single Cycle of HIV-1 Replication

“…Although it is widely believed that HIV-1 replication is exceptionally error-prone, we show here that the in vivo forward mutation rate of HIV-1, measured by the inactivation of lacZ␣, is approximately 1.4 ϫ 10 Ϫ5 mutations/bp/ cycle. This rate is approximately 20-fold lower than the in vitro error rate for RT measured using a DNA template (9,10,29,30,62,68) and 17-fold lower than the rate measured using an RNA template (29,30). Our reported mutation rate for HIV-1 in HOS cells is also about 3-fold lower than earlier reported rates in HeLa and CEM cells (42,46) and is similar to the average rate reported for other retroviruses (1.5 ϫ 10 Ϫ5 mutations/bp/cycle) (69).…”

“…Using an HIV-1 vector containing a lacZ␣ reporter gene, Mansky and Temin reported a mutation rate of 3.4 ϫ 10 Ϫ5 to 4 ϫ 10 Ϫ5 mutations/ bp/cycle (42,46), some 10-to 20-fold lower than the rates reported for purified HIV-1 RT in vitro (9,39,62,68) but within 3-fold of the retroviral average of 1.5 ϫ 10 Ϫ5 mutations/ bp/cycle (46,47,(54)(55)(56). To date, most of the measurements of HIV-1 fidelity in cell culture have been based solely on the frequency of phenotypic inactivation of LacZ␣; there has been relatively little information about the nature of the underlying mutations (15,(43)(44)(45).…”

“…These assays have identified RT residues involved in nucleotide discrimination, binding, and incorporation (4,9,39,65,68). In vitro, HIV-1 RT has a high rate of nucleotide misincorporation, in the range of 5.9 ϫ 10 Ϫ4 to 5.3 ϫ 10 Ϫ5 mutations/bp/cycle (reviewed in references 61 and 69).…”

“…In vitro, HIV-1 RT has a high rate of nucleotide misincorporation, in the range of 5.9 ϫ 10 Ϫ4 to 5.3 ϫ 10 Ϫ5 mutations/bp/cycle (reviewed in references 61 and 69). Despite the fact that many groups have used the same mutagenic target (lacZ␣), different error rates have been reported, and there has been little agreement on the proportions and frequencies of the specific types of mutations RT makes (4,8,9,30,68). A part of the problem is that HIV-1 RT fidelity in vitro depends both on the recombinant RT used and on the assay conditions (39,48).…”

“…A part of the problem is that HIV-1 RT fidelity in vitro depends both on the recombinant RT used and on the assay conditions (39,48). It has also been reported that recombinant HIV-1 RT is more error-prone than the RTs of other retroviruses (1,2,63,68,70). Together, these data have led to proposals that the low fidelity of RT makes HIV-1 replication particularly error-prone (50,59).…”