Accurate estimates of virus mutation rates are important to understand the evolution of the viruses and to combat them. However, methods of estimation are varied and often complex. Here, we critically review over 40 original studies and establish criteria to facilitate comparative analyses. The mutation rates of 23 viruses are presented as substitutions per nucleotide per cell infection (s/n/c) and corrected for selection bias where necessary, using a new statistical method. The resulting rates range from 10 ؊8 to10 ؊6 s/n/c for DNA viruses and from 10 ؊6 to 10 ؊4 s/n/c for RNA viruses. Similar to what has been shown previously for DNA viruses, there appears to be a negative correlation between mutation rate and genome size among RNA viruses, but this result requires further experimental testing. Contrary to some suggestions, the mutation rate of retroviruses is not lower than that of other RNA viruses. We also show that nucleotide substitutions are on average four times more common than insertions/deletions (indels). Finally, we provide estimates of the mutation rate per nucleotide per strand copying, which tends to be lower than that per cell infection because some viruses undergo several rounds of copying per cell, particularly double-stranded DNA viruses. A regularly updated virus mutation rate data set will be available at www.uv.es/rsanjuan/virmut.The mutation rate is a critical parameter for understanding viral evolution and has important practical implications. For instance, the estimate of the mutation rate of HIV-1 demonstrated that any single mutation conferring drug resistance should occur within a single day and that simultaneous treatment with multiple drugs was therefore necessary (72). Also, in theory, viruses with high mutation rates could be combated by the administration of mutagens (1,5,21,44,53,83). This strategy, called lethal mutagenesis, has proved effective in cell cultures or animal models against several RNA viruses, including enteroviruses (11,39,44), aphtoviruses (83), vesiculoviruses (44), hantaviruses (10), arenaviruses (40), and lentiviruses (15, 53), and appears to at least partly contribute to the effectiveness of the combined ribavirin-interferon treatment against hepatitis C virus (HCV) (13). The viral mutation rate also plays a role in the assessment of possible vaccination strategies (16), and it has been shown to influence the stability of live attenuated polio vaccines (91). Finally, at both the epidemiological and evolutionary levels, the mutation rate is one of the factors that can determine the risk of emergent infectious disease, i.e., pathogens crossing the species barrier (46). Slight changes of the mutation rate can also determine whether or not some virus infections are cleared by the host immune system and can produce dramatic differences in viral fitness and virulence (75,90), clearly stressing the need to have accurate estimates. However, our knowledge of viral mutation rates is somewhat incomplete, partly due to the inherent difficulty of measuring a rare and r...

The level of genetic variation of human immunodeficiency virus type 1 (HIV-1), a member of the lentivirus genus of the Retroviridae family, is high relative to that of retroviruses in some other genera. The high error rates of purified HIV-1 reverse transcriptase in cell-free systems suggest an explanation for this high genetic variation. To test whether the in vivo rate of mutation during reverse transcription of HIV-1 is as high as predicted by cell-free studies, and therefore higher than that rates of mutation of retroviruses in other genera, we developed an in vivo assay for detecting forward mutations in HIV-1, using the lacZ␣ peptide gene as a reporter for mutations. This system allows the rates and types of mutations that occur during a single cycle of replication to be studied. We found that the forward mutation rate for HIV-1 was 3.4 ؋ 10 ؊5 mutations per bp per cycle. Base substitution mutations predominated; G-to-A transition mutations were the most common base substitution. The in vivo mutation rates for HIV-1 are three and seven times higher than those previously reported for two other retroviruses, spleen necrosis virus and bovine leukemia virus, respectively. In contrast, our calculated in vivo mutation rate for HIV-1 is about 20-fold lower than the error rate of purified HIV-1 reverse transcriptase, with the same target sequence. This finding indicates that HIV-1 reverse transcription in vivo is not as error prone as predicted from the fidelity of purified reverse transcriptase in cell-free studies. Our data suggest that the fidelity of purified HIV-1 reverse transcriptase may not accurately reflect the level of genetic variation in a natural infection.

Encapsidation of host restriction factor APOBEC3G (A3G) into vif-deficient human immunodeficiency virus type 1 (HIV-1) blocks virus replication at least partly by C-to-U deamination of viral minus-strand DNA, resulting in G-to-A hypermutation. A3G may also inhibit HIV-1 replication by reducing viral DNA synthesis and inducing viral DNA degradation. To gain further insight into the mechanisms of viral inhibition, we examined the metabolism of A3G-exposed viral DNA. We observed that an overall 35-fold decrease in viral infectivity was accompanied by a five-to sevenfold reduction in viral DNA synthesis. Wild-type A3G induced an additional fivefold decrease in the amount of viral DNA that was integrated into the host cell genome and similarly reduced the efficiency with which HIV-1 preintegration complexes (PICs) integrated into a target DNA in vitro. The A3G C-terminal catalytic domain was required for both of these antiviral activities. Southern blotting analysis of PICs showed that A3G reduced the efficiency and specificity of primer tRNA processing and removal, resulting in viral DNA ends that are inefficient substrates for integration and plus-strand DNA transfer. However, the decrease in plus-strand DNA transfer did not account for all of the observed decrease in viral DNA synthesis associated with A3G. These novel observations suggest that HIV-1 cDNA produced in the presence of A3G exhibits defects in primer tRNA processing, plus-strand DNA transfer, and integration.

The Vpr protein of human immunodeficiency virus type 1 (HIV-1) influences the in vivo mutation rate of the virus. Since Vpr interacts with a cellular protein implicated in the DNA repair process, uracil DNA glycosylase (UNG), we have explored the contribution of this interaction to the mutation rate of HIV-1. Single-amino-acid variants of Vpr were characterized for their differential UNG-binding properties and used to trans complement vpr null mutant HIV-1. A striking correlation was established between the abilities of Vpr to interact with UNG and to influence the HIV-1 mutation rate. We demonstrate that Vpr incorporation into virus particles is required to influence the in vivo mutation rate and to mediate virion packaging of the nuclear form of UNG. The recruitment of UNG into virions indicates a mechanism for how Vpr can influence reverse transcription accuracy. Our data suggest that distinct mechanisms evolved in primate and nonprimate lentiviruses to reconcile uracil misincorporation into lentiviral DNA.