Rates of spontaneous mutation critically determine the genetic diversity and evolution of RNA viruses. Although these rates have been characterized in vitro and in cell culture models, they have seldom been determined in vivo for human viruses. Here, we use the intrapatient frequency of premature stop codons to quantify the HIV-1 genome-wide rate of spontaneous mutation in DNA sequences from peripheral blood mononuclear cells. This reveals an extremely high mutation rate of (4.1 ± 1.7) × 10⁻³ per base per cell, the highest reported for any biological entity. Sequencing of plasma-derived sequences yielded a mutation frequency 44 times lower, indicating that a large fraction of viral genomes are lethally mutated and fail to reach plasma. We show that the HIV-1 reverse transcriptase contributes only 2% of mutations, whereas 98% result from editing by host cytidine deaminases of the A3 family. Hypermutated viral sequences are less abundant in patients showing rapid disease progression compared to normal progressors, highlighting the antiviral role of A3 proteins. However, the amount of A3-mediated editing varies broadly, and we find that low-edited sequences are more abundant among rapid progressors, suggesting that suboptimal A3 activity might enhance HIV-1 genetic diversity and pathogenesis. The high levels of genetic diversity of the HIV-1 virus grant it the ability to escape the immune system, to rapidly evolve drug resistance, and to circumvent vaccination strategies. However, our knowledge of HIV-1 mutation rates has been largely restricted to in vitro and cell culture studies because of the inherent complexity of measuring these rates in vivo. Here, by analyzing the frequency of premature stop codons, we show that the HIV-1 mutation rate in vivo is two orders of magnitude higher than that predicted by in vitro studies, making it the highest reported mutation rate for any biological system. A large component of this rate is from host cellular cytidine deaminases, which induce mutations in the viral DNA as a defense mechanism. While the HIV-1 genome is hypermutated in blood cells, only a very small fraction of these mutations reach the plasma, indicating that many viruses are defective as a result of the extremely high mutation load. In addition, we find that the HIV-1 mutation rate tends to be higher in patients showing normal disease progression than in those undergoing rapid progression, emphasizing the negative impact on viral fitness of hypermutation by host cytidine deaminases. However, we also observe subpopulations of weakly-mutated viral genomes whose sequence diversity may influence viral pathogenesis. Our work highlights the fine balance for HIV-1 between enough mutation to evade host responses and too much mutation that can inactivate the virus.

Their extremely error-prone replication makes RNA viruses targets for lethal mutagenesis. In the case of hepatitis C virus (HCV), the standard treatment includes ribavirin, a base analog with an in vitro mutagenic effect, but the in vivo mode of action of ribavirin remains poorly understood. Here, we test the mutagenic effects of ribavirin plus interferon treatment in vivo using a new method to estimate mutation rates based on the analysis of nonsense mutations. We apply this methodology to a large HCV sequence database containing over 15,000 reverse transcription-PCR molecular clone sequences from 74 patients infected with HCV. We obtained an estimate of the spontaneous mutation rate of ca. 10⁻⁴substitutions per site or lower, a value within the typically accepted range for RNA viruses. A roughly threefold increase in mutation rate and a significant shift in mutation spectrum were observed in samples from patients undergoing 6 months of interferon plus ribavirin treatment. This result is consistent with the known in vitro mutagenic effect of ribavirin and suggests that the antiviral effect of ribavirin plus interferon treatment is at least partly exerted through lethal mutagenesis.

Mutational robustness is defined as the constancy of a phenotype in the face of deleterious mutations. Whether robustness can be directly favored by natural selection remains controversial. Theory and in silico experiments predict that, at high mutation rates, slow-replicating genotypes can potentially outcompete faster counterparts if they benefit from a higher robustness. Here, we experimentally validate this hypothesis, dubbed the “survival of the flattest,” using two populations of the vesicular stomatitis RNA virus. Characterization of fitness distributions and genetic variability indicated that one population showed a higher replication rate, whereas the other was more robust to mutation. The faster replicator outgrew its robust counterpart in standard competition assays, but the outcome was reversed in the presence of chemical mutagens. These results show that selection can directly favor mutational robustness and reveal a novel viral resistance mechanism against treatment by lethal mutagenesis. Understanding the conditions that favor the constancy of phenotypes in the face of deleterious mutation pressure—mutational robustness—is an outstanding question in evolutionary biology. Theoretical and in silico studies utilizing digital organisms predict that slow-replicating populations can outcompete those with higher individual fitness if the former show greater robustness. This “survival of the flattest” hypothesis sits in contrast to most models of natural selection based on individual fitness, and hence challenges the “survival of the fittest” paradigm. In this work, the authors use experimental populations of the rapidly evolving vesicular stomatitis RNA virus to provide the first evidence of natural selection for mutational robustness. Based on the analysis of fitness distributions, genetic variability, and the ability to tolerate mutation accumulation, two populations with different levels of robustness were characterized. At artificially enhanced mutation rates following the application of mutagens, the more robust viral population outcompeted the other population despite having a lower replication rate. This study has important implications for lethal mutagenesis—an antiviral strategy that consists of increasing viral mutation rates through the use of mutagenic drugs—since selectively favored mutational robustness may allow RNA viruses to evolve resistance to this form of treatment.

Genetic diversity enables a virus to colonize novel hosts, evade immunity, and evolve drug resistance. However, viral diversity is typically assessed at the population level. Given the existence of cell-to-cell variation, it is critical to understand viral genetic structure at the single-cell level. By combining single-cell isolation with ultra-deep sequencing, we characterized the genetic structure and diversity of a RNA virus shortly after single-cell bottlenecks. Full-length sequences from 881 viral plaques derived from 90 individual cells reveal that sequence variants pre-existing in different viral genomes can be co-transmitted within the same infectious unit to individual cells. Further, the rate of spontaneous virus mutation varies across individual cells, and early production of diversity depends on the viral yield of the very first infected cell. These results unravel genetic and structural features of a virus at the single-cell level, with implications for viral diversity and evolution.

Despite being silent with respect to protein sequence, synonymous nucleotide substitutions can be targeted by natural selection directly at the DNA or RNA level. However, there has been no systematic assessment of how frequent this type of selection is. Here, we have constructed 53 single random synonymous substitution mutants of the bacteriophages Qβ and ΦX174 by site-directed mutagenesis and assayed their fitness. Analysis of this mutant collection and of previous studies undertaken with a variety of single-stranded (ss) viruses demonstrates that selection at synonymous sites is stronger in RNA viruses than in DNA viruses. We estimate that this type of selection contributes approximately 18% of the overall mutational fitness effects in ssRNA viruses under our assay conditions and that random synonymous substitutions have a 5% chance of being lethal to the virus, whereas in ssDNA viruses, these figures drop to 1.4% and 0%, respectively. In contrast, the effects of nonsynonymous substitutions appear to be similar in ssRNA and ssDNA viruses.

Mutational fitness effects can be measured with relatively high accuracy in viruses due to their small genome size, which facilitates full-length sequencing and genetic manipulation. Previous work has shown that animal and plant RNA viruses are very sensitive to mutation. Here, we characterize mutational fitness effects in single-stranded (ss) DNA and ssRNA bacterial viruses. First, we performed a mutation-accumulation experiment in which we subjected three ssDNA (ΦX174, G4, F1) and three ssRNA phages (Qβ, MS2, and SP) to plaque-to-plaque transfers and chemical mutagenesis. Genome sequencing and growth assays indicated that the average fitness effect of the accumulated mutations was similar in the two groups. Second, we used site-directed mutagenesis to obtain 45 clones of ΦX174 and 42 clones of Qβ carrying random single-nucleotide substitutions and assayed them for fitness. In ΦX174, 20% of such mutations were lethal, whereas viable ones reduced fitness by 13% on average. In Qβ, these figures were 29% and 10%, respectively. It seems therefore that high mutational sensitivity is a general property of viruses with small genomes, including those infecting animals, plants, and bacteria. Mutational fitness effects are important for understanding processes of fitness decline, but also of neutral evolution and adaptation. As such, these findings can contribute to explain the evolution of ssDNA and ssRNA viruses. The fitness effects of mutations are the raw material for natural selection. It has been shown that point mutations typically have strongly deleterious effects in plant and animal RNA viruses, whereas cellular organisms are comparatively more robust. Here, we characterize the fitness effects of random mutations in DNA viruses and compare them with those found in RNA viruses, using six phage species of similar genome sizes. To achieve this goal, we introduced mutations by chemical and site-directed mutagenesis, identified the genetic changes by sequencing, and quantified their fitness effects using growth-rate assays. In all cases, mutations had a strong average impact on fitness. We conclude that mutational sensitivity is a general property of viruses with small genomes and discuss the evolutionary implications of these findings.