Highly heterogeneous mutation rates in the hepatitis C virus genome.

Spontaneous mutations are the ultimate source of genetic variation and have a prominent role in evolution. RNA viruses such as hepatitis C virus (HCV) have extremely high mutation rates, but these rates have been inferred from a minute fraction of genome sites, limiting our view of how RNA viruses create diversity. Here, by applying high-fidelity ultradeep sequencing to a modified replicon system, we scored >15,000 spontaneous mutations, encompassing more than 90% of the HCV genome. This revealed >1,000-fold differences in mutability across genome sites, with extreme variations even between adjacent nucleotides. We identify base composition, the presence of high- and low-mutation clusters and transition/transversion biases as the main factors driving this heterogeneity. Furthermore, we find that mutability correlates with the ability of HCV to diversify in patients. These data provide a site-wise baseline for interrogating natural selection, genetic load and evolvability in HCV, as well as for evaluating drug resistance and immune evasion risks.

Immune activation promotes evolutionary conservation of T-cell epitopes in HIV-1.

The immune system should constitute a strong selective pressure promoting viral genetic diversity and evolution. However, HIV shows lower sequence variability at T-cell epitopes than elsewhere in the genome, in contrast with other human RNA viruses. Here, we propose that epitope conservation is a consequence of the particular interactions established between HIV and the immune system. On one hand, epitope recognition triggers an anti-HIV response mediated by cytotoxic T-lymphocytes (CTLs), but on the other hand, activation of CD4(+) helper T lymphocytes (TH cells) promotes HIV replication. Mathematical modeling of these opposite selective forces revealed that selection at the intrapatient level can promote either T-cell epitope conservation or escape. We predict greater conservation for epitopes contributing significantly to total immune activation levels (immunodominance), and when TH cell infection is concomitant to epitope recognition (trans-infection). We suggest that HIV-driven immune activation in the lymph nodes during the chronic stage of the disease may offer a favorable scenario for epitope conservation. Our results also support the view that some pathogens draw benefits from the immune response and suggest that vaccination strategies based on conserved TH epitopes may be counterproductive.

Distribution of fitness effects caused by single-nucleotide substitutions in bacteriophage f1.

Empirical knowledge of the fitness effects of mutations is important for understanding many evolutionary processes, yet this knowledge is often hampered by several sources of measurement error and bias. Most of these problems can be solved using site-directed mutagenesis to engineer single mutations, an approach particularly suited for viruses due to their small genomes. Here, we used this technique to measure the fitness effect of 100 single-nucleotide substitutions in the bacteriophage f1, a filamentous single-strand DNA virus. We found that approximately one-fifth of all mutations are lethal. Viable ones reduced fitness by 11% on average and were accurately described by a log-normal distribution. More than 90% of synonymous substitutions were selectively neutral, while those affecting intergenic regions reduced fitness by 14% on average. Mutations leading to amino acid substitutions had an overall mean deleterious effect of 37%, which increased to 45% for those changing the amino acid polarity. Interestingly, mutations affecting early steps of the infection cycle tended to be more deleterious than those affecting late steps. Finally, we observed at least two beneficial mutations. Our results confirm that high mutational sensitivity is a general property of viruses with small genomes, including RNA and single-strand DNA viruses infecting animals, plants, and bacteria.