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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.

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Figure 1: Experimental system for the accumulation of spontaneous mutations in HCV sequences.
Figure 2: HCV genome-wide mutation frequency.
Figure 3: Relationship between replicon mutation frequencies and genetic diversity in patients.
Figure 4: HCV genome-wide mutational bias.


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The authors thank R. Bartenschlager for the HCV replicon, F. González-Candelas for the viral RNA, V. Sentandreu and A. Martínez for help with optimization of the Duplex Sequencing technique, R. Flores for suggestions, M. Binder for discussions on HCV replication models and S. Torres for laboratory assistance. This work was financially supported by grants from the European Research Council (ERC-2011-StG- 281191-VIRMUT) and the Spanish MINECO (BFU2013-41329) to R.San.

Author information




R.Ge. performed the experiments, including replicon construction, passaging, Sanger and Duplex Sequencing, and performed the bioinformatics analysis. U.E. and J.M.C. contributed to optimizing the Duplex Sequencing. I.A., J.-V.B. and J.M.C. performed Sanger sequencing of the replicon. R.Ga. performed Sanger sequencing of in vitro polymerization products. R.Sab., J.B.P. and A.M. performed in vitro polymerization assays. R.San. designed and supervised research, acquired funding, analysed the data and wrote the manuscript.

Corresponding author

Correspondence to Rafael Sanjuán.

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The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary Tables 1-2, Figures 1-4 and References (PDF 5452 kb)

Supplementary Dataset 1

Sequence of the seven cloned fragments and the fragment used for in vitro polymerization. (TXT 10 kb)

Supplementary Dataset 2

List of mutations obtained by Duplex Sequencing in the three replicon lines, mapped to annotated H77 reference sequence including sequencing coverage, patient sequence diversity, and SHAPE data. (XLSX 2594 kb)

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Geller, R., Estada, Ú., Peris, J. et al. Highly heterogeneous mutation rates in the hepatitis C virus genome. Nat Microbiol 1, 16045 (2016).

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