Silent mutations in coding regions of Hepatitis C virus affect patterns of HCV RNA structures and attenuate viral replication and pathogenesis

Vaccines based on live attenuated viruses are the most effective strategy for controlling infections, since they elicit long-lasting natural and effective immune response, but entail challenges for safety and virulence. Hepatitis C Virus (HCV) causes liver diseases and liver cancer, with millions infected each year and hundreds of thousands of annual fatalities; but no vaccine is currently available for the virus. Here, we present a novel computational approach for the accurate prediction of virus attenuation. We rationally design viral variants by inserting a large number of synonymous mutations in the NS5A/B coding region to disrupt the viral RNA’s secondary structure and regulatory sequences important for the viral life cycle. By measuring RNA levels and virus spread in an HCV infection model, we show that some variants have lower viral fitness relative to the wild-type virus, with gradient of attenuation in concordance with the prediction model. Deep sequencing of replicating viruses demonstrates relative genomic stability of the attenuated variant. Differential expression analysis and evaluation of cancer-related phenotypes reveal that some variants have a lower pathogenic influence on the host cells, compared to the wildtype virus. These rationally designed variants reveal novel information on key functional elements in HCV RNA important for virus fitness, that may be further considered as a promising direction for a viable HCV vaccine. Importantly, the computational approach described here is based on the most fundamental viral regulatory motifs and therefore may be applied for almost all viruses as a new strategy for vaccine development.