Experimental evolution of an RNA virus in cells with innate immunity defects

Experimental evolution studies have shown that RNA viruses respond rapidly to directional selection and thus can adapt efficiently to changes in host cell tropism, antiviral drugs, or other imposed selective pressures. However, the evolution of RNA viruses under relaxed selection has been less extensively explored. Here, we evolved vesicular stomatitis virus in mouse embryonic fibroblasts knocked-out for PKR, a protein with a central role in antiviral innate immunity. Vesicular stomatitis virus adapted to PKR-negative mouse embryonic fibroblasts in a gene-specific manner, since the evolved viruses exhibited little or no fitness improvement in PKR-positive cells. Full-length sequencing revealed the presence of multiple parallel nucleotide substitutions arising in independent evolution lines. However, site-directed mutagenesis showed that the effects of these substitutions were not PKR dependent. In contrast, we found evidence for sign epistasis, such that a given substitution which was positively selected was strongly deleterious when tested as a single mutation. Our results suggest that virus evolution in cells with specific innate immunity defects may drive viral specialization. However, this process is not deterministic at the molecular level, probably because the fixation of mutations which are tolerated under a relaxed selection regime is governed mainly by random genetic drift.