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

Abstract

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.

Experimental virus evolution in cancer cell monolayers, spheroids, and tissue explants

“…This makes it difficult to identify adaptive mutations by looking at parallel evolution events. Previous work with VSV has revealed greater levels of parallel sequence evolution than those observed here in some cases, but not in others ( Cuevas, Elena, and Moya 2002 ; Novella et al 2004 ; Remold, Rambaut, and Turner 2008 ; Cuevas, Moya, and Sanjuán 2009 ; Garijo et al 2014 , 2016 ; Hernández-Alonso et al 2015 ). The reasons for these differences might be attributed to effective population sizes (determined mainly by inoculum sizes), evolutionary time (number of transfers), and the strength of the selective pressures applied, among other possible factors ( Sanjuán and Grdzelishvili 2015 ).…”

“…Although evolutionary repeatability was relatively low, some interesting parallel evolution events can be identified by comparing across studies. First, the E254K substitution in gene G, which appeared in line E1, was previously reported in 4/5 WT lines evolved in MEFs ( Hernández-Alonso et al 2015 ). Interestingly, this mutation was shown to be deleterious when assayed individually by site-directed mutagenesis, suggesting epistasis with other mutations.…”

Host nutritional status affects alphavirus virulence, transmission, and evolution

“…It is possible that differences in the immune status of mice from each group result in different selective pressures on the virus population. Several studies have shown that the innate immune system is involved in viral evolution [59,60]. Finally, obese mice are expected to have more adipocytes (i.e., fat cells) while undernourished mice are expected to have fewer.…”

Experimental Evolution Generates Novel Oncolytic Vesicular Stomatitis Viruses with Improved Replication in Virus-Resistant Pancreatic Cancer Cells

“…Directed evolution and bioselection for more potent oncolytic viruses has been explored in other studies using a variety of oncolytic viruses and cancer types (50)(51)(52)(53)(54)(55)(56). VSV has been a widely used model to study viral evolution for several decades (57) and has been experimentally evolved for various purposes, such as understanding how viruses evade innate immune responses (58) and the generation of novel VSV-G protein variants used to pseudotype retroviral and lentiviral vectors for gene delivery (59) and to produce novel variants of foreign proteins encoded in VSV genome (60). Moreover, several previous studies have successfully used a directed evolution approach to improve VSV's oncolytic abilities (61)(62)(63)(64).…”