A single amino acid mutation in norovirus NS4 promotes viral spread

Viruses can rapidly adapt and evolve to new, unfavorable environments due to their decreased replication fidelity, large reproductive index, and short life cycle. Often, these adaptations that enable increased fitness in a new, specialized environment come with a trade-off of decreased fitness in a standard, general environment. Understanding the mechanistic basis for these fitness trade-offs has provided important insight into vaccine development, mechanism of action of antivirals, and function of viral proteins. Here, we sought to identify how a specialist murine norovirus (MNV) could be converted to a jack of all trades through a novel mutation without genetic reversion. Previously, we found that a mutation in MNV (NS6^F182C) overcame restriction by host protein Trim7 but decreased the efficiency of viral polyprotein NS6-7 cleavage and resulted in attenuation of this virus. Here, we find that a single valine-to-isoleucine mutation in MNV non-structural protein NS4 (NS4^V11I) is sufficient to rescue the attenuated replication of NS6^F182C over multiple cycles of replication. However, NS4^V11I did not affect the defective polyprotein cleavage; instead, the mutation facilitates faster viral spread in vitro independent of interferon signaling. The emergence of this mutation in NS4^V11I suggests an unappreciated connection between NS4 and NS6 during norovirus replication and provides a system to define the unknown role of norovirus NS4 during infection.IMPORTANCEViruses and hosts are involved in a continuous arms race for survival. Often, when viruses evolve to specialize in specific host environments, they lose their versatility and become specialists, only able to grow in one setting. This feature has been leveraged to create live-attenuated vaccines, identify the mechanism of action of antivirals, and uncover fundamental aspects of viral replication. Using murine norovirus as a model system, we aim to understand how a virus can adapt to overcome replication inefficiencies imposed by a previously acquired adaptive mutation. Here, we identify an unexpected connection between two murine norovirus non-structural proteins and uncover a role for the viral protein NS4 in viral spread. Taken together, these data provide new insight into viral evolution and the functions of norovirus proteins.