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 comes with a trade-off of decreased fitness in a standard, general environment. Understanding the tradeoffs of generalist and specialist viruses 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 generalist without a simple reversion of a genetic mutation. 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 specialist 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 specialist NS6 ^F182C over multiple cycles of replication. However, NS4 ^V11I did not affect the defective polyprotein cleavage but instead the NS4 ^V11I 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.