# A recurrent adaptive mutation in the transmembrane 2B protein of an insect picorna-like virus in a nonnative host

## TL;DR

The study shows that a specific mutation in the 2B protein of a virus helps it adapt to a new host, regardless of the host's immune response.

## Contribution

Identifies a key viral protein involved in cross-species adaptation independent of the host's innate immune system.

## Key findings

- The CrPV 2B D29N variant was selected in all six virus lineages in Drosophila, showing strong selection pressure.
- The 2B protein is associated with endomembranes and may play a role in replication organelle formation.
- The 2B protein's role in adaptation is independent of the cGAS-STING antiviral pathway.

## Abstract

Virus emergence is often due to cross-species transmission and adaptation to the new host. We studied the effect of innate immune responses on shaping virus populations in native and nonnative virus-host combinations, using as a model system Drosophila melanogaster infected with either Drosophila C virus (DCV) or cricket paralysis virus (CrPV). In this host, the cGAS-like receptor 1 senses viral double-stranded RNA and produces cyclic dinucleotides (CDNs) to activate the STING protein and induce an antiviral response. Both viruses were serially passaged in three host conditions differing in their cGAS-STING response: wild-type (WT) flies, Sting knock-out (KO) flies, and flies with a primed immune response by CDN injection. We found no immune-related effects on virus evolution, but we uncovered the CrPV nonstructural 2B protein as a key regulator of cross-species transmission. Nucleotide diversity specifically accumulated in the 2B gene during passage of CrPV in its nonnative Drosophila host, while 2B of the fly-adapted DCV displayed markedly lower and constant nucleotide diversity. In particular, the CrPV 2B D29N variant was selected in all six virus lineages evolved in WT and Sting KO flies, with an estimated selection coefficient greater than 0.2. This variant replicated faster and was more lethal than the parental virus in all three host backgrounds. 2B is a predicted transmembrane protein, which we found to be associated with cellular endomembranes and may be involved in replication organelle formation. Our findings suggest a role for the 2B protein in adaptation to a new host independent of the cGAS-STING pathway.

The forces driving virus evolution are central to understanding cross-species transmission and virus emergence. It is well established that the adaptive immune system drives virus evolution in mammals, but whether innate responses likewise drive virus evolution upon host shifts is less well understood. In this manuscript, we used Drosophila melanogaster as a model to study the evolution of a native and a nonnative pathogen under conditions in which innate antiviral immunity is either abolished or enhanced. Using an experimental evolution approach, we find little evidence for adaptive evolution of the natural pathogen Drosophila C virus. In contrast, we observed a recurrent adaptive mutation in the viral nonstructural 2B protein in the nonnative cricket paralysis virus, independent of the antiviral cGAS/STING pathway. Our work provides insights into viral adaptation to new hosts and the characteristics of the 2B protein of dicistroviruses, a family comprising important model insect viruses.