# Cytoplasmic domain of CM2 is involved in the replication of influenza C virus

## TL;DR

The cytoplasmic domain of CM2 protein is crucial for influenza C virus replication by aiding genome packaging and uncoating.

## Contribution

This study identifies specific cytoplasmic domain mutations in CM2 that impair influenza C virus replication and genome packaging.

## Key findings

- Alanine substitutions at positions 67–69, 73–75, and 85–87 in CM2's cytoplasmic domain reduced viral replication and CM2 protein levels.
- Mutant viruses showed lower viral RNA content in particles and reduced RNA-NP complex transport into the nucleus.
- Mutations at positions 113–115 prevented virus rescue and suppressed genome packaging and uncoating.

## Abstract

CM2 is a single-pass transmembrane protein located in the viral envelope and is essential for the growth of influenza C virus. We previously reported that CM2 is involved in viral genome packaging and uncoating. We also demonstrated that alanine substitutions at residues 47–48, 67–69, 73–75, 85–87 and 113–115 in the cytoplasmic domain of CM2 significantly reduced its expression levels. Herein, we analysed whether these mutations affected viral replication. The alanine-substituted recombinant viruses rCM2-Ala67–69, 73–75 and 85–87 exhibited significantly reduced replication compared with recombinant wild-type viruses (rWT), and the amount of CM2 protein in virions and infected cells was also reduced compared with that in rWT. In addition, the amount of viral RNA within the particles of these mutant viruses was significantly lower than that in rWT. Furthermore, the amount of viral RNA-NP complexes transported into the nucleus of infected cells was reduced in rCM2-Ala73–75 and 85–87 viruses. In contrast, recombinant viruses with mutations at positions 113–115 could not be rescued, and virus-like particles containing this mutation showed suppressed genome packaging and uncoating and reduced expression of the CM2 protein. These results suggested that the cytoplasmic region of CM2 contributes to efficient packaging and uncoating of the genome through its stable expression.