The Respiratory Syncytial Virus M2-2 protein is targeted for proteasome degradation and inhibits translation and stress granules assembly

The M2-2 protein from the respiratory syncytial virus (RSV) is a 10 kDa protein expressed by the second ORF of the viral gene M2. During infection, M2-2 has been described as the polymerase cofactor responsible for promoting genome replication. This function was first inferred by infection with a mutant virus lacking the M2-2 ORF, in which viral genome presented delayed accumulation in comparison to wild-type virus. In accordance with this phenotype, it has been recently shown that M2-2 promotes changes in interactions between the polymerase and other viral proteins at early stages of infection. Despite its well-explored role in the regulation of the polymerase activity, little has been made to investigate the relationship of M2-2 with cellular proteins. In fact, a previous report showed poor recruitment of M2-2 to viral structures, with the protein being mainly localized to the nucleus and cytoplasmic granules. To unravel which other functions M2-2 exerts during infection, we expressed the protein in HEK293T cells and performed proteomic analysis of co-immunoprecipitated partners, identifying enrichment of proteins involved with regulation of translation, protein folding and mRNA splicing. In approaches based on these data, we found that M2-2 expression downregulates eiF2α phosphorylation and inhibits stress granules assembly under arsenite induction. In addition, we also verified that M2-2 inhibits translation initiation, and is targeted for proteasome degradation, being localized to granules composed by defective ribosomal products at the cytoplasm. These results suggest that besides its functions in the regulation of genome replication, M2-2 may exert additional functions to contribute to successful RSV infection. Author summary Exploring how viruses take control of the cellular machinery is a common strategy to understand the infection process and to identify targets for inhibition of virus replication. In this work we investigated the cellular functions of the protein M2-2 from the respiratory syncytial virus. Although this virus is an important pathogen responsible for respiratory infections in immunocompromised individuals, currently there are no vaccines or effective treatments to inhibit its infection. Our findings showed that the protein M2-2 interferes with protein synthesis, being able to downregulate the assembly of stress granules during stress stimuli. Besides, we verified the relationship between M2-2 and the proteasome machinery, which is responsible for protein degradation and is also involved with protein synthesis. These results present new functions for the protein M2-2, indicating additional mechanisms utilized by the virus to facilitate infection, providing new perspectives for the search of antiviral targets. ### Competing Interest Statement The authors have declared no competing interest.