Mass spectrometry and enrichment analysis.

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, which occurs by the induction of 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. 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 performed proteomic analysis of co-immunoprecipitated cellular 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 both translation and stress granules assembly. Finally, we also verified that M2-2 is targeted for proteasome degradation, being localized to granules composed of defective ribosomal products at the cytoplasm. These results suggest that besides its functions in the replicative complex, M2-2 may exert additional functions to contribute to successful RSV infection.