PIV5 M Protein Interaction with Host Protein 14-3-3 Negatively Affects Virus Particle Formation

1 2 3 Paramyxovirus matrix (M) proteins organize virus assembly, linking viral glycoproteins 4 and viral ribonucleoproteins together at virus assembly sites on cellular membranes. 5 Using a yeast two-hybrid screening approach, we identified 14-3-3 as a binding partner for 6 the M protein of parainfluenza virus 5 (PIV5). Binding was confirmed by co7 immunoprecipitation in both transfected and PIV5-infected cells, and was mapped to a C8 terminal region within M protein, 366-KTKSLP-371. This sequence resembles known 149 3-3 binding sites in which the key residue for binding is a phosphorylated serine residue. 10 Mutation of S369 within PIV5 M protein disrupted 14-3-3 binding and improved the budding 11 of both virus-like particles (VLPs) and recombinant viruses, suggesting that 14-3-3 binding 12 impairs virus budding. 14-3-3 protein overexpression reduced the budding of VLPs. Using 13 P-labeling, phosphorylated M protein was detected in PIV5-infected cells, and this 14 phosphorylation was nearly absent in cells infected with recombinant virus harboring 15 S369A mutation within M protein. Assembly of M protein into clusters and filaments at 16 infected cell surfaces was enhanced in cells infected with recombinant virus defective in 17 14-3-3 binding. These findings support a model in which a portion of M protein within 18 PIV5-infected cells is phosphorylated at residue S369, binds 14-3-3 protein, and is held 19 away from sites of virus budding. 20 on N ovem er 3, 2017 by gest http/jvi.asm .rg/ D ow nladed fom