Oligomerization of the vesicular stomatitis virus phosphoprotein is dispensable for mRNA synthesis but facilitates RNA replication.

Non-segmented negative-strand (NNS) RNA viruses possess a ribonucleoprotein template in which the genomic RNA is sequestered within a homopolymer of nucleocapsid protein (N). The viral RNA dependent RNA polymerase (RdRP) resides within an approximately 250 kDa large protein (L), along with unconventional mRNA capping enzymes - a GDP:polyribonucleotidyltransferase (PRNT) and a dual specificity mRNA cap methylase (MT). To gain access to the N-RNA template and orchestrate the LRdRP, LPRNT and LMT, an oligomeric phosphoprotein (P) is required. Vesicular stomatitis virus (VSV) P is dimeric with an oligomerization domain (OD) separating two largely disordered regions followed by a globular C-terminal domain that binds template. P is also responsible for bringing new N protomers onto the nascent RNA during genome replication. We show VSV P lacking the OD (PDeltaOD) is monomeric but is indistinguishable from wild type P in supporting mRNA transcription in vitro. Recombinant virus VSV-PDeltaOD, exhibits a pronounced kinetic delay in progeny virus production. Fluorescence recovery after photobleaching demonstrate that PDeltaOD diffuses 6-fold more rapidly than wild type within viral replication compartments. A well characterized defective-interfering particle of VSV (DI-T) that is only competent for RNA replication, requires significantly higher levels of N to drive RNA replication in the presence of PDeltaOD We conclude P oligomerization is not required for mRNA synthesis but enhances genome replication by facilitating RNA encapsidation.IMPORTANCE All NNS RNA viruses, including the human pathogens rabies, measles, respiratory syncytial virus, Nipah and Ebola possess an essential L-protein co-factor, required to access the N-RNA template and coordinate the various enzymatic activities of L. The polymerase co-factors share a similar modular organization of a soluble N-binding domain, and a template-binding domain separated by a central oligomerization domain. Using a prototype of NNS RNA virus gene expression, vesicular stomatitis virus (VSV), we determined the importance of P oligomerization. We find that oligomerization of VSV P is not required for any step of viral mRNA synthesis but is required for efficient RNA replication. We present evidence that this likely occurs through the stage of loading soluble N onto the nascent RNA strand as it exits the polymerase during RNA replication. Interfering with the oligomerization of P may represent a general strategy to interfere with NNS RNA virus replication.