ABSTRACTThe mumps virus (MuV) genome encodes a phosphoprotein (P) that is important for viral RNA synthesis. P forms the viral RNA-dependent RNA polymerase with the large protein (L). P also interacts with the viral nucleoprotein (NP) and self-associates to form a homotetramer. The P protein consists of three domains, the N-terminal domain (PN), the oligomerization domain (PO), and the C-terminal domain (PC). While PNis known to relax the NP-bound RNA genome, the roles of POand PCare not clear. In this study, we investigated the roles of POand PCin viral RNA synthesis using mutational analysis and a minigenome system. We found that PNand PCfunctions can betrans-complemented. However, this complementation requires PO, indicating that POis essential for P function. Using thistrans-complementation system, we found that P forms parallel dimers (PNto PNand PCto PC). Furthermore, we found that residues R231, K238, K253, and K260 in POare critical for P's functions. We identified PCto be the domain that interacts with L. These results provide structure-function insights into the role of MuV P.IMPORTANCEMuV, a paramyxovirus, is an important human pathogen. The P protein of MuV is critical for viral RNA synthesis. In this work, we established a novel minigenome system that allows the domains of P to be complemented intrans. Using this system, we confirmed that MuV P forms parallel dimers. An understanding of viral RNA synthesis will allow the design of better vaccines and the development of antivirals.