Mumps virus (MuV) is a highly contagious pathogen, and despite extensive vaccination campaigns, outbreaks continue to occur worldwide. The virus has a negative-sense, single-stranded RNA genome that is encapsidated by the nucleocapsid protein (N) to form the nucleocapsid (NC). NC serves as the template for both transcription and replication. In this paper we solved an 18-Å-resolution structure of the authentic MuV NC using cryo-electron microscopy. We also observed the effects of phosphoprotein (P) binding on the MuV NC structure. The N-terminal domain of P (P NTD ) has been shown to bind NC and appeared to induce uncoiling of the helical NC. Additionally, we solved a 25-Å-resolution structure of the authentic MuV NC bound with the C-terminal domain of P (P CTD ). The location of the encapsidated viral genomic RNA was defined by modeling crystal structures of homologous negative strand RNA virus Ns in NC. Both the N-terminal and C-terminal domains of MuV P bind NC to participate in access to the genomic RNA by the viral RNA-dependent-RNA polymerase. These results provide critical insights on the structurefunction of the MuV NC and the structural alterations that occur through its interactions with P.replication | paramyxovirus | mononegavirale P aramyxoviruses are enveloped nonsegmented negative-strand RNA viruses (NSV) belonging to the order Mononegavirales. Mononegavirales also includes the Bornaviridae, Filoviridae, and Rhabdoviridae families. The Paramyxoviridae family includes several important human pathogens such as measles virus (MeV), respiratory syncytial virus (RSV), and mumps virus (MuV). Although vaccines exist for some paramyxoviruses, they are not available for others, such as RSV. In addition, no effective antiviral treatments have been developed.The MuV genome encodes 9 proteins, three of which are required for replication of the MuV genome; the nucleocapsid protein (N), phosphoprotein (P), and the large protein (L). N, P, and L have orthologs in a number of NSV. Studies on the roles of N, P, and L in viral RNA synthesis have shown that each can individually and differentially affect the processes of mRNA transcription and genome replication (1-10).Throughout the virus replication cycle, the genome of NSV always exists in the nucleocapsid (NC), a unique protein-RNA complex in which the viral RNA [viral genomic RNA (vRNA) or complementary genomic RNA (cRNA)] is completely sequestered by the N protein. NC is used as the functional template for RNA synthesis by the viral RNA dependent RNA polymerase (vRdRp), which includes L and P. The L protein contains all of the enzymatic activities needed for viral RNA synthesis, such as the ability to cap and polyadenylate mRNA transcripts. P acts as a cofactor to home vRdRp onto the NC template for RNA synthesis. In addition, the P protein chaperones monomeric and RNA-free N to encapsidate newly synthesized viral genomes during replication. The encapsidation of RNA by N is concomitant with the replication process.How the sequestered vRNA is accessed by vRdRp ...

Mumps virus (MuV), a paramyxovirus containing a negative-sense nonsegmented RNA genome, is a human pathogen that causes an acute infection with symptoms ranging from parotitis to mild meningitis and severe encephalitis. Vaccination against mumps virus has been effective in reducing mumps cases. However, recently large outbreaks have occurred in vaccinated populations. There is no anti-MuV drug. Understanding replication of MuV may lead to novel antiviral strategies. MuV RNA-dependent RNA polymerase minimally consists of the phosphoprotein (P) and the large protein (L). The P protein is heavily phosphorylated. To investigate the roles of serine (S) and threonine (T) residues of P in viral RNA transcription and replication, P was subjected to mass spectrometry and mutational analysis. P, a 392-amino acid residue protein, has 64 S and T residues. We have found that mutating nine S/T residues significantly reduced and mutating residue T at 101 to A (T101A) significantly enhanced activity in a minigenome system. A recombinant virus containing the P-T101A mutation (rMuV-P-T101A) was recovered and analyzed. rMuV-P-T101A grew to higher titers and had increased protein expression at early time points. Together, these results suggest that phosphorylation of MuV-P-T101 plays a negative role in viral RNA synthesis. This is the first time that the P protein of a paramyxovirus has been systematically analyzed for S/T residues that are critical for viral RNA synthesis. IMPORTANCE Mumps virus (MuV) is a reemerging paramyxovirus that caused large outbreaks in the UnitedStates, where vaccination coverage is very high. There is no anti-MuV drug. In this work, we have systematically analyzed roles of Ser/Thr residues of MuV P in viral RNA synthesis. We have identified S/T residues of P critical for MuV RNA synthesis and phosphorylation sites that are important for viral RNA synthesis. This work leads to a better understanding of viral RNA synthesis as well as to potential novel strategies to control mumps. Mumps virus (MuV) is a human pathogen that causes acute parotitis and is highly neurotropic (1). Invasion of the central nervous system is evident in almost half of all clinical cases, with asceptic meningitis occurring in approximately 10% of cases and encephalitis in less than 1% (1). Even though the mumps vaccine has dramatically reduced disease incidence, large outbreaks have recently occurred in vaccinated populations (2, 3). Over 5,700 mumps cases were reported in a 2006 outbreak that originated at a university in Iowa and spread to 10 other states (2). A mumps outbreak occurred in New York and New Jersey in 2009 to 2010 where 88% of the patients had one dose of mumps vaccine and 75% of patients had two doses (3). There is no antiviral drug for MuV infection. Understanding functions of viral proteins will aid development of antiviral strategies. In this study, a strain of MuV from a recent outbreak in Iowa in 2006 (4), MuV Iowa/US/06 (referred to here as MuV), was used to examine the role of phosphorylation of the M...

The 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 (P N ), the oligomerization domain (P O ), and the C-terminal domain (P C ). While P N is known to relax the NP-bound RNA genome, the roles of P O and P C are not clear. In this study, we investigated the roles of P O and P C in viral RNA synthesis using mutational analysis and a minigenome system. We found that P N and P C functions can be trans-complemented. However, this complementation requires P O , indicating that P O is essential for P function. Using this trans-complementation system, we found that P forms parallel dimers (P N to P N and P C to P C ). Furthermore, we found that residues R231, K238, K253, and K260 in P O are critical for P's functions. We identified P C to 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 in trans. 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. Mumps virus (MuV) is a human pathogen of the Rubulavirus genus of the Paramyxoviridae family that causes an acute infection with symptoms ranging from parotitis to mild meningitis and severe encephalitis (1). The nonsegmented, negativestranded RNA genome of MuV contains 15,384 nucleotides and encodes nine viral proteins (1). The viral RNA is encapsidated by the nucleoprotein (NP), and this helical nucleocapsid (RNP) functions as the template for viral RNA synthesis. Together, the large protein (L) and the phosphoprotein (P) make up the viral RNA-dependent RNA polymerase (vRdRp) (2). The enzymatic activities of the L protein involve the initiation, elongation, and termination of RNA synthesis, as well as mRNA capping (3). While P is not known to have intrinsic enzymatic activity, P is an essential cofactor of the polymerase. P oligomerizes by itself and forms complexes with L, NP, and RNP. It is thought that P docks the vRdRP to RNP (4).The P proteins of paramyxoviruses are modular and consist of N-terminal (P N ), oligomerization (P O ), and C-terminal (P C ) domains with flexible linkers between adjoining domains. The selfassociation of P is observed throughout negative-stranded RNA viruses (NSVs). The oligomerization domain of Sendai virus (SeV) P was the first to be crystallized, and those studies revealed a parallel coiled-coil tetramer (5, 6). The self-association of P is required for transcriptional activity, and the binding site for SeV L was found to neighbor the oligomerizat...

Although mumps vaccines have been used for several decades, protective immune correlates have not been defined. Recently, mumps outbreaks have occurred in vaccinated populations. To better understand the causes of the outbreaks and to develop means to control outbreaks in mumps vaccine immunized populations, defining protective immune correlates will be critical. Unfortunately, no small animal model for assessing mumps immunity exists. In this study, we evaluated use of type I interferon (IFN) alpha/beta receptor knockout mice (IFN-α/βR−/−) for such a model. We found these mice to be susceptible to mumps virus administered intranasally and intracranially. Passive transfer of purified IgG from immunized mice protected naïve mice from mumps virus infection, confirming the role of antibody in protection and demonstrating the potential for this model to evaluate mumps immunity.

Delivery of a gene of interest to target cells is highly desirable for translational medicine, such as gene therapy, regenerative medicine, vaccine development, and studies of gene function. Parainfluenza virus 5 (PIV5), a paramyxovirus with a negative-sense RNA genome, normally infects cells without causing obvious cytopathic effect, and it can infect many cell types. To exploit these features of PIV5, we established a system generating self-amplifying, virus-like particles (AVLP). Using enhanced green fluorescent protein (EGFP) as a reporter, AVLP encoding EGFP (AVLP–EGFP) successfully delivered and expressed the EGFP gene in primary human cells, including stem cells, airway epithelial cells, monocytes, and T cells. To demonstrate the application of this system for vaccine development, we generated AVLPs to express the HA and M1 antigens from the influenza A virus strain H5N1 (AVLP–H5 and AVLP–M1H5). Immunization of mice with AVLP–H5 and AVLP–M1H5 generated robust antibody and cellular immune responses. Vaccination with a single dose of AVLP–H5 and M1H5 completely protected mice against lethal H5N1 challenge, suggesting that the AVLP-based system is a promising platform for delivery of desirable genes.