Structural and Functional Characterization of the Mumps Virus Phosphoprotein

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In a negative-strand RNA virus, the genomic RNA is sequestered inside the nucleocapsid when the viral RNA-dependent RNA polymerase uses it as the template for viral RNA synthesis. It must require a conformational change in the nucleocapsid protein (N) to make the RNA accessible to the viral polymerase during this process. The structure of an empty mumps virus (MuV) nucleocapsid-like particle was determined to 10.4-Å resolution by cryo-electron microscopy (cryo-EM) image reconstruction. By modeling the crystal structure of parainfluenza virus 5 into the density, it was shown that the ␣-helix close to the RNA became flexible when RNA was removed. Point mutations in this helix resulted in loss of polymerase activities. Since the core of N is rigid in the nucleocapsid, we suggest that interactions between this region of the mumps virus N and its polymerase, instead of large N domain rotations, lead to exposure of the sequestered genomic RNA. Some pathogens appear to reemerge in spite of available vaccines, such as mumps virus and measles virus (3-5). Effective controls are needed to combat these pathogens. In order to develop more effective countermeasures, the mechanism of NSV replication should be better understood. One of the unique features in NSVs is that the genomic RNA is sequestered in the nucleocapsid (6). During transcription and replication, the viral RNA-dependent RNA polymerase (vRdRp) must be able to gain access to the sequestered genomic RNA in order to use it as the template. For Rhabdoviridae and Paramyxoviridae, the virus encodes a single nucleocapsid protein (N) that polymerizes as a linear capsid to encapsidate the genomic RNA (7). The viral polymerase complex consists of the large protein (L) and the phosphoprotein (P). IMPORTANCE Mumps virus (MuVThe structure of the nucleocapsid or a nucleocapsid-like particle has been solved for several members of Rhabdoviridae and Paramyxoviridae by X-ray crystallography or cryo-electron microscopy (cryo-EM) three-dimensional (3D) reconstruction (8-12). The common features among various structures are that the N protein has an N-terminal domain and C-terminal domain in its core, composed mostly of ␣-helices. When the N subunits assemble into a polymeric capsid, they are aligned in parallel in a linear fashion (13). There are extensive side-by-side interactions between the neighboring domains and domain swaps of extended loops and long termini. The genomic RNA is encapsidated in a cavity formed between the two core domains. Most of the RNA bases are stacked, some of which face the exterior and some the interior of the N protein core. The tight assembly of the nucleocapsid clearly suggests that vRdRp must open the N protein core in order to unveil the genomic RNA. How this action is carried out remains to be discovered. The interaction of the polymerase cofactor P with the nucleocapsid may provide some insights on this subject. The C-terminal domain of vesicular stomatitis virus (VSV) P protein binds between the extended loops in the C-terminal domains ...

Section: Resultsmentioning

confidence: 85%

Releasing the Genomic RNA Sequestered in the Mumps Virus Nucleocapsid

Severin

Terrell

Zengel

et al. 2016

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“…The self-association of P is required for transcriptional activity, and the binding site for SeV L was found to neighbor the oligomerization region (5,(7)(8)(9). Tetrameric P structures have also been observed for other paramyxoviruses, with crystallization of the P oligomerization domains being found for measles virus, human metapneumovirus, and mumps virus (10)(11)(12)(13)(14)(15)(16).…”

mentioning

confidence: 85%

“…The last 49 amino acids (aa) of MuV P (aa 343 to 391) were found to directly mediate binding to the nucleocapsid through their interaction with the assembly domain of NP (27). This nucleocapsid-binding domain is conserved, but MuV P is unique in that the N-terminal domain also binds to the nucleocapsid (16). Electron microscopy revealed uncoiling of the helical nucleocapsid by the N-terminal domain of P, which resulted in enhanced viral RNA synthesis in a minigenome system (28).…”

mentioning

confidence: 99%

Oligomerization of Mumps Virus Phosphoprotein

Pickar

Elson

Yang

et al. 2015

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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...

“…However, we considered this unlikely, since knockdown of Hsp72 showed little, if any, effect on MuV propagation in cultured cells. Recent studies revealed that the MuV P protein forms a unique tetramer structure different from those of other paramyxoviruses (31,32). Thus, MuV might differ from MV and RSV in the requirements for viral RNA replication and propagation.…”

Section: Discussionmentioning

confidence: 99%

“…However, Hsp72 was not essential for V protein degradation. The MuV P protein contains a multimerization domain at the unique C-terminal region and forms a homotetramer (31), while the V protein lacks the multimerization domain and functions as a monomer (42,43). The rate-limiting step of proteasomal degradation is the unfolding of substrates (44).…”

Section: Discussionmentioning

confidence: 99%

Heat Shock Protein 70 Regulates Degradation of the Mumps Virus Phosphoprotein via the Ubiquitin-Proteasome Pathway

Katoh

Kubota

Kita

et al. 2015

Mumps virus (MuV) infection induces formation of cytoplasmic inclusion bodies (IBs). Growing evidence indicates thatIBs are the sites where RNA viruses synthesize their viral RNA. However, in the case of MuV infection, little is known about the viral and cellular compositions and biological functions of the IBs. In this study, pulldown purification and Nterminal amino acid sequencing revealed that stress-inducible heat shock protein 70 (Hsp72) was a binding partner of MuV phosphoprotein (P protein), which was an essential component of the IB formation. Immunofluorescence and immunoblotting analyses revealed that Hsp72 was colocalized with the P protein in the IBs, and its expression was increased during MuV infection. Knockdown of Hsp72 using small interfering RNAs (siRNAs) had little, if any, effect on viral propagation in cultured cells. Knockdown of Hsp72 caused accumulation of ubiquitinated P protein and delayed P protein degradation. These results show that Hsp72 is recruited to IBs and regulates the degradation of MuV P protein through the ubiquitin-proteasome pathway. IMPORTANCE Formation of cytoplasmic inclusion bodies (IBs One of the characteristic features of mononegavirus infection is formation of cytoplasmic inclusion bodies (IBs), which can be observed by light microscopy (1), fluorescence microscopy (2-6), and electron microscopy (7-9). It is well known that IBs contain nucleocapsid-like structures, but the detailed compositions and biological functions of IBs remain to be elucidated. In the case of Ebola virus (family Filoviridae, order Mononegavirales), the IBs have been reported to be the sites of viral RNA replication (6). Similar findings were also reported for other RNA viruses, including rabies virus (RV) (4) and vesicular stomatitis virus (VSV) (5) (both in the family Rhabdoviridae, order Mononegavirales). In regard to the significance of IB formation, it is currently considered that the IBs concentrate the machinery for viral RNA synthesis. In the present study, we studied mumps virus (MuV), which is also known to form IBs.MuV is the causative agent of mumps, a common childhood illness characterized by fever and swelling of the salivary glands (10). It often causes neurological complications, including aseptic meningitis, encephalitis, and deafness. MuV belongs to the genus Rubulavirus within the family Paramyxoviridae (order Mononegavirales) (11). The viral nonsegmented negative-strand RNA genome encodes eight viral proteins: the nucleocapsid (N), V, phospho-(P), matrix (M), hemagglutinin-neuraminidase (HN), fusion (F), large (L), and small hydrophobic (SH) proteins. The genome is encapsidated by the N protein and forms an active template for RNA replication and transcription, a viral ribonucleoprotein (vRNP), with viral polymerases composed of the P and L proteins (12). The F and HN proteins are envelope glycoproteins, and the M protein is an intravirion protein that associates with the cytoplasmic tails of the envelope glycoproteins and vRNP. The SH protein is also a structura...