Phosphoprotein of the Rinderpest Virus Forms a Tetramer through a Coiled Coil Region Important for Biological Function

The phosphoprotein (P protein) of vesicular stomatitis virus (VSV) is an essential subunit of the viral RNA-dependent RNA polymerase complex and plays a central role in viral transcription and replication. Using both the yeast two-hybrid system and coimmunoprecipitation assays, we confirmed the self-association of the P protein of Indiana serotype (Pind) and heterotypic interaction between Pind and the P protein of New Jersey serotype (Pnj). Furthermore, by using various truncation and deletion mutants of Pind, the self-association domain of the Pind protein was mapped to amino acids 161 to 210 within the hinge region. The self-association domain of Pind protein is not required for its binding to nucleocapsid and large proteins. We further demonstrated that the self-association domain of Pind protein is essential for VSV transcription in a minireplicon system and that a synthetic peptide spanning amino acids 191 to 210 in the self-association domain of Pind protein strongly inhibited the transcription of the VSV genome in vitro in a dose-dependent manner. These results indicated that the self-association domain of Pind protein plays a critical role in VSV transcription.Vesicular stomatitis virus (VSV) is a negative-stranded RNA virus belonging to family Rhabdoviridae. It packages within the mature virion an RNA-dependent RNA polymerase that transcribes the negative-sense genome RNA into mRNAs (2) in the infected cells that are subsequently translated into five proteins: the glycoprotein (G protein), the matrix protein (M protein), the nucleoprotein (N protein), the RNA-dependent RNA polymerase large protein (L protein), and the phosphoprotein (P protein) (42). The N-RNA complex and the P and L proteins, which form the ribonucleoprotein (RNP) core complex, can be separately isolated and purified from the virions and can effectively reconstitute mRNA synthesis in vitro when these components are mixed (17,20,21). L protein by itself fails to transcribe the N-RNA template; P protein is absolutely required to elicit its RNA polymerase activity (17,21,36). P protein thus seems to act as a transcription factor for L protein. In addition, it may also help to stabilize the L protein from proteolytic degradation (7) and may be required for productive virion assembly (13). P protein also associates with newly synthesized N protein during infection to keep the latter in a soluble, replication-competent form (16,34,39) to enwrap the nascent RNA during replication (34). P protein also forms a tripartite replicase complex with L and N proteins, as L-N-P, which has been shown to convert N-RNA template to positive-sense genome RNP in vivo.Pind protein has been arbitrarily divided into three domains, domains I, II, and III. Domain I (residues 1 to 137) is highly acidic in nature, and binds to the L protein (22). Domain I contains three phosphorylation sites (Ser-60, Thr-62, and Ser-64) that are phosphorylated by cellular casein kinase II and are indispensable for the transcriptional role of the P protein (5,6,9,14,28,38,44,46). D...

Section: Discussionsupporting

confidence: 75%

Mapping and Functional Role of the Self-Association Domain of Vesicular Stomatitis Virus Phosphoprotein

Chen

Ogino

Banerjee

2006

“…This result suggests that only complexes composed of functional VP35 proteins are able to support transcription and replication. Further support for this idea comes from results published by Rahaman et al showing that homo-oligomerization-competent but functionally negative mutants of the rinderpest virus P protein display a dominant-negative phenotype (37). Possibly, this feature can be addressed in the quest to develop antivirals against MARV.…”

Section: Discussionmentioning

confidence: 53%

Homo-Oligomerization of Marburgvirus VP35 Is Essential for Its Function in Replication and Transcription

Möller

Pariente

Klenk

et al. 2005

The nucleocapsid protein VP35 of Marburgvirus, a filovirus, acts as the cofactor of the viral polymerase and plays an essential role in transcription and replication of the viral RNA. VP35 forms complexes with the genome encapsidating protein NP and with the RNA-dependent RNA polymerase L. In addition, a trimeric complex had been detected in which VP35 bridges L and the nucleoprotein NP. It has been presumed that the trimeric complex represents the active polymerase bound to the nucleocapsid. Here we present evidence that a predicted coiled-coil domain between amino acids 70 and 120 of VP35 is essential and sufficient to mediate homo-oligomerization of the protein. Substitution of leucine residues 90 and 104 abolished (i) the probability to form coiled coils, (ii) homo-oligomerization, and (iii) the function of VP35 in viral RNA synthesis. Further, it was found that homo-oligomerization-negative mutants of VP35 could not bind to L. Thus, it is presumed that homo-oligomerization-negative mutants of VP35 are unable to recruit the polymerase to the NP/RNA template. In contrast, inability to homo-oligomerize did not abolish the recruitment of VP35 into inclusion bodies, which contain nucleocapsid-like structures formed by NP. Finally, transcriptionally inactive mutants of VP35 containing the functional homo-oligomerization domain displayed a dominant-negative phenotype. Inhibition of VP35 oligomerization might therefore represent a suitable target for antiviral intervention.Marburgvirus (MARV) and the closely related Ebolavirus (EBOV) together make up the family Filoviridae, which is classified in the order Mononegavirales. MARV causes a fulminant hemorrhagic fever in humans and nonhuman primates with high fatality rates (28). To date, neither a vaccine nor a curative treatment for MARV infection in humans is available. However, live attenuated recombinant vaccines have been described which protected nonhuman primates against MARV and EBOV infections (23). These might represent promising candidate vaccines for human use as well. The recent outbreak of MARV disease in Angola underlines the emerging potential of this pathogen (4). The MARV particle is composed of seven structural proteins. Four of them, NP, VP35, VP30, and L, constitute the nucleocapsid complex of MARV, which surrounds the viral genome. NP, the major nucleocapsid protein, self-assembles into tubular nucleocapsid-like structures, which are found intracellularly in large inclusions (25,29). The formation of the tubular structures by NP is presumed to represent the first step in the assembly of the nucleocapsid. NP interacts with VP35, which in turn interacts with the RNAdependent RNA polymerase L (3). The complex of VP35 and L represents the active RNA-dependent RNA polymerase, with VP35 serving as a polymerase cofactor (33). Additionally, a trimeric complex was observed consisting of NP, VP35, and L, with VP35 connecting L and NP (3). Three of the four nucleocapsid proteins, NP, VP35, and L, are essential for transcription and replication of the viral ...

“…In the present study, we confirmed that all P domains are required for full P function. P multimerization has been found to be essential for viral transcription of rinderpest virus and VSV (10,19). Interestingly, when MuV P truncations lacking either the N-terminal domain (P OC ) or the C-terminal domain (P NO ) were transfected together, P activity was restored due to trans-complementation through P O .…”

Section: Discussionmentioning

confidence: 93%

“…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: 84%

Oligomerization of Mumps Virus Phosphoprotein

Pickar

Elson

Yang

et al. 2015

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...