Basic Amino Acid Residues at the Carboxy-Terminal Eleven Amino Acid Region of the Phosphoprotein (P) Are Required for Transcription but Not for Replication of Vesicular Stomatitis Virus Genome RNA

The phosphoprotein (P) of vesicular stomatitis virus (VSV) is an essential subunit of the viral RNAdependent RNA polymerase (RdRp) complex. It is phosphorylated at two different domains. Using defective interfering (DI) RNA or minigenomic RNA templates, we previously demonstrated that phosphorylation within the amino-terminal domain I is essential for transcription, whereas phosphorylation within the carboxyterminal domain II is necessary for replication. For the present study, we examined the role of the phosphorylation of residues in these domains in the life cycle of VSV. Various mutant P coding sequences were inserted into a full-length cDNA clone of VSV, and the virus recovery, kinetics of growth, and mRNA and protein synthesis were examined. We observed that virus recovery was completely abolished when all three phosphate acceptor sites in domain I or both sites in domain II were replaced with alanine. Single or double mutations in domain I (with the exception of P60/64) or single mutations in domain II had no adverse effect on virus recovery. VSVP227, carrying alanine at position 227, showed reduced kinetics of virus growth but increased kinetics of viral mRNA synthesis in infected cells. More interestingly, this particular virus exhibited a significantly reduced cytopathic effects and apoptosis in infected cells, implying that P may be involved in these processes. Furthermore, we found that DI RNAs of different sizes were generated by high-multiplicity passaging of various mutant VSVs, indicating that the viral RdRp may play a significant role in the process of DI particle generation. Taken together, our results suggest that the phosphorylation of residues in domains I and II of VSV P is indispensable for virus growth.Vesicular stomatitis virus (VSV) is the prototypic rhabdovirus and has a negative-stranded RNA genome of 11,161 nucleotides (50). Within the virus and in infected cells, the genomic RNA is tightly encapsidated by the nucleocapsid (N) protein, forming the viral nucleocapsid, which serves as the template for transcription and replication by the associated viral RNA-dependent RNA polymerase (RdRp) (50). The RdRp is a complex that contains the virally encoded large (L) protein and the phosphoprotein (P protein). While the L protein contains the catalytic center for the polymerization of nucleotides, the P protein serves as an essential subunit of the RdRp. The P protein is multifunctional: in addition to playing a major role in polymerase functions, it binds to the L protein and stabilizes it from proteolytic degradation (5, 12), it complexes with the newly synthesized N protein for the efficient encapsidation of nascent RNA (9, 39, 48), and it interacts with terminal sequences of the viral genome for viral RNA synthesis (27, 29).Through mutational and biochemical studies, three functionally homologous domains have been identified in the P proteins (Fig. 1A) of both the Indiana (P I ) and New Jersey (P NJ ) serotypes of VSV (20,47). The amino-terminal domain I (residues 1 to approximately 150) ...

supporting

confidence: 82%

Section: Vol 78 2004 P Phosphorylation Is Essential For Vsv Growth mentioning

confidence: 99%

Phosphorylation of Vesicular Stomatitis Virus Phosphoprotein P Is Indispensable for Virus Growth

Das

Pattnaik

2004

“…The phosphorylation of P in nonsegmented negative-stranded RNA virus is thought to play a critical role in virus RNA synthesis (19). Mutations within P of vesicular stomatitis virus that selectively affect viral RNA replication or viral mRNA transcription have been identified, suggesting that the protein plays an important role in regulating viral RNA replication and viral mRNA transcription (7). However, the roles of phosphorylation of paramyxovirus P proteins in viral RNA synthesis have been less defined.…”

mentioning

confidence: 99%

A Single Amino Acid Residue Change in the P Protein of Parainfluenza Virus 5 Elevates Viral Gene Expression

Timani

Sun

et al. 2008

Parainfluenza virus 5 (PIV5) is a prototypical paramyxovirus. The V/P gene of PIV5 encodes two mRNA species through a process of pseudotemplated insertion of two G residues at a specific site during transcription, resulting in two viral proteins, V and P, whose N termini of 164 amino acid residues are identical. Previously it was reported that mutating six amino acid residues within this identical region results in a recombinant PIV5 (rPIV5-CPI؊) that exhibits elevated viral protein expression and induces production of cytokines, such as beta interferon and interleukin 6. Because the six mutations correspond to the shared region of the V protein and the P protein, it is not clear whether the phenotypes associated with rPIV5-CPI؊ are due to mutations in the P protein and/or mutations in the V protein. To address this question, we used a minigenome system and recombinant viruses to study the effects of mutations on the functions of the P and V proteins. We found that the P protein with six amino acid residue changes (Pcpi؊) was more efficient than wild-type P in facilitating replication of viral RNA, while the V protein with six amino acid residue changes (Vcpi؊) still inhibits minigenome replication as does the wild-type V protein. These results indicate that elevated viral gene expression in rPIV5-CPI؊ virus-infected cells can be attributed to a P protein with an increased ability to facilitate viral RNA synthesis. Furthermore, we found that a single amino acid residue change at position 157 of the P protein from Ser (the residue in the wild-type P protein) to Phe (the residue in Pcpi؊) is sufficient for elevated viral gene expression. Using mass spectrometry and 33 P labeling, we found that residue S157 of the P protein is phosphorylated. Based on these results, we propose that phosphorylation of the P protein at residue 157 plays an important role in regulating viral RNA replication.Parainfluenza virus 5 (PIV5), formerly known as simian virus 5, is a prototypical paramyxovirus in the Rubulavirus genus of Paramyxovirinae (4). The paramyxovirus family includes many important human and animal pathogens, such as Sendai virus, mumps virus, human parainfluenza viruses 1, 2, 3, and 4, Newcastle disease virus, measles virus, and emerging viruses, such as Hendra virus and Nipah virus (19). Paramyxoviruses contain nonsegmented negative-stranded RNA genomes, which are encapsidated by nucleocapsid protein (NP). The gene order within the paramyxovirus genomes is 3Ј-NP-P(V/W/C)-M-F-(SH)-HN-L-5Ј, where genes in parenthesis are not found in all species (reviewed in reference 19). The viral RNA-dependent RNA polymerase of paramyxoviruses minimally consists of two proteins, phosphoprotein (P) and the large (L) polymerase protein (11). The L proteins of paramyxoviruses have masses of 220 to 250 kDa. They have the capacity to initiate, elongate, and terminate transcription. In addition, they have the capacity to insert nontemplated G residues at selected sites within viral mRNAs during transcription and to add cap structures to t...

“…The last 11 amino acids of this domain are highly conserved and contain a number of basic residues that are important for P protein function in mediating the binding of P protein to N-RNA template (15).…”

mentioning

confidence: 99%

Role of the Hypervariable Hinge Region of Phosphoprotein P of Vesicular Stomatitis Virus in Viral RNA Synthesis and Assembly of Infectious Virus Particles

Das

Pattnaik

2005

The phosphoprotein (P protein) of vesicular stomatitis virus (VSV) is an essential subunit of the viral RNAdependent RNA polymerase and has multiple functions residing in its different domains. In the present study, we examined the role of the hypervariable hinge region of P protein in viral RNA synthesis and recovery of infectious VSV by using transposon-mediated insertion mutagenesis and deletion mutagenesis. We observed that insertions of 19-amino-acid linker sequences at various positions within this region affected replication and transcription functions of the P protein to various degrees. Interestingly, one insertion mutant was completely defective in both transcription and replication. Using a series of deletion mutants spanning the hinge region of the protein, we observed that amino acid residues 201 through 220 are required for the activity of P protein in both replication and transcription. Neither insertion nor deletion had any effect on the interaction of P protein with N or L proteins. Infectious VSVs with a deletion in the hinge region possessed retarded growth characteristics and exhibited small-plaque morphology. Interestingly, VSV containing one P protein deletion mutant (P⌬7, with amino acids 141 through 200 deleted), which possessed significant levels of replication and transcription activity, could be amplified only by passage in cells expressing the wild-type P protein. We conclude that the hypervariable hinge region of the P protein plays an important role in viral RNA synthesis. Furthermore, our results provide a previously unidentified function for the P protein: it plays a critical role in the assembly of infectious VSV.Vesicular stomatitis virus (VSV) is the prototypic member of the family Rhabdoviridae in the order Mononegavirales. VSV is an enveloped virus with a negative-stranded RNA genome of 11,161 nucleotides. The viral envelope consists of a lipid bilayer with the spike glycoprotein protruding out and the matrix (M) protein lying underneath the lipid bilayer. The viral genome which is present in the virion core is tightly encapsidated by the nucleocapsid protein (N protein) to form the ribonucleoprotein complex (6). The viral ribonucleoprotein serves as the template for transcription and replication by the viral RNA-dependent RNA polymerase, which is a complex of the large protein (L protein) and phosphoprotein (P protein). The L protein is the catalytic subunit of the polymerase complex responsible for polymerization of nucleotides, capping, methylation of capped mRNAs, and polyadenylation of mRNAs (reviewed in references 1 and 45). P protein, on the other hand, acts as an accessory subunit of the viral polymerase (1, 45). In addition to having a role in polymerase functions, it binds to the L protein and stabilizes it from proteolytic degradation (7, 17), it complexes with the newly synthesized N protein for the efficient encapsidation of nascent RNA (16,36,42), and it interacts with terminal sequences of viral genome for viral RNA synthesis (29,30).The P proteins of the two s...