Viral proteins required for the in Vitro replication of vesicular stomatitis virus defective interfering particle genome RNA

Peluso, Richard W.; Moyer, Sue A.

doi:10.1016/0042-6822(88)90477-1

Cited by 116 publications

(130 citation statements)

References 19 publications

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“…Upon infection, these proteins transcribe viral genome to generate the viral mRNAs to produce more viral proteins. Accumulation of the soluble NP-P complex is thought to trigger the switch from RNA transcription to genome replication (15)(16)(17). However, the fact that P and mutant T101A interacted with NP at similar levels indicates that the mutant did not exert its impact on viral mRNA synthesis through its interaction with NP.…”

Section: Discussionmentioning

confidence: 99%

“…To further investigate the mechanism of accelerated viral protein expression in rMuV-P-T101A-infected cells, real-time PCR was used to compare the levels of viral mRNA and viral genomic RNA in Vero cells. The cells were infected with MuV or rMuV-P-T101A at an MOI of 0.01, and total RNA was purified from infected cells at 2,4,8,12,16, and 20 hpi. Interestingly, more viral mRNAs were detected in rMuV-P-T101A-infected cells at earlier time points (4 and 8 hpi) (Fig.…”

Section: Figmentioning

confidence: 99%

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Roles of Serine and Threonine Residues of Mumps Virus P Protein in Viral Transcription and Replication

Pickar

Elson

et al. 2014

J Virol

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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...

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Section: Discussionmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

Roles of Serine and Threonine Residues of Mumps Virus P Protein in Viral Transcription and Replication

Pickar

Elson

et al. 2014

J Virol

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“…An NP ! -P complex has also been demonstrated for other paramyxoviruses, including MV (Huber et al, 1991), respiratory syncytial virus (RSV) (Garcia et al, 1993), parainfluenza virus (PIV) types 2 and 3 (Nishio et al, 1996 ;Zhao & Banerjee, 1995), simian virus 5 (Precious et al, 1995), as well as for the rhabdoviruses, VSV and rabies virus (Davis et al, 1986 ;Peluso & Moyer, 1988 ;Fu et al, 1994 ;Chenik et al, 1994).…”

Section: Introductionmentioning

confidence: 99%

Identification of nucleocapsid protein residues required for Sendai virus nucleocapsid formation and genome replication.

Myers

Smallwood

Moyer

1999

Journal of General Virology

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Alanine substitution mutations in the Sendai virus nucleocapsid (NP) protein have defined highly conserved hydrophobic and charged residues from amino acids (aa) 362 to 371 that are essential for function of the protein in RNA replication. Mutant NP362, which had the change F362A, was incapable of supporting in vitro RNA replication. NP362 expressed alone formed extended oligomers which exhibited an abnormal morphology and density suggesting that these particles were not associated with any RNA. Mutant NP364, which had changes L362A and G365A, was also inactive in RNA replication ; however, this was because the protein was unstable and did not form NP-NP complexes. Mutant NP370 mutant, which had changes K370A and D371A, was inactive in in vitro replication, although it could form the required NP 0 -P and NP-NP protein complexes. The self-assembled nucleocapsid-like particles formed by NP370 alone had a morphology like that of wild-type NP and banded in CsCl as ribonucleoprotein particles, suggesting that they contained cellular RNA. These data suggest that the replication defect of NP370 may be in the ability to specifically encapsidate Sendai virus genome RNA. Mutant NP373, where nonconserved charged residues at aa 373 and 375 were substituted with alanine, gave a wild-type phenotype. Thus these amino acids are not required for either protein-protein interactions or in vitro Sendai virus RNA replication.

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“…The active RNA polymerase complex of VSV is composed of the large protein (L) and its cofactor, the phosphoprotein (P) (1), and both are required for the transcription and replication of the viral negative-sense single-stranded RNA genome, which is tightly encapsidated by nucleoprotein (N) (2,3). P is a multifunctional protein: it associates with newly synthesized N during infection to prevent N from binding to cellular RNAs and maintains the replication-competent form of N by specifically encapsidating the nascent viral genomic/antigenomic RNA (4)(5)(6). On the other hand, it may also help to protect the L protein from proteolytic degradation (7).…”

mentioning

confidence: 99%

N-Terminal Phosphorylation of Phosphoprotein of Vesicular Stomatitis Virus Is Required for Preventing Nucleoprotein from Binding to Cellular RNAs and for Functional Template Formation

Chen

Zhang

Banerjee

et al. 2013

J Virol

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bThe phosphoprotein (P) of vesicular stomatitis virus (VSV) plays essential roles in viral RNA synthesis. It associates with nascent nucleoprotein (N) to form N 0 -P (free of RNAs), thereby preventing the N from binding to cellular RNAs and maintaining the N in a viral genomic RNA encapsidation-competent form for transcription and replication. The contributions of phosphorylation of P to transcription and replication have been studied intensively, but a concrete mechanism of action still remains unclear. In this study, using a VSV minigenome system, we demonstrated that a mutant of P lacking N-terminal phosphorylation (P3A), in which the N-terminal phosphate acceptor sites are replaced with alanines (S60/A, T62/A, and S64/A), does not support transcription and replication. However, results from protein interaction assays showed that P3A self-associates and interacts with N and the large protein (L) as efficiently as P does. Furthermore, purified recombinant P3A from Sf21 cells supported transcription in an in vitro transcription reconstitution assay. We also proved that P3A is not distributed intranuclearly in vivo. CsCl gradient centrifugation showed that P3A is incapable of preventing N from binding to cellular RNAs and therefore prevents functional template formation. Taken together, our results demonstrate that N-terminal phosphorylation is indispensable for P to prevent N from binding to nonviral RNAs and to maintain the N-specific encapsidation of viral genomic RNA for functional template formation. V esicular stomatitis virus (VSV) is a nonsegmented negativestrand RNA virus that serves as the prototype of the rhabdovirus group. The active RNA polymerase complex of VSV is composed of the large protein (L) and its cofactor, the phosphoprotein (P) (1), and both are required for the transcription and replication of the viral negative-sense single-stranded RNA genome, which is tightly encapsidated by nucleoprotein (N) (2, 3). P is a multifunctional protein: it associates with newly synthesized N during infection to prevent N from binding to cellular RNAs and maintains the replication-competent form of N by specifically encapsidating the nascent viral genomic/antigenomic RNA (4-6). On the other hand, it may also help to protect the L protein from proteolytic degradation (7). In addition, P also forms a tripartite replicase complex with L and N (L-N-P) to convert the N-RNA template to positive-sense genomic RNA in vivo. Via mutational and biochemical studies, P of the VSV Indiana serotype has been divided arbitrarily into three domains, i.e., N-terminal domain I (residues 1 to 137), domain II (residues 211 to 244), and domain III (residues 245 to 265), and a hypervariable hinge region (residues 138 to 210).Like the P's of many other negative-strand RNA viruses, the P of VSV is phosphorylated in infected cells and virions (8-10). The P of the VSV Indiana serotype has been shown to be phosphorylated in two different domains: N-terminal domain I, in which phosphorylation sites were mapped to S60, T62, and S64, and ...

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Viral proteins required for the in Vitro replication of vesicular stomatitis virus defective interfering particle genome RNA

Cited by 116 publications

References 19 publications

Roles of Serine and Threonine Residues of Mumps Virus P Protein in Viral Transcription and Replication

Roles of Serine and Threonine Residues of Mumps Virus P Protein in Viral Transcription and Replication

Identification of nucleocapsid protein residues required for Sendai virus nucleocapsid formation and genome replication.

N-Terminal Phosphorylation of Phosphoprotein of Vesicular Stomatitis Virus Is Required for Preventing Nucleoprotein from Binding to Cellular RNAs and for Functional Template Formation

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