Phosphorylated States of Vesicular Stomatitis Virus P Proteinin Vitroandin Vivo

Chen, Jin Lian; Das, Tapas; Banerjee, Amiya K.

doi:10.1006/viro.1996.8401

Cited by 38 publications

(63 citation statements)

References 38 publications

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“…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 C-terminal domain II, in which phosphorylation sites were mapped to S226 and S227. Since P was shown to be phosphorylated in specific domains involved in regulating transcription in vitro, phosphorylation of P and its role in transcription have been investigated intensively (11)(12)(13)(14)(15)(16)(17)(18). The conclusions derived from these investigations have demonstrated that the phosphorylation of P is important for its functionality in various ways.…”

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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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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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“…Phosphorylation sites of the VSV P protein have been located to acidic domain I in the N-terminal half and domain II at the C-terminus. Additionally, stepwise phosphorylations at domains I and then II have been proposed for the P protein, in which cellular casein kinase (CK) II and the viral L protein-or nucleocapsidassociated protein kinase activity have been suggested to be involved, respectively (2,3,6). More recently, CK II was shown to be involved in the phosphorylation of domain I, and interconversion of P1 and P2 is a reflection of phosphorylation and dephosphorylation of the phosphate groups in domain I (6).…”

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Studies on the Rabies Virus RNA Polymerase: 2. Possible Relationships between the Two Forms of the Non‐Catalytic Subunit (P Protein)

Takamatsu

Asakawa

Morimoto

et al. 1998

Microbiology and Immunology

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We investigated the relationship between the two forms of rabies virus P protein, a non‐catalytic subunit of rabies virus RNA polymerase. The two displayed different electrophoretic mobilities as 37‐ and 40‐kDa polypeptides, hence termed as p37 and p40, respectively. Double labeling experiments with [3H]leucine and [32P]orthophosphate demonstrated that p40 was much more phosphorylated than p37. Treatment of the virion proteins with alkaline phosphatase eliminated only p40, and not 37‐kDa polypeptide. The p37 was a major product of the P gene, and was accumulated in the infected cell and incorporated into the virion. On the other hand, p40 was apparently detected only in the virion, and little detected in the cells. Treatment of infected cells with okadaic acid, however, resulted in significant accumulation of p40 in the cell, suggesting that p40 was continuously produced in the cell but dephosphorylated quickly. We detected both 37‐ and 40‐kDa products in P cDNA‐transfected animal cells, while only a 37‐kDa product was produced in Escherichia coli. Incubation of 37‐kDa products from E. coli with the lysates of animal cells in vitro resulted in the production of a 40‐kDa product, which was also shown to be suppressed by the heparin. From these results, it is suggested that p40 is produced by the hyperphosphorylation of a 37‐kDa polypeptide, which depends on certain heparin‐sensitive cellular enzyme(s) and occurs even in the absence of the other viral gene products, and that p40 is reverted quickly to p37 in the infected cells, probably being dependent on some virus‐induced factor(s).

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“…In the presence of cellular casein kinase II, it becomes phosphorylated and functions in transcription in vitro (2,22). By mutational and biochemical studies, the phosphate acceptor sites were mapped to Ser-59 and Ser-61 in P NJ and Ser-60, Thr-62, and Ser-64 in P I (6,53,54). Further studies suggested that the phosphorylation of these residues results in multimerization of the P protein, which in turn facilitates its binding to the L protein for transcription (7,16,17).…”

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“…For P NJ , domain I residues must be phosphorylated prior to the phosphorylation of domain II residues (3), but for P I , domain II residues can be phosphorylated without the prior phosphorylation of domain I residues (6). Since phosphorylation in domain I is essential and sufficient for transcription, it seems that the phosphorylation of domain II may not be involved in transcription (16,38).…”

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

Das

Pattnaik

2004

J Virol

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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) ...

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Phosphorylated States of Vesicular Stomatitis Virus P Proteinin Vitroandin Vivo

Cited by 38 publications

References 38 publications

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

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

Studies on the Rabies Virus RNA Polymerase: 2. Possible Relationships between the Two Forms of the Non‐Catalytic Subunit (P Protein)

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

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