Constitutive phosphorylation of the vesicular stomatitis virus P protein modulates polymerase complex formation but is not essential for transcription or replication

Spadafora, Domenico; Canter, D.M.; Jackson, R L; Perrault, Jacques

doi:10.1128/jvi.70.7.4538-4548.1996

Cited by 52 publications

(27 citation statements)

References 32 publications

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“…From the results documented here, we conclude that although phosphorylation of P protein in the domain I residues is not essential for replication function of the protein, this modification is critical for its transcriptional activity. Using a similar panel of mutant P proteins, Spadafora et al recently concluded that phosphorylation of the domain I residues of the Indiana serotype P protein does not play a major role in DI RNA replication in vivo (50). Our results presented in this report confirm their results of in vivo DI RNA replication studies.…”

Section: Discussionsupporting

confidence: 88%

“…Further increase in the amount of P 60/62/64 plasmid in transfection did not result in increased levels of transcription (data not shown). It should noted that with increasing amounts of the plasmid transfected (up to 9 g), a corresponding increase in the P-protein expression was observed (data not shown), which is consistent with our previous observation (46) and those obtained recently by Spadafora et al (50).…”

Section: Expression and Stability Of Wt And Mutant P Proteins In Transupporting

confidence: 93%

“…However, under in vitro phosphorylation conditions, the mutant protein could be readily phosphorylated at T-62, which led to the suggestion that the mutant protein is phosphorylated at T-62 when expressed in vivo but perhaps is selectively dephosphorylated by the phosphatase(s) present in the cell extracts (13). When this mutant protein was tested for its ability to support transcription under in vitro conditions, it was found to be much less active (Յ5% of the wt P level) at low concentrations but was active (about 28% of the wt P level) at higher concentrations of the P protein (50). We therefore wished to determine if this mutant protein could rescue transcription in vivo with increased concentrations of the protein.…”

Section: Expression and Stability Of Wt And Mutant P Proteins In Tranmentioning

confidence: 99%

“…The bacterially expressed inactive P protein, however, becomes transcriptionally active if it is first phosphorylated by casein kinase II (CKII) (4,26). By various mutagenesis and biochemical studies, the acceptor sites for CKII-mediated phosphorylation of P protein have been mapped to Ser-59 and Ser-61 for New Jersey serotype (51) and Ser-60, Thr-62, and Ser-64 for Indiana serotype of VSV (13,50). Phosphorylation of these residues which are located in the amino-terminal acidic domain I of the protein (see Fig.…”

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confidence: 99%

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Phosphorylation within the amino-terminal acidic domain I of the phosphoprotein of vesicular stomatitis virus is required for transcription but not for replication

Pattnaik

Hwang

et al. 1997

J Virol

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Phosphorylation by casein kinase II at three specific residues (S-60, T-62, and S-64) within the acidic domain I of the P protein of Indiana serotype vesicular stomatitis virus has been shown to be critical for in vitro transcription activity of the viral RNA polymerase (P-L) complex. To examine the role of phosphorylation of P protein in transcription as well as replication in vivo, we used a panel of mutant P proteins in which the phosphate acceptor sites in domain I were substituted with alanines or other amino acids. Analyses of the alanine-substituted mutant P proteins for the ability to support defective interfering RNA replication in vivo suggest that phosphorylation of these residues does not play a significant role in the replicative function of the P protein since these mutant P proteins supported replication at levels >70% of the wild-type P-protein level. However, the transcription function of most of the mutant proteins in vivo was severely impaired (2 to 10% of the wild-type P-protein level). The level of transcription supported by the mutant P protein (P 60/62/64 ) in which all phosphate acceptor sites have been mutated to alanines was at best 2 to 3% of that of the wild-type P protein. 81% of the wild-type level), substitution with phenylalanine (P FFF ) rendered the protein much less active in transcription (<5%). Substitution with arginine residues led to significantly reduced activity in replication (6%), whereas glutamic acid substituted P protein (P EEE ) supported replication (42%) and transcription (86%) well. In addition, the mutant P proteins that were defective in replication (P RRR ) or transcription (P 60/62/64 ) did not behave as transdominant repressors of replication or transcription when coexpressed with wild-type P protein. From these results, we conclude that phosphorylation of domain I residues plays a major role in in vivo transcription activity of the P protein, whereas in vivo replicative function of the protein does not require phosphorylation. These findings support the contention that different phosphorylated states of the P protein regulate the transcriptase and replicase functions of the polymerase protein, L. Increasing the amount of P 60/62/64 expression in transfected cells did not rescue significant levels of transcription. Substitution with other amino acids at these sites had various effects on replication and transcription. While substitution with threonine residues (P TTT ) had no apparent effect on transcription (113% of the wild-type level) or replication (

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Section: Discussionsupporting

confidence: 88%

Section: Expression and Stability Of Wt And Mutant P Proteins In Transupporting

confidence: 93%

Section: Expression and Stability Of Wt And Mutant P Proteins In Tranmentioning

confidence: 99%

mentioning

confidence: 99%

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Phosphorylation within the amino-terminal acidic domain I of the phosphoprotein of vesicular stomatitis virus is required for transcription but not for replication

Pattnaik

Hwang

et al. 1997

J Virol

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“…Importantly, D70 is present in a region of VSV-P that is heavily phosphorylated (phosphorylation of residues 60, 62 and 64 has been described) (51). Phosphorylation of these sites has been proposed to be important in P-protein dimerization and interaction with the L-protein (large polymerase protein) (52–56) and phosphorylation of this region is essential for functional RNA dependent RNA polymerase activity (57). Because tyrosine residues are known to be phosphorylated in the VSV-P protein (58), it is therefore tempting to speculate that a tyrosine at position 70 could be phosphorylated, and that this phosphorylation somehow overcomes the block mediated by TRIM69.…”

Section: Discussionmentioning

confidence: 99%

TRIM69 inhibits Vesicular Stomatitis Indiana Virus (VSIV)

Rihn¹,

Aziz²,

Stewart³

et al. 2019

Preprint

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word count: 210 16 Text character count: 58,351 17 18 ABSTRACT 19Vesicular Stomatitis Indiana Virus (VSIV) is a model virus that is exceptionally 20 sensitive to the inhibitory action of interferons. Interferons induce an antiviral state by 21 stimulating the expression of hundreds of interferon stimulated genes (ISGs). These 22 ISGs constrain viral replication, limit tissue tropism, reduce pathogenicity and inhibit 23 viral transmission. Because VSIV is used as a backbone for multiple oncolytic and 24 vaccine strategies, understanding how ISGs restrict VSIV, not only helps in 25 understanding VSIV-pathogenesis, but helps evaluate and understand the safety and 26 efficacy of VSIV-based therapies. Thus there is a need to identify and characterize 27 the ISGs that possess anti-VSIV activity. Using arrayed ISG expression screening, 28 solely responsible for limiting VSIV replication in other organs (15). Thus, other ISGs 85 must play key roles in limiting VSIV tissue tropism. Importantly, VSIV causes 86 neurological disease in multiple species following intracranial inoculation (17, 18), 87suggesting that the ability of ISGs to prevent VSIV from initially accessing the CNS is 88 the cornerstone in limiting VSIV neuropathology across multiple species (19). 89VSIV's low pathogenicity in humans, its rapid replication, and ease of genetic 90 manipulation have made this virus the basis of multiple therapeutic strategies. For 91 example, VSIV can be modified to express antigens from heterologous viruses that 92 can be utilised as vaccine strategies (20). This approach has achieved recent notable 93 success in conferring protection from Ebola virus infection (21). Similarly, VSIV has 94 been used as the backbone of multiple oncolytic strategies (22). Just like natural 95 VSIV infection, IFNs and ISGs appear to be critical for preventing oncolytic viruses 96 from invading healthy tissues (23, 24) and could be critical determinants governing 97 whether oncolytic vesiculoviruses will be efficacious (25). Furthermore, ISGs likely 98 play a key role in limiting the replication of VSIV-based vaccines and are an 99 important safety feature of this immunization strategy. 100The key roles that ISGs play in constraining VSIV pathogenesis and limiting 101 VSIV replication (in natural infection, oncolytic therapies and vaccine strategies) 102 means there is a need to better understand how ISGs inhibit VSIV. Using arrayed 103 ISG expression screening, we identified that TRIM69, a relatively poorly 104 characterised TRIM protein, has anti-VSIV activity. Through exogenous expression 105 and CRISPR Cas9 knockout, we demonstrate that both exogenous and endogenous 106 TRIM69 have potent anti-VSIV activity. Importantly, the inhibition is highly specific 107 for VSIV and multiple other viruses were not inhibited by TRIM69. Notably, TRIM69 108 shows strong signatures of positive selection and multiple common alleles circulate in 109 human populations. Interestingly, murine orthologues of TRIM69 had no detectable 110 anti-VSIV activity whereas ...

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Phosphorylation of Serine-Rich Protein Encoded by Open Reading Frame 3 of the TT Virus Genome

Asabe

Nishizawa

Iwanari³

et al. 2001

Biochemical and Biophysical Research Communications

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Constitutive phosphorylation of the vesicular stomatitis virus P protein modulates polymerase complex formation but is not essential for transcription or replication

Cited by 52 publications

References 32 publications

Phosphorylation within the amino-terminal acidic domain I of the phosphoprotein of vesicular stomatitis virus is required for transcription but not for replication

Phosphorylation within the amino-terminal acidic domain I of the phosphoprotein of vesicular stomatitis virus is required for transcription but not for replication

TRIM69 inhibits Vesicular Stomatitis Indiana Virus (VSIV)

Phosphorylation of Serine-Rich Protein Encoded by Open Reading Frame 3 of the TT Virus Genome

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