The Major Phosphorylation Sites of the Respiratory Syncytial Virus Phosphoprotein Are Dispensable for Virus Replication In Vitro

Lü, Bin; Ma, Chien-Hui; Brazas, Robert; Ji, Hong

doi:10.1128/jvi.76.21.10776-10784.2002

Cited by 56 publications

(73 citation statements)

References 33 publications

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“…Previous studies have indicated that the P's of respiratory syncytial virus (RSV) (25) and Sendai virus (SeV) (26) play roles in the transcription and replication of these viruses. A recent study also showed that a phosphorylation site within the P of parainfluenza virus 5 plays a positive role in viral mRNA synthesis and virus growth (27).…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…Thus, P protein phosphorylation may have a role in the distinction between vRdRpR and vRdRpT (Collins et al, 2001 Phosphorylation at S237 in vitro has been suggested (Mazumder et al, 1994). Phosphorylation at these residues is not required for viral transcription or replication, either to support M2-1 transcriptional activities or for the viral growth cycle (Asenjo et al, 2005;Lu et al, 2002;Villanueva et al, 2000).Nevertheless, in a P protein variant with all of these residues replaced by non-phosphorylatable residues (VPm30) (Fig. 1a), phosphorylation was detected at S and T residues when it was expressed transiently in HEp-2 cells and labelled with [ 32 P]orthophosphate in the presence of 1 mM okadaic acid (OKA) to inhibit cellular phosphatases PP2A and PP1 completely (Bialojan & Takai, 1988) (Fig.…”

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“…Highturnover phosphates are added to S30, S39, S45, S54 and perhaps T46 (Asenjo et al, 2005), those with intermediate turnover modify S116, S117 and/or S119 (Asenjo et al, 2005;Navarro et al, 1991) Phosphorylation at S237 in vitro has been suggested (Mazumder et al, 1994). Phosphorylation at these residues is not required for viral transcription or replication, either to support M2-1 transcriptional activities or for the viral growth cycle (Asenjo et al, 2005;Lu et al, 2002;Villanueva et al, 2000).…”

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Phosphorylation of human respiratory syncytial virus P protein at threonine 108 controls its interaction with the M2-1 protein in the viral RNA polymerase complex

Asenjo

Calvo

Villanueva

2006

Journal of General Virology

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The human respiratory syncytial virus (HRSV) P protein is phosphorylated, with different turnover rates, at several serine (S) and threonine (T) residues. The role of phosphothreonines in viral RNA synthesis was studied by using P protein substitution variants and the HRSV-based minigenome pM/SH. By using liquid chromatography coupled to ion-trap mass spectrometry, it was found that P protein T108 was phosphorylated by addition of a high-turnover phosphate group. This phosphorylation occurs in P protein expressed transiently and during HRSV infection. The results suggest that phosphorylation at P protein T108 affects M2-1 transcriptional activities, because this modification prevents interaction between the P and M2-1 proteins. Therefore, P protein phosphorylation-dephosphorylation at T108 could distinguish the role of the P protein in viral transcription and replication.Human respiratory syncytial virus (HRSV), a pneumovirus of the family Paramyxoviridae, causes the majority of acute respiratory-tract infections affecting babies, the immunocompromised and elderly adults (Hall, 2001). Live vaccines (Collins et al., 1999), humanized monoclonal antibodies (mAbs; Meissner et al., 1999) and specific antiviral compounds (Bitko et al., 2005;Pastey et al., 2000) are suitable prophylactic measures to control HRSV infections.The development of specific antiviral compounds requires a molecular understanding of viral protein functions. The viral ribonucleoproteins (RNPs) are ideal targets for antiviral compounds. They are multifunctional and involved in distinctive viral processes, as they are structural virion components and the functional units for viral RNA synthesis. The RNPs include the helical nucleocapsid, containing viral (v) RNA bound to N protein, the L protein (or polymerase) and their cofactors, the phosphoproteins P and M2-1 (Collins et al., 2001). Viral transcription and replication are distinct processes. In the first, discontinuous mRNA synthesis is driven by the gene-start (GS) and -stop (GE) signals present in each gene. In the second, continuous RNA synthesis occurs from the first nucleotide located at the vRNA 39 end to the last one at the 59 end (Lamb & Kolakofsky, 2001). The L protein displays nucleotide-polymerizing activity during viral RNA synthesis, but it requires P protein as an essential, non-catalytic cofactor. The P-L complex allows the L protein to contact the nucleocapsid (the template for viral transcription and replication) through L-P-N-RNA interactions. P-N interactions are essential to render the N protein competent for RNA encapsidation during replication (Collins et al., 2001). P-M2-1 interactions through P protein residues L101, Y102 and F109 are needed for M2-1 protein anti-termination and elongation transcriptional activities (Mason et al., 2003).It is not clear how the viral RNA polymerase (vRdRp) is involved differentially in transcription (vRdRpT) and replication (vRdRpR) (Gubbay et al., 2001). The essential interactions established by the P protein during these processes could...

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“…22 Furthermore, although PP5 is present in both nuclear and cytoplasmic compartments, its subcellular localization predominates in the nucleus, 19,23 a site where dephosphorylation of FKBP52 is most likely to occur. And finally, as PP5 contains a C-terminal catalytic domain structurally related to the PP1/PP2A/PP2B family and an N-terminal regulatory domain consisting of three tetratricopeptide repeats (TPRs) that usually mediate protein-protein interaction, 24 it has been demonstrated that PP5 interacts with multiple unrelated target proteins through the TPR domain, [25][26][27][28][29][30][31] and FKBP52 is known to contain a TPR domain. 32,33 In the current study, we used various known inhibitors of protein Ser/Thr phosphatases to pretreat an established human embryonic kidney cell line 293, in which FKBP52 is present predominantly in the Ser/Thr phosphorylated form.…”

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Adeno-associated virus 2-mediated gene transfer: role of a cellular serine/threonine protein phosphatase in augmenting transduction efficiency

Zhao

Zhong

et al. 2006

Gene Ther

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We have documented that a cellular chaperone protein, FKBP52, when phosphorylated at tyrosine and/or serine/ threonine (Ser/Thr) residues, interacts with the D-sequence in the inverted terminal repeats of the adeno-associated virus 2 (AAV) genome, inhibits the viral second-strand DNA synthesis, and leads to inefficient transgene expression from recombinant AAV vectors in certain cell types. We have also demonstrated that FKBP52 is dephosphorylated at tyrosine residues by T-cell protein tyrosine phosphatase (TC-PTP), and that deliberate overexpression of TC-PTP leads to more efficient viral second-strand DNA synthesis, and increased transgene expression. However, the identity of the putative Ser/Thr protein phosphatase that dephosphorylates FKBP52 at Ser/Thr residues has remained elusive. Using known inhibitors of Ser/Thr phosphatases, we have now identified protein phosphatase 5 (PP5) to be a candidate enzyme.Deliberate overexpression of PP5 in 293 cells, which does not influence cellular growth, leads to B5-fold increase in the transduction efficiency of conventional single-stranded AAV vectors, but no significant enhancement in the transduction efficiency of self-complementary AAV vectors, suggesting that PP5 plays a role in AAV second-strand DNA synthesis. Electrophoretic mobility-shift assays show that in cells overexpressing PP5, the extent of the complex formation between FKBP52 and the AAV D-sequence is significantly reduced. These studies suggest that PP5-mediated dephosphorylation of FKBP52 at Ser/Thr residues augments viral second-strand DNA synthesis and enhances AAV transduction efficiency, which has implications in the optimal use of these vectors in human gene therapy.

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The Major Phosphorylation Sites of the Respiratory Syncytial Virus Phosphoprotein Are Dispensable for Virus Replication In Vitro

Cited by 56 publications

References 33 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

Phosphorylation of human respiratory syncytial virus P protein at threonine 108 controls its interaction with the M2-1 protein in the viral RNA polymerase complex

Adeno-associated virus 2-mediated gene transfer: role of a cellular serine/threonine protein phosphatase in augmenting transduction efficiency

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