Phosphorylation of measles virus phosphoprotein at S86 and/or S151 downregulates viral transcriptional activity

Sugai, Akihiro; Sato, Hiroki; Yoneda, Misako; Kai, Chieko

doi:10.1016/j.febslet.2012.09.021

Cited by 32 publications

(20 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We previously demonstrated that the major phosphorylation sites of N protein (S479 and S510) are involved in various stages of the viral life cycle (28,29). Additionally, we found that a mutant with mutation of both major phosphorylation sites remained phosphorylated but to a lesser extent, suggesting that unidentified phosphorylation sites exist within the N protein.…”

Section: Discussionmentioning

confidence: 81%

“…Whether N proteins were expressed by a plasmid or during viral replication (46), the phosphorylation rate of the threonine residue in NC was almost the same. Thus, T279 phosphorylation of the N protein is required for the maintenance of the herringbone-like structure of NC, and T279 is different from the major phosphorylation sites that take part in the functional regulation of viral gene expression (27)(28)(29). Electron microscopy demonstrated that NC has a helical structure with approximately 13 to 14 N proteins per turn (15,50,51).…”

Section: Discussionmentioning

confidence: 99%

“…Precise methods for the minigenome experiment were described previously (27,28). Briefly, 293 cells in 24-well plates were transfected with N, P, and L protein expression plasmids using Lipofectamine LTX reagent (Invitrogen) and Plus reagent (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

“…Additionally, the functional significance of the major phosphorylation sites of N protein to the viral life cycle includes viral gene expression, viral genome RNA stability, and regulation of P-protein phosphorylation (28,29). However, despite double mutation of the major phosphorylation sites of MV N protein, it was still phosphorylated, albeit to a lesser extent.…”

mentioning

confidence: 99%

See 3 more Smart Citations

Newly Identified Minor Phosphorylation Site Threonine-279 of Measles Virus Nucleoprotein Is a Prerequisite for Nucleocapsid Formation

Sugai¹,

Sato²,

Hagiwara³

et al. 2014

J Virol

Self Cite

View full text Add to dashboard Cite

Measles virus nucleoprotein is the most abundant viral protein and tightly encapsidates viral genomic RNA to support viral transcription and replication. Major phosphorylation sites of nucleoprotein include the serine residues at locations 479 and 510. Minor phosphorylation residues have yet to be identified, and their functions are poorly understood. In our present study, we identified nine putative phosphorylation sites by mass spectrometry and demonstrated that threonine residue 279 (T279) is functionally significant. Minigenome expression assays revealed that a mutation at the T279 site caused a loss of activity. Limited proteolysis and electron microscopy suggested that a T279A mutant lacked the ability to encapsidate viral RNA but was not denatured. Furthermore, dephosphorylation of the T279 site by alkaline phosphatase treatment caused deficiencies in nucleocapsid formation. Taken together, these results indicate that phosphorylation at T279 is a prerequisite for successful nucleocapsid formation. Measles virus (MV), a member of the Morbillivirus genus in the Paramyxoviridae family, is an important human pathogen that causes disease characterized by fever, cough, coryza, conjunctivitis, and a maculopapular rash. Although the use of effective vaccines has decreased global mortality from measles, it remains a major cause of high mortality among children in developing countries (1-3). MV has a nonsegmented negativestranded RNA genome containing six structural genes encoding nucleoprotein (N), phosphoprotein (P), matrix (M) protein, fusion (F) protein, hemagglutinin (H) protein, and large (L) protein (4), and the P gene produces two accessory proteins, known as V and C (5, 6). N proteins encapsidate viral genomic RNA to support viral transcription and replication by an RNA-dependent RNA polymerase (RdRp) L protein. The P protein is a multifunctional protein (7,8) that assists with viral transcription and replication as a cofactor of the L protein (9, 10). The C and V accessory proteins suppress host immune responses (11,12). The M protein helps virus assembly, and the F and H proteins are required for membrane fusion and binding to the host cellular receptor, respectively (13).N protein is the most abundant viral protein in infected cells (14) and is mainly required for viral transcription and replication. N proteins tightly associate with the viral genome and antigenome to form an N-RNA complex with a herringbone-like structure (15,16). N proteins associate with every 6 bases of the 15,894-nucleotide viral genome and fully cover the genome RNA (17). This tight encapsidation allows the viral genome to be resistant to RNases and small interfering RNAs (18,19). Viral transcription and replication occur on the N-RNA complex in association with viral RdRp (vRdRp), composed of L and P proteins. This complex of N-RNA, P protein, and L protein is called the nucleocapsid (NC) and comprises 2,649 copies of the N protein, about 300 copies of the P protein, and about 20 to 30 copies of the L protein (20)(21)(22). Singly...

show abstract

Section: Discussionmentioning

confidence: 81%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Newly Identified Minor Phosphorylation Site Threonine-279 of Measles Virus Nucleoprotein Is a Prerequisite for Nucleocapsid Formation

Sugai¹,

Sato²,

Hagiwara³

et al. 2014

J Virol

Self Cite

View full text Add to dashboard Cite

show abstract

“…We also showed that the major phosphorylation sites of the N protein are indirectly involved in the regulation of viral transcription by changing the phosphorylation status of the P protein (17). In the present study, we rescued recombinant MV (rMV) HL strains (18) of these N-phosphorylation sites (S479A, S510A, and S479A/S510A) using a reverse genetics system that we had previously established (19) and investigated their influence on virus growth.…”

Section: Easles Virus (Mv) Amentioning

confidence: 85%

Phosphorylation of Measles Virus Nucleoprotein Affects Viral Growth by Changing Gene Expression and Genomic RNA Stability

Sugai

Sato

Yoneda

et al. 2013

J Virol

Self Cite

View full text Add to dashboard Cite

The measles virus (MV) nucleoprotein associates with the viral RNA genome to form the N-RNA complex, providing a template for viral RNA synthesis. In our previous study, major phosphorylation sites of the nucleoprotein were identified as S479 and S510. However, the functions of these phosphorylation sites have not been clarified. In this study, we rescued recombinant MVs (rMVs) whose phosphorylation sites in the nucleoprotein were substituted (rMV-S479A, rMV-S510A, and rMV-S479A/S510A) by reverse genetics and used them in subsequent analyses. In a one-step growth experiment, rMVs showed rapid growth kinetics compared with wild-type MV, although the peak titer of the wild-type MV was the same as or slightly higher than those of the rMVs. Time course analysis of nucleoprotein accumulation also revealed that viral gene expression of rMV was enhanced during the early phase of infection. These findings suggest that nucleoprotein phosphorylation has an important role in controlling viral growth rate through the regulation of viral gene expression. Conversely, multistep growth curves revealed that nucleoprotein-phosphorylation intensity inversely correlated with viral titer at the plateau phase. Additionally, the phosphorylation intensity of the wild-type nucleoprotein in infected cells was significantly reduced through nucleoprotein-phosphoprotein binding. Excessive nucleoprotein-phosphorylation resulted in lower stability against RNase and faster turnover of viral genomic RNA. These results suggest that nucleoprotein-phosphorylation is also involved in viral genomic RNA stability.

show abstract

Measles: The Disease That Refuses to Be Vanquished

Talekar

Porotto

2023

Emerging Human Viral Diseases, Volume I

View full text Add to dashboard Cite

Phosphorylation of measles virus phosphoprotein at S86 and/or S151 downregulates viral transcriptional activity

Cited by 32 publications

References 38 publications

Newly Identified Minor Phosphorylation Site Threonine-279 of Measles Virus Nucleoprotein Is a Prerequisite for Nucleocapsid Formation

Newly Identified Minor Phosphorylation Site Threonine-279 of Measles Virus Nucleoprotein Is a Prerequisite for Nucleocapsid Formation

Phosphorylation of Measles Virus Nucleoprotein Affects Viral Growth by Changing Gene Expression and Genomic RNA Stability

Measles: The Disease That Refuses to Be Vanquished

Contact Info

Product

Resources

About