The bulk of the phosphorylation of human respiratory syncytial virus phosphoprotein is not essential but modulates viral RNA transcription and replication

The ability of variants of the human respiratory syncytial virus (HRSV) phosphoprotein (P protein) to support RNA transcription and replication has been studied by using HRSV-based subgenomic replicons. The serine residues normally phosphorylated in P during HRSV infection have been replaced by other residues. The results indicate that the bulk of phosphorylation of P (98%) is not essential for viral RNA transcription or replication but that phosphorylation can modulate these processes.

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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

Journal of General Virology ◽

10.1099/vir.0.82165-0 ◽

2006 ◽

Vol 87 (12) ◽

pp. 3637-3642 ◽

Cited By ~ 36

Author(s):

Ana Asenjo ◽

Enrique Calvo ◽

Nieves Villanueva

Keyword(s):

Respiratory Syncytial Virus ◽

Ion Trap ◽

Human Respiratory Syncytial Virus ◽

Viral Rna ◽

Turnover Rates ◽

High Turnover ◽

P Protein ◽

Syncytial Virus ◽

Transcription And Replication

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.

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Respiratory Syncytial Virus Phosphoprotein Residue S156 Plays a Role in Regulating Genome Transcription and Replication

Journal of Virology ◽

10.1128/jvi.01206-21 ◽

2021 ◽

Author(s):

Ashley C. Beavis ◽

Kim C. Tran ◽

Enrico R. Barrozo ◽

Shannon I. Phan ◽

Michael N. Teng ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Viral Rna ◽

Growth Defect ◽

Compensatory Mutation ◽

Phosphorylation Sites ◽

Viral Growth ◽

P Protein ◽

Genome Transcription ◽

Syncytial Virus ◽

Transcription And Replication

Respiratory syncytial virus (RSV) is a single-stranded, negative-sense, RNA virus in the family Pneumoviridae and genus Orthopneumoviridae that can cause severe disease in infants, immunocompromised adults, and the elderly. The RSV viral RNA-dependent RNA polymerase (vRdRp) complex is composed of the phosphoprotein (P) and the large polymerase protein (L). The P protein is constitutively phosphorylated by host kinases and has 41 serine (S) and threonine (T) residues as potential phosphorylation sites. To identify important phosphorylation residues in the P protein, we systematically and individually mutated all serine S and T residues to alanine (A) and first analyzed their effect on genome transcription and replication using a minigenome system. We found that the mutation of eight residues resulted in significantly reduced minigenome activity compared to wild-type P. We then incorporated these mutations (T210A, S203A, T151A, S156A, T160A, S23A, T188A, and T105A) into full-length genome cDNA to rescue recombinant RSV. We were able to recover four recombinant viruses (T151A, S156A, T160A, and S23A), suggesting RSV-P residues T210, S203, T188, and T105 are essential for viral RNA replication. Among the four rescued, rRSV-T160A caused a minor growth defect compared to its parental virus while rRSV-S156A had severely restricted replication due to decreased levels of genomic RNA. During infection, P-S156A phosphorylation was decreased, and when passaged, the S156A virus acquired a known compensatory mutation in L (L795I) that enhanced both WT-P and P-S156A minigenome activity and was able to partially rescue the S156A viral growth defect. This work demonstrates that residues T210, S203, T188, and T105 are critical for RSV replication, and S156 plays a critical role in viral RNA synthesis. Importance RSV-P is a heavily phosphorylated protein that is required for RSV replication. In this study, we identified several residues, including P-S156, as phosphorylation sites that play critical roles in efficient viral growth and genome replication. Future studies to identify the specific kinase(s) that phosphorylate these residues can lead to kinase inhibitors and anti-viral drugs for this important human pathogen.

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