Serine 3 is critical for phosphorylation at the N-terminal end of the nucleoprotein of influenza virus A/Victoria/3/75
- PMID: 8648669
- PMCID: PMC190210
- DOI: 10.1128/JVI.70.6.3385-3391.1996
Serine 3 is critical for phosphorylation at the N-terminal end of the nucleoprotein of influenza virus A/Victoria/3/75
Abstract
The influenza A virus nucleoprotein (NP) is a phosphoprotein that encapsidates the viral genomic RNA. To map the in vivo phosphorylation site(s) of this protein, 32P-labeled NP was purified from cell cultures infected with influenza virus A/Victoria/3/75 by immunoaffinity chromatography. The purified protein was then subjected to chemical digestion with formic acid, which cleaves proteins at Asp-Pro bonds, and the resulting products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two of the phosphorylated products obtained were identified as fragments corresponding to the N-terminal 88 amino acids and to the C-terminal 196 residues of the NP. To identify the phosphate acceptor site(s) at the N-terminal phosphorylated region of NP, each of the seven serines within this region was individually changed to alanine by site-directed mutagenesis. The mutant proteins were then transiently expressed in mammalian cells and analyzed for their phosphorylation state. It was observed that the S-to-A mutation at position 3 drastically reduced the amount of 32P label incorporated into NP, whereas the other substitutions did not have a discernible effect on the phosphorylation level of the protein. In addition, all serine-altered proteins were tested for their functionality in an artificial system in which expression of a synthetic chloramphenicol acetyl-transferase RNA molecule is driven by influenza virus proteins synthesized from cloned genes. The results obtained demonstrate that all mutant proteins were competent to cooperate with the subunits of the viral polymerase for expression of the synthetic virus-like chloramphenicol acetyltransferase RNA in vivo. These data are discussed regarding the possible roles of NP phosphorylation for the viral replicative cycle.
Similar articles
-
Application of bioinformatics-coupled experimental analysis reveals a new transport-competent nuclear localization signal in the nucleoprotein of influenza A virus strain.BMC Cell Biol. 2008 Apr 28;9:22. doi: 10.1186/1471-2121-9-22. BMC Cell Biol. 2008. PMID: 18442378 Free PMC article.
-
Identification of an RNA binding region within the N-terminal third of the influenza A virus nucleoprotein.J Virol. 1995 Jun;69(6):3799-806. doi: 10.1128/JVI.69.6.3799-3806.1995. J Virol. 1995. PMID: 7745727 Free PMC article.
-
Mutational analysis of influenza A virus nucleoprotein: identification of mutations that affect RNA replication.J Virol. 1999 Feb;73(2):1186-94. doi: 10.1128/JVI.73.2.1186-1194.1999. J Virol. 1999. PMID: 9882320 Free PMC article.
-
Analysis of influenza A virus nucleoproteins for the assessment of molecular genetic mechanisms leading to new phylogenetic virus lineages.Arch Virol. 1993;131(3-4):237-50. doi: 10.1007/BF01378629. Arch Virol. 1993. PMID: 8347076 Review.
-
Structure and sequence analysis of influenza A virus nucleoprotein.Sci China C Life Sci. 2009 May;52(5):439-49. doi: 10.1007/s11427-009-0064-x. Epub 2009 May 27. Sci China C Life Sci. 2009. PMID: 19471866 Review.
Cited by
-
Structural phosphoprotein M2-1 of the human respiratory syncytial virus is an RNA binding protein.J Virol. 2000 Nov;74(21):9858-67. doi: 10.1128/jvi.74.21.9858-9867.2000. J Virol. 2000. PMID: 11024112 Free PMC article.
-
Phosphorylation of the porcine reproductive and respiratory syndrome virus nucleocapsid protein.J Virol. 2002 Oct;76(20):10569-76. doi: 10.1128/jvi.76.20.10569-10576.2002. J Virol. 2002. PMID: 12239338 Free PMC article.
-
FDA-Approved Inhibitors of RTK/Raf Signaling Potently Impair Multiple Steps of In Vitro and Ex Vivo Influenza A Virus Infections.Viruses. 2022 Sep 16;14(9):2058. doi: 10.3390/v14092058. Viruses. 2022. PMID: 36146864 Free PMC article.
-
Phosphorylation controls the nuclear-cytoplasmic shuttling of influenza A virus nucleoprotein.J Virol. 2015 Jun;89(11):5822-34. doi: 10.1128/JVI.00015-15. Epub 2015 Mar 18. J Virol. 2015. PMID: 25787277 Free PMC article.
-
The N-terminal extension of the influenza B virus nucleoprotein is not required for nuclear accumulation or the expression and replication of a model RNA.J Virol. 1998 Jun;72(6):5307-12. doi: 10.1128/JVI.72.6.5307-5312.1998. J Virol. 1998. PMID: 9573310 Free PMC article.
References
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Miscellaneous