Determination of a phosphorylation site in Nipah virus nucleoprotein and its involvement in virus transcription.

Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.
Journal of General Virology (Impact Factor: 3.13). 05/2011; 92(Pt 9):2133-41. DOI: 10.1099/vir.0.032342-0
Source: PubMed

ABSTRACT Many viruses use their host's cellular machinery to regulate the functions of viral proteins. The phosphorylation of viral proteins is known to play a role in genome transcription and replication in paramyxoviruses. The paramyxovirus nucleoprotein (N), the most abundant protein in infected cells, is a component of the N-RNA complex and supports the transcription and replication of virus mRNA and genomic RNA. Recently, we reported that the phosphorylation of measles virus N is involved in the regulation of viral RNA synthesis. In this study, we report a rapid turnover of phosphorylation in the Nipah virus N (NiV-N). The phosphorylated NiV-N was hardly detectable in steady-state cells, but was detected after inhibition of cellular protein phosphatases. We identified a phosphorylated serine residue at Ser451 of NiV-N by peptide mass fingerprinting by electrospray ionization-quadrupole time-of-flight mass spectrometry. In the NiV minigenome assay, using luciferase as a reporter gene, the substitution of Ser451 for alanine in NiV-N resulted in a reduction in luciferase activity of approximately 45 % compared with the wild-type protein. Furthermore, the substitution of Ser451 for glutamic acid, which mimics a phosphoserine, led to a more significant decrease in luciferase activity - approximately 81 %. Northern blot analysis showed that both virus transcription and replication were reduced by these mutations. These results suggest that a rapid turnover of the phosphorylation of NiV-N plays an important role in virus transcription and replication.

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    ABSTRACT: Nipah virus (NiV) is a non-segmented single-stranded negative-sense RNA virus belonging to the genus Henipavirus, family Paramyxoviridae. NiV causes acute encephalitis and respiratory disease in humans, is associated with high mortality, and poses a threat in southern Asia. The genomes of henipaviruses are about 18,246 nucleotides (nt) long, which is longer than those of other paramyxoviruses (around 15,384 nt). This difference is caused by the noncoding RNA region, particularly the 3' untranslated region (UTR), which occupies more than half of the noncoding RNA region. To determine the function(s) of the NiV noncoding RNA region, we investigated the effects of NiV 3' UTRs on reporter gene expression. The NiV N 3' UTR (nt 1-100) demonstrated strong repressor activity associated with hnRNP D protein binding to that region. Mutation of the hnRNP D binding site or knockdown of hnRNP D resulted in increased expression of the NiV N 3' UTR reporter. Our findings suggest that NiV N expression is repressed by hnRNP D through the NiV N 3' UTR, and demonstrated the involvement of post-transcriptional regulation in the NiV life cycle. To the best of our knowledge, this provides the first report of the functions of the NiV noncoding RNA region.
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    ABSTRACT: When HEp-2 cells are infected by human respiratory syncytial virus (HRSV) its N protein becomes phosphorylated at tyrosine (Y) Y38, in a strictly regulated way. To determine how this phosphorylation affects nucleocapsid (NC) template activity during viral RNA synthesis, N protein variants were analysed in which Y38 and nearby Y residues were substituted by phenylalanine (F; Y23F, Y38F and Y69F) or aspartic acid (D; Y23D and Y38D). While the capacity of these proteins to form the NC and to interact with the P protein was maintained, their NC template activity was altered affecting distinctly viral transcription and replication of HRSV based minigenomes. Thus, Y38 phosphorylation of the HRSV N protein modulates NC template activity probably by altering the interactions of the monomeric components of the NC.
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    ABSTRACT: Measles virus nucleoprotein is the most abundant viral protein that 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 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.
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