14th ICID - Poster Abstracts - International Society for Infectious ...

When citing these abstracts please use the following reference:
Author(s) of abstract. Title of abstract [abstract]. Int J Infect Dis 2010;14S1: Abstract number.
Please note that the official publication of the International Journal of Infectious Diseases 2010, Volume 14, Supplement 1
is available electronically on http://www.sciencedirect.com
Final Abstract Number: 84.030
Session: Virology and Viral Infections (Non-HIV)
Date: Friday, March 12, 2010
Time: 12:30-13:30
Room: Poster & Exhibition Area/Ground Level
Type: Poster Presentation
Analysis of phosphorylation residues on Nipah virus nucleoprotein and role of the phosphorylation
M. Huang, H. Sato, K. Hagiwara, A. watanabe, F. Ikeda, M. Oyama, M. Yoneda, C. Kai
The Institute of Medical Science, Tokyo, Japan
Background: Nipah virus (NiV) is a recently emerged zoonotic virus that causes encephalitic and
respiratory illness in humans and livestock, with a high mortality rate (40-70%) in humans. NiV is
a negative single-stranded RNA virus that belongs to the family Paramyxoviridae and is
composed of six structural proteins. The viral nucleoprotein (N) encapsidates the genomic RNA
and forms a nucleocapsid. This serves as a template for viral replication and transcription, which
is catalyzed by an RNA-dependent RNA polymerase that is composed of viral phosphoprotein (P)
and large (L) proteins. NiV is related closely to Measles virus (MV), which has a wellcharacterized
N protein and also belongs to the family Paramyxoviridae. It has been reported that
MV-N undergoes phosphorylation modification. Recently, we identified the phosphorylation sites
of MV-N, and the phosphorylation was required for efficient viral transcription. However,
phosphorylation of NiV-N has not been reported. In this study, we investigated that if NiV-N also
undergoes phosphorylation.
Methods: NiV-N was expressed in COS-7 cells in the presence of 32P and immunoprecipitated,
followed by SDS-PAGE and autoradiography. Alternatively, after expression in COS-7 cell,
cesium chloride density-gradient centrifugation was performed to purify the nucleocapsid
complex. The phosphorylation site of NiV-N was subsequently determined by ESI-Q-TOF MS
analysis. To evaluate the role of the phosphorylation for viral transcription and replication, NiV
minigenome assay was utilized.
Results: We demonstrated that the NiV-N is phosphorylated and dephosphorylated in a shortspan
turnover in steady-state cells. Furthermore, we succeeded in identification of the
phosphorylated site on NiV-N. In the minigenome assay, introduction of mutation on this site
resulted in the reduction of reporter activity by approximately 45% as compared with the wild-type
protein. In addition, a mimic of phosphoserine mutation, showed more reduction. Northern blotting
showed that the transcription level was affected more than replication level by mimic of
phosphoserine mutation. On the other hand, mutation of the phosphorylation site of NiV-N did not
affect N-P complex formation.
Conclusion: In this study, we revealed that NiV-N undergoes phosphorylation in a short-span
turnover. In addition, the instant phosphorylation of NiV-N was important for efficient replication,
and rapid dephosphorylation for transcription.