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Journal of Virology, August 2008, p. 7846-7862, Vol. 82, No. 16
0022-538X/08/$08.00+0     doi:10.1128/JVI.00789-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

A Single N-Linked Glycosylation Site in the Japanese Encephalitis Virus prM Protein Is Critical for Cell Type-Specific prM Protein Biogenesis, Virus Particle Release, and Pathogenicity in Mice {triangledown} ,{dagger}

Jeong-Min Kim,1,{ddagger} Sang-Im Yun,1,{ddagger} Byung-Hak Song,1,{ddagger} Youn-Soo Hahn,2 Chan-Hee Lee,3 Hyun-Woo Oh,4 and Young-Min Lee1*

Department of Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, South Korea,1 Department of Pediatrics, College of Medicine and Clinical Research Institute, Chungbuk National University, Cheongju, South Korea,2 Division of Life Sciences, College of Natural Sciences and Research Institute for Genetic Engineering, Chungbuk National University, Cheongju, South Korea,3 Biological Resources Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, South Korea4

Received 13 April 2008/ Accepted 23 May 2008

The prM protein of Japanese encephalitis virus (JEV) contains a single potential N-linked glycosylation site, N15-X16-T17, which is highly conserved among JEV strains and closely related flaviviruses. To investigate the role of this site in JEV replication and pathogenesis, we manipulated the RNA genome by using infectious JEV cDNA to generate three prM mutants (N15A, T17A, and N15A/T17A) with alanine substiting for N15 and/or T17 and one mutant with silent point mutations introduced into the nucleotide sequences corresponding to all three residues in the glycosylation site. An analysis of these mutants in the presence or absence of endoglycosidases confirmed the addition of oligosaccharides to this potential glycosylation site. The loss of prM N glycosylation, without significantly altering the intracellular levels of viral RNA and proteins, led to an {approx}20-fold reduction in the production of extracellular virions, which had protein compositions and infectivities nearly identical to those of wild-type virions; this reduction occurred at the stage of virus release, rather than assembly. This release defect was correlated with small-plaque morphology and an N-glycosylation-dependent delay in viral growth. A more conservative mutation, N15Q, had the same effect as N15A. One of the four prM mutants, N15A/T17A, showed an additional defect in virus growth in mosquito C6/36 cells but not human neuroblastoma SH-SY5Y or hamster BHK-21 cells. This cell type dependence was attributed to abnormal N-glycosylation-independent biogenesis of prM. In mice, the elimination of prM N glycosylation resulted in a drastic decrease in virulence after peripheral inoculation. Overall, our findings indicate that this highly conserved N-glycosylation motif in prM is crucial for multiple stages of JEV biology: prM biogenesis, virus release, and pathogenesis.

* Corresponding author. Mailing address: Department of Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University, 12 Gaeshin-Dong, Heungduk-Ku, Cheongju-Si, South Korea. Phone: 82-43-261-2863. Fax: 82-43-272-1603. E-mail: ymlee{at}chungbuk.ac.kr

{triangledown} Published ahead of print on 4 June 2008.

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/.

{ddagger} These authors contributed equally to this work.

Journal of Virology, August 2008, p. 7846-7862, Vol. 82, No. 16
0022-538X/08/$08.00+0     doi:10.1128/JVI.00789-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Yun, S.-I., Choi, Y.-J., Song, B.-H., Lee, Y.-M. (2009). 3' cis-Acting Elements That Contribute to the Competence and Efficiency of Japanese Encephalitis Virus Genome Replication: Functional Importance of Sequence Duplications, Deletions, and Substitutions. J. Virol. 83: 7909-7930 [Abstract] [Full Text]  


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