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Archives of Virology

, Volume 150, Issue 4, pp 771–786 | Cite as

Deglycosylation of the NS1 protein of dengue 2 virus, strain 16681: Construction and characterization of mutant viruses

  • M. B. Crabtree
  • R. M. Kinney
  • B. R. Miller
Article

Summary.

The dengue 2 virus (DENV-2) NS1 glycoprotein contains two potential sites for N-linked glycosylation at Asn-130 and Asn-207. NS1 produced in infected cells is glycosylated at both of these sites. We used site-directed mutagenesis of a DENV-2, strain 16681, full length infectious clone to create mutant viruses lacking the Asn-130, Asn-207 or both of these NS1 glycosylation sites in order to investigate the effects of deglycosylation. Ablation of both NS1 glycosylation sites resulted in unstable viruses that acquired numerous additional mutations; these viruses were not further characterized. Viruses altered at the Asn-130 site exhibited growth characteristics similar to the wild-type (WT) 16681 virus in LLC-MK2 cells and reduced growth in C6/36 cells. Viruses mutated at the Asn-207 site achieved similar titers in LLC-MK2 cells compared to WT, however, the appearance of cytopathic effect was delayed and growth of these viruses in C6/36 cells was also reduced compared to WT virus. The plaque size of mutant viruses altered at the Asn-130 site did not differ from that of the WT virus, while mutants altered at the Asn-207 site exhibited a reduced and mixed plaque size. Temperature sensitivity studies comparing the growth of the viruses at 37 °C and 39 °C showed no significant differences compared to the WT virus. Immunofluorescent antibody staining of infected cells showed that for WT 16681 virus or the Asn-130 site mutant viruses NS1 was located throughout the cytoplasm, however, Asn-207 site mutant virus NS1 protein appeared to be localized to the perinuclear region. Viruses deglycosylated at either site exhibited a significant reduction in mouse neurovirulence compared to the WT virus. The results of our studies indicate that glycosylation of the DENV-2 virus NS1 protein may influence NS1 protein processing/transport as well as the pathogenicity of the virus.

Keywords

Mutant Virus Perinuclear Region Plaque Size Infectious Clone Immunofluorescent Antibody 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer-Verlag/Wien 2004

Authors and Affiliations

  • M. B. Crabtree
    • 1
  • R. M. Kinney
    • 1
  • B. R. Miller
    • 1
  1. 1.Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, U.S. Department of Health and Human ServicesFort CollinsU.S.A.

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