Growth restriction of dengue virus type 2 by site-specific mutagenesis of virus-encoded glycoproteins

J Gen Virol. 1998 Nov;79 ( Pt 11):2631-9. doi: 10.1099/0022-1317-79-11-2631.

Abstract

The three flavivirus glycoproteins prM, E and NS1 are formed by post-translational cleavage and are glycosylated by the addition of N-linked glycans. NS1 may form homodimers, whereas E may form homodimers, homotrimers or heterodimers (prM-E). Modification of these processes by mutagenesis of the proteins has the potential to generate viruses that are restricted in growth and are possible vaccine candidates. Using an SV40-based expression system, we previously analysed dimerization and secretion of the NS1 protein of dengue virus type 2 (DEN-2) with mutations in the conserved Cys residues, or within hydrophilic or hydrophobic regions, or at glycosylation sites. In this study, mutations which reduce cleavage at the DEN-2 prM/E signalase cleavage site are described. On the basis of earlier and current results with transient expression, six mutations which reduced NS1 dimerization and two mutations which inhibited prM/E cleavage were analysed individually for their effects on virus growth using a genomic length cDNA clone. Two viruses were obtained that showed reduced growth in cell culture and attenuation of neurovirulence when inoculated into 3-day-old mice. One of these viruses encoded NS1 that lacked the second glycosylation site, the other encoded a Ser --> Ile change at the -3 position of the prM/E cleavage site. A third virus encoding a mutation in NS1 within a hydrophilic region grew as well as the parental virus. No virus was detected for the remaining five mutations.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Dengue Virus / genetics
  • Dengue Virus / growth & development*
  • Gene Expression Regulation, Viral / physiology*
  • Genes, Viral*
  • Mice
  • Mutagenesis, Site-Directed
  • Point Mutation
  • Viral Nonstructural Proteins / physiology*

Substances

  • NS1 protein, Dengue virus type 2
  • Viral Nonstructural Proteins