title: Growth restriction of dengue virus type 2 by site-specific mutagenesis of virus-encoded glycoproteins.
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.
authors.affiliation: Department of Microbiology, Monash University, Clayton, Victoria, Australia.
authors.forename: M J
authors.initials: MJ
authors.lastname: Pryor
authors.forename: R C
authors.initials: RC
authors.lastname: Gualano
authors.forename: B
authors.initials: B
authors.lastname: Lin
authors.forename: A D
authors.initials: AD
authors.lastname: Davidson
authors.forename: P J
authors.initials: PJ
authors.lastname: Wright
mesh_terms: D000818:Animals; D003716:Dengue Virus / Q000235:genetics / Q000254:growth & development*; D015967:Gene Expression Regulation, Viral / Q000502:physiology*; D005814:Genes, Viral; D051379:Mice; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017361:Viral Nonstructural Proteins / Q000502:physiology*
chemical_list: C079340:NS1 protein, Dengue virus type 2; D017361:Viral Nonstructural Proteins