Changes in the Dengue Virus Major Envelope Protein on Passaging and Their Localization on the Three-Dimensional Structure of the Protein

Abstract

To help define the molecular events involved in dengue virus adaptation during serial passagein vivoand in cultured cells, we have sequenced the structural protein genes of three dengue type 3 isolates after intracerebral passage in mice and after passage in cultured monkey kidney (Vero) andAedes albopictus(mosquito) cells. Passaging in each host selected for amino acid changes in the envelope protein E and occasionally in prM but not in the capsid protein. Most changes were first apparent within five passages. Nineteen of twenty mutations in the structural protein genes resulted in amino acid changes concentrated on 12 residues; 9 of the 12 amino acid changes were at residues which are conserved between the four dengue virus serotypes. Certain amino acid changes were repeatedly selected on passage in cell culture. In six independent Vero cell passage series, changes were observed in E at residues 191 (four times), 202 (twice), 266 and 268 (three times), and 291; change in prM was seen in two passage series at residue 26. Two independent passage series in mosquito cells each resulted in the loss of a conserved glycosylation site at Asn153 in E. Passage in mouse brain selected for mutations at E residues 18, 54, 277, 401, and 403. Residues which altered on passaging have been localized on the three-dimensional structure of the tick-borne encephalitis virus E protein soluble fragment (F. A. Rey,et al.,1995,Nature375, 291–298). Residues 54, 191, 202, 266, 268, and 277 map to a postulated “hinge” region between domains I and II which may be involved in fusion of flaviviruses with cell membranes. The oligosaccharide at Asn153 also appears to be involved in flavivirus fusion. Changes in the fusion characteristics of the passaged viruses were demonstrated.