CHANGES IN THE DENGUE VIRUS MAJOR ENVELOPE PROTEIN ON PASSAGING AND THEIR LOCALIZATION ON THE 3-DIMENSIONAL STRUCTURE OF THE PROTEIN

To help define the molecular events involved in dengue virus adaptatio n during serial passage in vivo and in cultured cells, we have sequenc ed the structural protein genes of three dengue type 3 isolates after intracerebral passage in mice and after passage in cultured monkey kid ney (Vero) and Aedes albopictus (mosquito) cells. Passaging in each ho st selected for amino acid changes in the envelope protein E and occas ionally in prM but not in the capsid protein. Most changes were first apparent within five passages. Nineteen of twenty mutations in the str uctural protein genes resulted in amino acid changes concentrated on 1 2 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 in dependent Vero cell passage series, changes were observed in E at resi dues 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 inde pendent passage series in mosquito cells each resulted in the loss of a conserved glycosylation site al Asn153 in E. Passage in mouse brain selected for mutations at E residues 18, 54, 277, 401, and 403. Residu es which altered on passaging have been localized on the three-dimensi onal structure of the tick-borne encephalitis virus E protein soluble fragment (F. A. Rey, et al., 1995, Nature 375, 291-298). Residues 54, 191, 202, 266, 268, and 277 map to a postulated ''hinge'' region betwe en domains I and II which may be involved in fusion of flaviviruses wi th cell membranes. The oligosaccharide al Asn153 also appears to be in volved in flavivirus fusion. Changes in the fusion characteristics of the passaged viruses were demonstrated. (C) 1997 Academic Press.