| Flaviviruses are mosquito- and tick-transmitted RNA viruses that cause epidemic-scale human illnesses, such as hemorrhagic fever and acute encephalitis. For many of these viruses including yellow fever virus (YFV), dengue viruses, Japanese encephalitis virus, West Nile virus, and the tick-borne encephalitis virus either vaccines, or antiviral therapies are needed.;The envelope (E) protein of these viruses is a critical protein in the virus life cycle, and a key virulence factor during viral pathogenesis. Despite considerable understanding of the 3-dimensional structure of this protein and abundant evidence that mutations in the protein can modulate virulence properties, the functional effects of virulence determinants on the properties of this viral protein during interaction with host cells remain largely uncharacterized. To approach this question, we derived mutants of YFV by passage of several different YF17D strains on mouse neuroblastoma (NB41A3) cells and selected for small-plaque variants to identify the molecular determinants of their altered phenotype. All variant viruses contained aspartic acid or glutamic acid instead of glycine at locus 360--362 in the putative receptor-binding region (Domain III) of the E protein, which conferred upon the virus defects in growth kinetics, cell attachment, penetration, and spread through cell monolayers, but showed no difference from parental virus in limited studies of mouse neurovirulence. Further analysis of position 360 by site-directed mutagenesis revealed that the locus is tolerant of other amino acids with neutral and/or negative charge, but most of them were deleterious for the virus. These defects could be compensated in part by one or more second-site suppressing mutations in domain II and/or domain III, as revealed by genetic analysis of plaque-revertant viruses. These studies indicate that mutations at locus E360--362 affect multiple properties of YF 17D virus in cell culture, presumably through coordinated interactions among different regions of the protein during cell-attachment and penetration that depend on a negative charge at E360. These studies increase our understanding of the virus-cell interactions at the level of virus entry, and may be of benefit to the design of live-attenuated viral vaccines using genetic engineering. |
