The goal of this project is the identification of nonlethal mutations in the DEN virus noncoding and structural gene regions and their characterization in the context of the full-length infectious dengue-2 cDNA. (1) Membrane integration of the capsid (C) protein. In 1997, a paper was published in Virology (see Bibliography). (2) Interaction of the flavivirus C with other viral proteins during virion morphogenesis. Full-length chimeric D2 cDNAs containing appropriate mutations of the internal hydrophobic domain in the D2 C were constructed. The internal hydrophobic domain in C was shown to be strictly required for virus replication. Substitution of Arg/55 by hydrophobic aas, including Leu, was lethal in the context of D2 replication. We infer that Leu in the YF sequence context is required for interaction of C with another YF protein during virion morphogenesis.(3) Effects of mutations in the 3'-SL on replication of dengue-2 virus. Flavivirus genomic RNAs contain a conserved stem-loop structure (3'-SL) within the 3'-noncoding region. A mutant containing a substitution of the 3'-terminal 7 nt of the D2 sequence by analogous WN nts, was severely restricted for growth in mosquito cells. This is a desireable property of a live, attenuated vaccine virus, and this mutation is being incorporated into vaccine candidate mutant viruses. INDs exist for the development of new live, attenuated virus vaccines against several flaviviruses, alphaviruses, and arenaviruses. These are all positive-stranded RNA viruses that employ analogous mechanisms of replication involving the processing of a polycistronic protein or """"""""polyprotein"""""""" precursor. Our studies of possible attenuating mutations in the dengue virus genome aid in the critical analysis of these potential new products, as well as being relevant to the development of a live dengue virus vaccine in our laboratory and adding to scientific knowledge of the biology of flaviviruses. We are interested in determining the effects of mutations upon replication of dengue virus in the context of our full-length """"""""infectious"""""""" cDNA copy of the dengue-2 genome. In this way we hope to develop attenuated mutant viruses that might be useful in vaccine development and/or in understanding dengue virus virulence.
