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 94-95, we demonstrated that the flavivirus C contains a conserved internal 20-aa hydrophobic domain which functions as a signal-anchor (SA) to mediate its membrane integration. This year, a MS was submitted to J Virol, and it was returned for revision. Additional experiments performed in 95-96 included the demonstration that C was membrane inserted in vivo by IFA for C and an ER resident protein, BiP. The MS is to be re-submitted. (2) Interaction of the flavivirus C with other viral proteins during virion morphogenesis. The conserved internal hydrophobic domain in C contains an interrupting hydrophilic residue (Lys or Arg) in all published flavivirus sequences except for that of yellow fever virus (YFV), which contains Leu in the position usually occupied by Lys or Arg (aa 55 in the D2 sequence). In vitro studies of membrane integration of the D4 C, revealed that substitution of Met for the conserved Arg (aa 54 for D4) had no detectable effect on membrane integration of C. However, replacement of D2 Arg-55 by Met or Leu was lethal for infectivity of RNAs derived from full-length D2 cDNAs containing these mutations. (Abrogation of the hydrophobic domain in C by substitution mutation was also lethal in the context of the full-length D2 cDNA, showing that membrane integration of C is an essential step in virus replication.) We hypothesize that the conserved hydrophilic residue within the hydrophobic segment in the flavivirus C is required for an interaction of C with other viral proteins, most likely the structural proteins prM and/or E, during virion morphogenesis. To test this hypothesis initially, full-length chimeric D2 cDNAs containing the Arg-55/Leu substitution mutation and the entire YFV prM and/or E, instead of the D2 prM and/or E, are being constructed. Initially, we will determine whether RNAs derived from these chimeric cDNAs are infectious. If so, we would infer that Leu in the YF sequence context is required for interaction of C with the YF prM and/or E during virion morphogenesis. It should then be possible to map the locus of this interaction within the prM and/or E sequence. (3) Effects of mutations in the 3'-SL on replication of dengue-2 virus. Flavivirus genomic RNAs contain a conserved stem-loop structure within the 3'-noncoding region (3'-SL). Existing data suggest that the 3'-SL is required for RNA replication. We constructed a chimeric full-length cDNA in which the D2 3'-SL was replaced by the West Nile virus (WNV) 3'-SL. The chimeric D2/WN mutant virus was markedly defective for growth in vitro, showing that nucleotide sequence differences between the D2 and the WN 3'-SLs were significant for D2 replication. A set of chimeric mutants was then constructed in order to demonstrate that a small 4-bp region within the D2 3'-SL was essential for near wt growth kinetics.
