title: Analyses of mutations selected by passaging a chimeric flavivirus identify mutations that alter infectivity and reveal an interaction between the structural proteins and the nonstructural glycoprotein NS1.
abstract: We previously described a single-cycle dengue vaccine (RepliVAX D2) engineered from a capsid (C) gene-deleted West Nile virus (WNV) expressing dengue virus serotype 2 (DENV2) prM/E genes in place of the corresponding WNV genes. That work demonstrated that adaptation of RepliVAX D2 to grow in WNV C-expressing cells resulted in acquisition of non-synonymous mutations in the DENV2 prM/E and WNV NS2A/NS3 genes. Here we demonstrate that the prM/E mutations increase the specific infectivity of chimeric virions and the NS2A/NS3 mutations independently enhance packaging. Studies with the NS2A mutant demonstrated that it was unable to produce a larger form of NS1 (NS1'), suggesting that the mutation had been selected to eliminate a ribosomal frame-shift "slippage site" in NS2A. Evaluation of a synonymous mutation at this slippage site confirmed that genomes that failed to make NS1' were packaged more efficiently than WT genomes supporting a role for NS1/NS1' in orchestrating virion assembly.
authors: Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0436, USA.
authors: Evandro R
authors: ER
authors: Winkelmann
authors: Douglas G
authors: DG
authors: Widman
authors: Ryosuke
authors: R
authors: Suzuki
authors: Peter W
authors: PW
authors: Mason
mesh_terms: D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003716:Dengue Virus / Q000235:genetics* / Q000472:pathogenicity / Q000502:physiology*; D018965:Frameshifting, Ribosomal; D009154:Mutation; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000378:metabolism*; D019065:Virus Assembly / Q000235:genetics*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D020365:RNA Helicases; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; D015678:Viral Structural Proteins; C100315:NS1 protein, Flavivirus; C079341:NS2A protein, Dengue virus type 2; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases