title: A novel in vitro replication system for Dengue virus. Initiation of RNA synthesis at the 3'-end of exogenous viral RNA templates requires 5'- and 3'-terminal complementary sequence motifs of the viral RNA.
abstract: Positive strand viral replicases are membrane-bound complexes of viral and host proteins. The mechanism of viral replication and the role of host proteins are not well understood. To understand this mechanism, a viral replicase assay that utilizes extracts from dengue virus-infected mosquito (C6/36) cells and exogenous viral RNA templates is reported in this study. The 5'- and 3'-terminal regions (TR) of the template RNAs contain the conserved elements including the complementary (cyclization) motifs and stem-loop structures. RNA synthesis in vitro requires both 5'- and 3'-TR present in the same template molecule or when the 5'-TR RNA was added in trans to the 3'-untranslated region (UTR) RNA. However, the 3'-UTR RNA alone is not active. RNA synthesis occurs by elongation of the 3'-end of the template RNA to yield predominantly a double-stranded hairpin-like RNA product, twice the size of the template RNA. These results suggest that an interaction between 5'- and 3'-TR of the viral RNA that modulates the 3'-UTR RNA structure is required for RNA synthesis by the viral replicase. The complementary cyclization motifs of the viral genome also seem to play an important role in this interaction.
authors.affiliation: Department of Biochemistry, University of Kansas Medical Center, Kansas City, Kansas 66160-7421, USA.
authors.forename: S
authors.initials: S
authors.lastname: You
authors.forename: R
authors.initials: R
authors.lastname: Padmanabhan
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D009033:Culicidae / Q000821:virology; D017931:DNA Primers; D003716:Dengue Virus / Q000235:genetics / Q000502:physiology*; D007700:Kinetics; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D013698:Templates, Genetic; D014779:Virus Replication
chemical_list: D020413:3' Untranslated Regions; D017931:DNA Primers; D012367:RNA, Viral