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.
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.
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Department of Biochemistry, University of Kansas Medical Center, Kansas City, Kansas 66160-7421, USA.
Padmanabhan
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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
D020413:3' Untranslated Regions; D017931:DNA Primers; D012367:RNA, Viral