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*

Shihyun You and R. Padmanabhan‡

J. Bio. Chem. 274, 33714-33722 (1999)



 

Abstract

  The mosquito-brone dengue viruses , members of positive strand RNA viruses of Flavivirus family , are human pathogens that cause dengue fever , dengue hemorrhagic fever(DHF)/dengue shock syndrome(DSS). 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 cell-free extracts of DEN2-infected mosquito (C6/36) cells and exogenous subgenomic RNA templates containing 5'- and/or 3'- terminal regions of the viral genome. The results indicate that there is an interaction between 5'- and 3'- terminal regions of the viral RNA that is required for RNA synthesis in vitro. This interaction is modulated by the complementary cyclization motifs. RNA synthesis occurs by the 3'- end elongation of the template RNA to yield a predominantly double-stranded RNA hairpin with a limited single-stranded loop region. The kinetics of the formation of the RNA hairpin product indicates that the template RNA is first modified yielding a RNase A-sensitive intermediate that is then converted to the hairpin product twice the size of the template RNA. This in vitro RdRP (RNA-dependent RNA polymerase) assay will be useful to study the sequence and protein requirements for RNA synthesis in vitro.


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