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.
Highlights
Positive strand viral replicases are membrane-bound complexes of viral and host proteins
Cytoplasmic Extracts from Dengue virus type 2 (DEN2)-infected Mosquito (C6/36) Cells Are Active in the Synthesis of RNA from the Exogenous Templates—Previous studies showed that flavivirus RNA-dependent RNA polymerases (RdRP) activity was tightly associated with intracellular membranes in the cytoplasmic fractions of flavivirus-infected mammalian (monkey kidney cell line (Vero), or baby hamster kidney cell line (BHK-21)) cells [37,38,39,40, 62, 63]
We describe the first flaviviral replicase assay system that utilizes cytoplasmic extracts from dengue virusinfected mosquito (C6/36) cells and exogenously added subgenomic RNA templates to study the mechanism of viral replication in vitro
Summary
INITIATION OF RNA SYNTHESIS AT THE 3Ј-END OF EXOGENOUS VIRAL RNA TEMPLATES REQUIRES 5Ј- AND 3Ј-TERMINAL COMPLEMENTARY SEQUENCE MOTIFS OF THE VIRAL RNA*. Template-dependent and template-specific in vitro replication systems have been developed to study mechanisms of some plant and a few eukaryotic positive strand RNA viruses The in vitro RdRP assays that have been developed to study flavivirus replication utilize membrane-bound complexes isolated from the infected cell lysates [37,38,39,40] These studies have examined incorporation of radiolabeled nucleotides into the three RNA species on endogenous viral RNA templates. To study the mechanism of viral replication in molecular detail, it is crucial to develop an in vitro RdRP assay that can utilize exogenous RNA templates containing essential regulatory elements of the viral genome involved in viral replication. For initiation of (Ϫ)- and (ϩ)-strand RNA synthesis, conserved RNA sequences with intrinsic stem-loop structures from the 3Ј
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