In vitro RNA synthesis from exogenous dengue viral RNA templates requires long range interactions between 5'- and 3'-terminal regions that influence RNA structure.
Viral replicases of many positive-strand RNA viruses are membrane-bound complexes of cellular and viral proteins that include viral RNA-dependent RNA polymerase (RdRP). The in vitro RdRP assay system that utilizes cytoplasmic extracts from dengue viral-infected cells and exogenous RNA templates was developed to understand the mechanism of viral replication in vivo. Our results indicated that in vitro RNA synthesis at the 3'-untranslated region (UTR) required the presence of the 5'-terminal region (TR) and the two cyclization (CYC) motifs suggesting a functional interaction between the TRs. In this study, using a psoralen-UV cross-linking method and an in vitro RdRP assay, we analyzed structural determinants for physical and functional interactions. Exogenous RNA templates that were used in the assays contained deletion mutations in the 5'-TR and substitution mutations in the 3'-stem-loop structure including those that would disrupt the predicted pseudoknot structure. Our results indicate that there is physical interaction between the 5'-TR and 3'-UTR that requires only the CYC motifs. RNA synthesis at the 3'-UTR, however, requires long range interactions involving the 5'-UTR, CYC motifs, and the 3'-stem-loop region that includes the tertiary pseudoknot structure.
You
S
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Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160-7421, USA.
Falgout
B
B
Markoff
L
L
Padmanabhan
R
R
D020413:3' Untranslated Regions / Q000737:chemistry* / Q000235:genetics; D020121:5' Untranslated Regions / Q000737:chemistry* / Q000235:genetics; D000330:Aedes; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003593:Cytoplasm / Q000821:virology; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D003716:Dengue Virus / Q000235:genetics*; D011564:Furocoumarins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D017384:Sequence Deletion; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014466:Ultraviolet Rays; D014764:Viral Proteins; D014902:West Nile virus / Q000235:genetics
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D011564:Furocoumarins; D012367:RNA, Viral; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases