title: VPg-primed RNA synthesis of norovirus RNA-dependent RNA polymerases by using a novel cell-based assay.
abstract: Molecular studies of human noroviruses (NoV) have been hampered by the lack of a permissive cell culture system. We have developed a sensitive and reliable mammalian cell-based assay for the human NoV GII.4 strain RNA-dependent RNA polymerase (RdRp). The assay is based on the finding that RNAs synthesized by transiently expressed RdRp can stimulate retinoic acid-inducible gene I (RIG-I)-dependent reporter luciferase production via the beta interferon promoter. Comparable activities were observed for the murine norovirus (MNV) RdRp. RdRps with mutations at divalent metal ion binding residues did not activate RIG-I signaling. Furthermore, both NoV and MNV RdRp activities were stimulated by the coexpression of their respective VPg proteins, while mutations in the putative site of nucleotide linkage on VPg abolished most of their stimulatory effects. Sequencing of the RNAs linked to VPg revealed that the cellular trans-Golgi network protein 2 (TGOLN2) mRNA was the template for VPg-primed RNA synthesis. Small interfering RNA knockdown of RNase L abolished the enhancement of signaling that occurred in the presence of VPg. Finally, the coexpression of each of the other NoV proteins revealed that p48 (also known as NS1-2) and VP1 enhanced and that VP2 reduced the RdRp activity. The assay should be useful for the dissection of the requirements for NoV RNA synthesis as well as the identification of inhibitors of the NoV RdRp.
authors: Department of Molecular and Cellular Biochemistry, 205C Simon Hall, 212 S. Hawthorne Drive, Indiana University, Bloomington, IN 47405, USA.
authors: Chennareddy V
authors: CV
authors: Subba-Reddy
authors: Ian
authors: I
authors: Goodfellow
authors: C Cheng
authors: CC
authors: Kao
mesh_terms: D019677:Artificial Gene Fusion; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D029322:Norovirus / Q000201:enzymology*; D011401:Promoter Regions, Genetic; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D014773:Virology / Q000379:methods*
chemical_list: D008156:Luciferases; D012324:RNA-Dependent RNA Polymerase