The murine norovirus core subgenomic RNA promoter consists of a stable stem-loop that can direct accurate initiation of RNA synthesis.
All members of the Caliciviridae family of viruses produce a subgenomic RNA during infection. The subgenomic RNA typically encodes only the major and minor capsid proteins, but in murine norovirus (MNV), the subgenomic RNA also encodes the VF1 protein, which functions to suppress host innate immune responses. To date, the mechanism of norovirus subgenomic RNA synthesis has not been characterized. We have previously described the presence of an evolutionarily conserved RNA stem-loop structure on the negative-sense RNA, the complementary sequence of which codes for the viral RNA-dependent RNA polymerase (NS7). The conserved stem-loop is positioned 6 nucleotides 3' of the start site of the subgenomic RNA in all caliciviruses. We demonstrate that the conserved stem-loop is essential for MNV viability. Mutant MNV RNAs with substitutions in the stem-loop replicated poorly until they accumulated mutations that revert to restore the stem-loop sequence and/or structure. The stem-loop sequence functions in a noncoding context, as it was possible to restore the replication of an MNV mutant by introducing an additional copy of the stem-loop between the NS7- and VP1-coding regions. Finally, in vitro biochemical data suggest that the stem-loop sequence is sufficient for the initiation of viral RNA synthesis by the recombinant MNV RNA-dependent RNA polymerase, confirming that the stem-loop forms the core of the norovirus subgenomic promoter.


OBJECTIVE
Noroviruses are a significant cause of viral gastroenteritis, and it is important to understand the mechanism of norovirus RNA synthesis. Here we describe the identification of an RNA stem-loop structure that functions as the core of the norovirus subgenomic RNA promoter in cells and in vitro. This work provides new insights into the molecular mechanisms of norovirus RNA synthesis and the sequences that determine the recognition of viral RNA by the RNA-dependent RNA polymerase.
Section of Virology, Faculty of Medicine, Imperial College London, London, United Kingdom Advanced Medical & Dental Institute, Universiti Sains Malaysia, Bertam, Pulau Pinang, Malaysia.
Muhammad Amir
MA
Yunus
Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, USA.
Xiaoyan
X
Lin
Section of Virology, Faculty of Medicine, Imperial College London, London, United Kingdom School of Immunity and Infection, University of Birmingham, Birmingham, United Kingdom.
Dalan
D
Bailey
Section of Virology, Faculty of Medicine, Imperial College London, London, United Kingdom Laboratory of Molecular Virology, Hellenic Pasteur Institute, Athens, Greece.
Ioannis
I
Karakasiliotis
Division of Virology, Department of Pathology, University of Cambridge, Addenbrookes Hospital, Cambridge, United Kingdom.
Yasmin
Y
Chaudhry
Division of Virology, Department of Pathology, University of Cambridge, Addenbrookes Hospital, Cambridge, United Kingdom.
Surender
S
Vashist
Section of Virology, Faculty of Medicine, Imperial College London, London, United Kingdom.
Guo
G
Zhang
Division of Virology, Department of Pathology, University of Cambridge, Addenbrookes Hospital, Cambridge, United Kingdom.
Lucy
L
Thorne
Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, USA.
C Cheng
CC
Kao
Section of Virology, Faculty of Medicine, Imperial College London, London, United Kingdom Division of Virology, Department of Pathology, University of Cambridge, Addenbrookes Hospital, Cambridge, United Kingdom ig299@cam.ac.uk.
Ian
I
Goodfellow
D000818:Animals; D002460:Cell Line; D008264:Macrophages / Q000821:virology; D051379:Mice; D050296:Microbial Viability; D029322:Norovirus / Q000235:genetics* / Q000502:physiology*; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D012367:RNA, Viral / Q000096:biosynthesis* / Q000737:chemistry*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism; D014779:Virus Replication
D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase