Abstract
The replication enzyme of RNA viruses must preferentially recognize their RNAs in an environment that contains an abundance of cellular RNAs. The factors responsible for specific RNA recognition are not well understood, in part because viral RNA synthesis takes place within enzyme complexes associated with modified cellular membrane compartments. Recombinant RNA-dependent RNA polymerases (RdRps) from the human norovirus and the murine norovirus (MNV) were found to preferentially recognize RNA segments that contain the promoter and a short template sequence for subgenomic RNA synthesis. Both the promoter and template sequence contribute to stable RdRp binding, accurate initiation of the subgenomic RNAs and efficient RNA synthesis. Using a method that combines RNA crosslinking and mass spectrometry, residues near the template channel of the MNV RdRp were found to contact the hairpin RNA motif. Mutations in the hairpin contact site in the MNV RdRp reduced MNV replication and virus production in cells. This work demonstrates that the specific recognition of the norovirus subgenomic promoter is through binding by the viral RdRp.
Highlights
Noroviruses are the major causative agents of nonbacterial epidemic gastroenteritis worldwide [1,2,3,4]
We demonstrate that chemically synthesized RNAs that contain the putative subgenomic core promoter and a short template derived from the GII.4 human norovirus or murine norovirus (MNV) can accurately and direct the initiation of RNA synthesis by the respective recombinant RNA-dependent RNA polymerases (RdRps) of the two viruses
Norovirus RdRps can synthesize RNA from their subgenomic core promoters We hypothesized that the norovirus RdRp is the component in the replicase complex that recognizes core promoters for subgenomic RNA (sgRNA) viral RNA synthesis
Summary
Noroviruses are the major causative agents of nonbacterial epidemic gastroenteritis worldwide [1,2,3,4]. The majority of noroviruses express three conserved open reading frames named ORF1–3 while MNV has an additional ORF4 [12,13]. ORF1 encodes a polyprotein which is processed by proteolysis to generate six non-structural (NS) proteins that replicate and transcribe the viral genome. These include NS7, the RNA-dependent RNA polymerase (RdRp) [14]. ORF4 of MNV is translated from an alternative reading frame within ORF2 to produce virulence factor 1 that targets innate immune responses [13]. The proteins from ORF1 are translated from the norovirus genomic RNA. The proteins from ORF2, ORF3 and ORF4 are translated from one polyadenylated RNA of ca. The proteins from ORF2, ORF3 and ORF4 are translated from one polyadenylated RNA of ca. 2.5 kb in length, the subgenomic RNA (sgRNA) [12,13]
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