Saltar al contenido
Merck
  • The murine norovirus core subgenomic RNA promoter consists of a stable stem-loop that can direct accurate initiation of RNA synthesis.

The murine norovirus core subgenomic RNA promoter consists of a stable stem-loop that can direct accurate initiation of RNA synthesis.

Journal of virology (2014-11-14)
Muhammad Amir Yunus, Xiaoyan Lin, Dalan Bailey, Ioannis Karakasiliotis, Yasmin Chaudhry, Surender Vashist, Guo Zhang, Lucy Thorne, C Cheng Kao, Ian Goodfellow
RESUMEN

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. 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.

MATERIALES
Referencia del producto
Marca
Descripción del producto

Sigma-Aldrich
Glicerol, ACS reagent, ≥99.5%
Sigma-Aldrich
Glicerol, for molecular biology, ≥99.0%
Sigma-Aldrich
HEPES, ≥99.5% (titration)
Sigma-Aldrich
Glicerol, ReagentPlus®, ≥99.0% (GC)
Sigma-Aldrich
HEPES, BioPerformance Certified, ≥99.5% (titration), suitable for cell culture
Sigma-Aldrich
Imidazol, ReagentPlus®, 99%
Sigma-Aldrich
Cloruro de sodio, for molecular biology, DNase, RNase, and protease, none detected, ≥99% (titration)
Sigma-Aldrich
Magnesium chloride solution, for molecular biology, 1.00 M±0.01 M
Sigma-Aldrich
Magnesium chloride, anhydrous, ≥98%
Sigma-Aldrich
DL-Ditiotreitol solution, BioUltra, for molecular biology, ~1 M in H2O
Supelco
DL-Ditiotreitol solution, 1 M in H2O
Sigma-Aldrich
Sodium chloride solution, 0.9% in water, BioXtra, suitable for cell culture
Sigma-Aldrich
Sodium chloride solution, 5 M in H2O, BioReagent, for molecular biology, suitable for cell culture
Sigma-Aldrich
Cloruro de sodio, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99%
Sigma-Aldrich
Imidazol, for molecular biology, ≥99% (titration)
Sigma-Aldrich
HEPES, BioUltra, for molecular biology, ≥99.5% (T)
SAFC
Sodium chloride solution, 5 M
Sigma-Aldrich
Glicerol solution, 83.5-89.5% (T)
Sigma-Aldrich
HEPES buffer solution, 1 M in H2O
Sigma-Aldrich
Imidazole buffer Solution, BioUltra, 1 M in H2O
Sigma-Aldrich
Glicerol, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for electrophoresis, ≥99% (GC)
Sigma-Aldrich
Glicerol, BioUltra, for molecular biology, anhydrous, ≥99.5% (GC)
Sigma-Aldrich
Glicerol, puriss., anhydrous, 99.0-101.0% (alkalimetric)
Sigma-Aldrich
Magnesium chloride, powder, <200 μm
Sigma-Aldrich
Imidazol, BioUltra, ≥99.5% (GC)
SAFC
HEPES
USP
Glicerol, United States Pharmacopeia (USP) Reference Standard
Sigma-Aldrich
Sodium chloride solution, BioUltra, for molecular biology, ~5 M in H2O
Sigma-Aldrich
Cloruro de sodio, 99.999% trace metals basis
Sigma-Aldrich
Glicerol, FCC, FG