Identification of amino acids within norovirus polymerase involved in RNA binding and viral replication.
Han, Kang Rok
Alhatlani, Bader Y
Kim, Kyung Hyun
Yang, Jai Myung
J Gen Virol
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Han, K. R., Alhatlani, B. Y., Cho, S., Lee, J., Hosmillo, M., Goodfellow, I., Kim, K. H., & et al. (2017). Identification of amino acids within norovirus polymerase involved in RNA binding and viral replication.. J Gen Virol, 98 (6), 1311-1315. https://doi.org/10.1099/jgv.0.000826
Until recently, molecular studies on human norovirus (HuNoV), a major causative agent of gastroenteritis, have been hampered by the lack of an efficient cell culture system. Murine norovirus-1 (MNV-1) has served as a surrogate model system for norovirus research, due to the availability of robust cell culture systems and reverse genetics. To identify amino acids involved in RNA synthesis by the viral RNA-dependent RNA polymerase (NS7), we constructed NS7 mutants in which basic amino acids surrounding the catalytic site were substituted with alanine. Electrophoretic mobility shift assay revealed that these residues are important for RNA binding, particularly R396. Furthermore, in vitro RNA synthesis and reverse genetics were used to identify conserved amino acids essential for RNA synthesis and viral replication. These results provide additional functional insights into highly conserved amino acids in NS7 and provide potential methods of rational attenuation of norovirus replication.
Cell Line, Animals, Norovirus, Amino Acids, RNA, Viral, Electrophoretic Mobility Shift Assay, Amino Acid Substitution, Mutagenesis, Site-Directed, DNA Mutational Analysis, Virus Replication, Binding Sites, Protein Binding, Cricetinae, Reverse Genetics, RNA-Dependent RNA Polymerase
This study was supported by grants from the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs (A085119) and Basic Science Research Programs through NRF funded by the Ministry of Education (NRF-2013R1A1A2064940, L.J.-H. and NRF-2016R1A6A3A01012238, K.R.H.). BA was supported by funding from Qassim University, Saudi Arabia, and the work in the lab of IG is supported by the Wellcome Trust (097997/Z/11/Z). K.R.H. was a recipient of postdoctoral fellowship from the BK21+ program. IG is a Wellcome Senior Fellow.
Wellcome Trust (097997/Z/11/Z)
External DOI: https://doi.org/10.1099/jgv.0.000826
This record's URL: https://www.repository.cam.ac.uk/handle/1810/265892