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Journal of Virology, October 2008, p. 9306-9317, Vol. 82, No. 19
0022-538X/08/$08.00+0     doi:10.1128/JVI.00301-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Leader of the Capsid Protein in Feline Calicivirus Promotes Replication of Norwalk Virus in Cell Culture{triangledown}

Kyeong-Ok Chang,1* David W. George,1 John B. Patton,1 Kim Y. Green,2 and Stanislav V. Sosnovtsev2

Kansas State University, Manhattan, Kansas 66506,1 National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland 208922

Received 11 February 2008/ Accepted 10 July 2008

The inability to grow human noroviruses in cell culture has greatly impeded the studies of their pathogenesis and immunity. Vesiviruses, in the family Caliciviridae, grow efficiently in cell culture and encode a unique protein in the subgenomic region designated as leader of the capsid protein (LC). We hypothesized that LC might be associated with the efficient replication of vesiviruses in cell culture and promote the replication of human norovirus in cells. To test this hypothesis, a recombinant plasmid was engineered in which the LC region of feline calicivirus (FCV) was placed under the control of the cytomegalovirus promoter (pCI-LC) so that the LC protein could be provided in trans to replicating calicivirus genomes bearing a reporter gene. We constructed pNV-GFP, a recombinant plasmid containing a full-length NV genome with a green fluorescent protein (GFP) in the place of VP1. The transfection of pNV-GFP in MVA-T7-infected cells produced few GFP-positive cells detected by fluorescence microscopy and flow cytometry analysis. When pNV-GFP was cotransfected with pCI-LC in MVA-T7-infected cells, we observed an increase in the number of GFP-positive cells (ca. 3% of the whole-cell population). Using this cotransfection method with mutagenesis study, we identified potential cis-acting elements at the start of subgenomic RNA and the 3' end of NV genome for the virus replication. We conclude that LC may be a viral factor which promotes the replication of NV in cells, which could provide a clue to growing the fastidious human noroviruses in cell culture.

* Corresponding author. Mailing address: Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, 1800 Denison Ave., Manhattan, KS 66506. Phone: (785) 532-3849. Fax: (785) 532-4039. E-mail: kchang{at}vet.ksu.edu

{triangledown} Published ahead of print on 16 July 2008.

Journal of Virology, October 2008, p. 9306-9317, Vol. 82, No. 19
0022-538X/08/$08.00+0     doi:10.1128/JVI.00301-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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