Recovery of infectious murine norovirus using pol II-driven expression of full-length cDNA

doi:10.1073/pnas.0700336104

Recovery of infectious murine norovirus using pol II-driven expression of full-length cDNA

^†Department of Microbiology and Immunology, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand;
^‡Molecular Microbiology Group, University of Southampton Medical School, Southampton General Hospital, Southampton SO16 6YD, United Kingdom; and
^¶Departments of Pathology and Immunology and
^‖Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110

Edited by Mary K. Estes, Baylor College of Medicine, Houston, TX, and accepted by the Editorial Board May 16, 2007 (received for review January 12, 2007)

Abstract

Noroviruses are the major cause of nonbacterial gastroenteritis in humans. These viruses have remained refractory to detailed molecular studies because of the lack of a reverse genetics system coupled to a permissive cell line for targeted genetic manipulation. There is no permissive cell line in which to grow infectious human noroviruses nor an authentic animal model that supports their replication. In contrast, murine norovirus (MNV) offers a tractable system for the study of noroviruses with the recent discovery of permissive cells and a mouse model. The lack of a reverse genetic system for MNV has been a significant block to understanding the biology of noroviruses. We report recovery of infectious MNV after baculovirus delivery of viral cDNA to human hepatoma cells under the control of an inducible DNA polymerase (pol) II promoter. Recovered virus replicated in murine macrophage (RAW264.7) cells, and the recovery of MNV from DNA was confirmed through recovery of virus containing a marker mutation. This pol II promoter driven expression of viral cDNA also generated infectious virus after transfection of HEK293T cells, thus providing both transduction and transfection systems for norovirus reverse genetics. We used norovirus reverse genetics to demonstrate by mutagenesis of the protease–polymerase (pro–pol) cleavage site that processing of pro–pol is essential for the recovery of infectious MNV. This represents the first infectious reverse genetics system for a norovirus, and should provide approaches to address fundamental questions in norovirus molecular biology and replication.

Footnotes

^§To whom correspondence should be addressed. E-mail: vernon.ward{at}stonebow.otago.ac.nz
Author contributions: V.K.W. and C.J.M. contributed equally to this work; V.K.W., C.J.M., I.N.C., O.S., L.B.T., and P.R.L. designed research; V.K.W., C.J.M., O.S., C.E.W., L.B.T., and P.R.L. performed research; C.J.M. and H.W.V. contributed new reagents/analytic tools; V.K.W., C.J.M., I.N.C., O.S., C.E.W., L.B.T., H.W.V., and P.R.L. analyzed data; and V.K.W., C.J.M., I.N.C., O.S., C.E.W., L.B.T., H.W.V., and P.R.L. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. M.K.E. is a guest editor invited by the Editorial Board.
This article contains supporting information online at www.pnas.org/cgi/content/full/0700336104/DC1.
Abbreviations:

CPE,

cytopathic effect;

HδV,

hepatitis delta virus;

HepG2,

human hepatocellular carcinoma cell line G2;

MNV,

murine norovirus;

pol,

DNA polymerase;

pro,

viral protease;

pol,

viral polymerase;

RACE,

rapid amplification of cDNA ends;

RAW264.7,

Murine macrophage cell line 264.7;

Sf9,

Spodoptera frugiperda cell line Sf9;

VPg,

MNV viral protein genomic.