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. 2007 Jun 26;104(26):11050-5.
doi: 10.1073/pnas.0700336104. Epub 2007 Jun 20.

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

Vernon K Ward  1 , Christopher J McCormick, Ian N Clarke, Omar Salim, Christiane E Wobus, Larissa B Thackray, Herbert W Virgin 4th, Paul R Lambden
Affiliations

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

Vernon K Ward et al. Proc Natl Acad Sci U S A. .

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Recovery of recombinant MNV in HepG2 and RAW 264.7 cells. (A–C) HepG2 cells were transduced with 100 pfu of BACtTA baculovirus (A), BACTET-MNV baculovirus (B), or both baculoviruses simultaneously (C) and observed 24 h after transduction. (D) Viral plaques of passaged recMNV (105-fold dilution) in RAW264.7 cells are shown at 24 h after infection. (E and F) Mock-infected RAW264.7 cells at 0 (E) and 24 h (F). (G and H) recMNV-infected RAW264.7 cells (MOI = 0.25) at 0 (G) and 24 h (H) after infection. (I–N) Expression of nonstructural and structural proteins in 20 h postinfection RAW 264.7 cells was tested by immunofluorescence of mock or infected cells with capsid (I and J), VPg (K and L), and N-term (M and N) monoclonal antibodies. (O and P) No primary antibody RAW264.7 or HEK293T cell controls. (Q–T) Expression of nonstructural and structural proteins in mock or pMNV* transfected HEK293T cells was tested by immunofluorescence with VPg (Q and R) and N-term (S and T) monoclonal antibodies. (U and V) No primary antibody pMNV* and pMNV*/pro–pol transfection controls. (W and X) Expression of VPg (W) and N-term (X) in HEK293T cells transfected with pro–pol cleavage mutation in pMNV*. Cell nuclei were stained with DAPI (I–X) and primary antibodies detected by fluorescence of goat anti-mouse FITC.
Fig. 2.
Fig. 2.
Detection of MNV in infected cells and recovery of marked recombinant MNV. (A) Immunoblot detection of MNV capsid protein in uninfected RAW264.7 cells (RAW) and recombinant MNV-1 infected RAW264.7 cells (recMNV-1). (B) Alignment of a 20-base region adjacent to nucleotide 1000 of the MNV genome. Sequences are derived from the fully sequenced MNV-1 (1; AY228235), MNV-2 (2; DQ223041), MNV-3 (3; DQ223042), MNV-4 (4; DQ223043), Berlin strain of MNV (B; DQ911368), and 12 other partial MNV sequences from a range of isolates, including nine sequences that are identical (9x). Bases that vary from the consensus of all 17 sequences are in bold. Base 1001 is boxed for all strains. The modification of base 1001 to an A to create an EcoRV restriction site (underlined) is indicated. The amino acid sequence in this region is shown. (C) Total RNA from RAW 264.7 cells infected with recombinant MNV-1 (rM) or recombinant MNV containing an introduced EcoRV site (M*) was subjected to RT-PCR of an 841-bp region encompassing the EcoRV mutation point. The resulting products were separated by agarose gel electrophoresis as uncut products (PCR) or digested with EcoRV before electrophoresis to detect the presence of an EcoRV site 167 bp from one end of the PCR product. No reverse transcriptase PCR (RT-) controls for both template RNA samples plus RT-PCR of an uninfected cellular RNA sample (U) are included as controls.
Fig. 3.
Fig. 3.
Expression and processing of N-term in transfected HEK293T cells. HEK293T cells were transfected with pMNV* (A) or pro–pol cleavage mutations (PPKO) in pFBTETMNV* or pMNV* (B) and subjected to Western blot analysis with N-term monoclonal antibody. Mock transfections were included as controls.
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References

    1. Mead PS, Slutsker L, Dietz V, McCaig LF, Bresee JS, Shapiro C, Griffin PM, Tauxe RV. Emerg Infect Dis. 1999;5:607–625. - PMC - PubMed
    1. Clarke IN, Lambden PR. In: Topley and Wilson's Microbiology and Microbial Infections: Virology. Mahy BWJ, ter Meulen W, editors. Vol 2. London: Hodder Arnold; 2005. pp. 911–931.
    1. Fankhauser RL, Noel JS, Monroe SS, Ando T, Glass RIM. J Infect Dis. 1998;178:1571–1578. - PubMed
    1. Green KY, Chanock RM, Kapikian AZ. In: Fields Virology. Knipe DM, Howley PM, editors. Philadelphia: Lippincott Williams & Wilkins; 2001. pp. 841–874.
    1. Hutson AM, Atmar RL, Estes MK. Trends Microbiol. 2004;12:279–287. - PMC - PubMed

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