room temperature. Cells were permeabilised and blocked in saponin buffer, also used as staining buffer, (0.1% saponin, 10% foetal calf serum, 0.1% sodium azide) for 1 hr at 4uC. Cells were stained using an anti-V5 monoclonal antibody (Invitrogen) followed by an anti-mouse Alexafluor 488 conjugated secondary antibody (Molecular Probes) at the recommended dilution in staining buffer for 30 min in the dark. Cells were then stained for 30 min in the dark with a Wheat Germ Agglutinin Alexafluor 594 nm conjugate (Molcular Probes) to allow identification of plasma and Golgi membranes. Coverslips were washed and mounted onto slides using Vectashield containing DAPI (Vector Labs). Microscopy was performed using an inverted Leica TCS- NT confocal laser scanning microscope. The anti-V5 antibody was also used to detect V5-tagged protein by Western blot. Cell lysates were prepared following transfection using lysis buffer (0.15 M sodium chloride, 0.5% (v/v) sodium deoxycholate, 0.1% (w/v) SDS, 50 mM Tris-Cl pH 8.0) and protease inhibitor cocktail (Sigma). Lysates were incubated for 15 min on ice followed by sonication to shear genomic DNA. Following Bradford analysis equal protein content from JV V5 and JV FLC lysates were run on a 10% SDS-PAGE gel and subsequently transferred onto Immobilon-P PVDF membrane (Millipore) according to the manufacturer’s recommendations. References 1. Gu¨nther H, Otto P, Heilman P (1984) Studies into diarrhoea of young calves. Sixth communication: Detection and determination of pathogenicity of a bovine Corona virus and an undefined icosahedric virus. Archives of experimental Veterinary medicine, Leipzig 38: 781–792. 2. Gu¨ nther H, Otto P (1987) Studies into diarrhoea of young calves-Seventh Communication: ‘‘Zackenvirus’’ (Jena-Agens 117/80)-A new diarrhoea pathogen to calf. Archives of experimental Veterinary medicine, Leipzig 41: 934–938. 3. Oliver SL, Asobayire E, Charpilienne A, Cohen J, Bridger JC (2007) Complete genomic characterization and antigenic relatedness of genogroup III, genotype 2 bovine noroviruses. Arch Virol 152: 257–272. 4. Oliver SL, Batten CA, Deng Y, Elschner M, Otto P, Charpilienne A, Clarke IN, Bridger JC, Lambden PR (2006) Genotype 1 and Genotype 2 Bovine Noroviruses Are Antigenically Distinct but Share a Cross-Reactive Epitope with Human Noroviruses. J Clin Microbiol 44: 992–998. 5. Bridger JC, Hall GA, Brown JF (1984) Characterization of a calici-like virus (Newbury agent) found in association with astrovirus in bovine diarrhea. Infect Immun 43: 133–138. 6. Pelosi E, Lambden PR, Caul EO, Liu BL, Dingle KE, Deng Y, Clarke IN (1999) The Seroepidemiology of genogroup 1 and genogroup 2 Norwalk-like viruses in Italy. J Med Virol 58: 93–99. 7. Liu BL, Lambden PR, Gu¨nther H, Otto P, Elschner M, Clarke IN (1999) Molecular characterization of a bovine enteric calicivirus: relationship to the Norwalk-like viruses. J Virol 73: 819–825. 8. Glass PJ, Zeng CQ, Estes MK (2003) Two nonoverlapping domains on the Norwalk virus open reading frame 3 (ORF3) protein are involved in the formation of the phosphorylated 35K protein and in ORF3-capsid protein interactions. J Virol 77: 3569–3577. 9. Sosnovtsev SV, Garfield M, Green KY (2002) Processing map and essential cleavage sites of the nonstructural polyprotein encoded by ORF1 of the feline calicivirus genome. J Virol 76: 7060–7072. 10. Meyers G, Wirblich C, Thiel H-J, Thumfart JO (2000) Rabbit Hemorrhagic Disease Virus: Genome Organisation and Polyprotein Processing of a Calicivirus Studied after Transient Expression of cDNA Constructs. Virology 276: 349–363. 11. Clarke IN, Lambden PR (2000) Organisation and Expression of Calicivirus Genes. J Infect Dis 181: S309–S316. 12. Sosnovtsev SV, Belliot G, Chang KO, Prikhodko VG, Thackray LB, Wobus CE, Karst SM, Virgin HW, Green KY (2006) Cleavage map and proteolytic processing of the murine norovirus nonstructural polyprotein in infected cells. J Virol 80: 7816–7831. 13. Hardy ME (2005) Norovirus protein structure and function. FEMS Microbiol Lett 253: 1–8. 14. Seah EL, Marshall JA, Wright PJ (1999) Open reading frame 1 of the Norwalk- like virus Camberwell: Completion of sequence and expression in mammalian cells. J Virol 73: 10531–10535. 15. Fernandez-Vega V, Sosnovtsev SV, Belliot G, King AD, Mitra T, Gorbalenya A, Green KY (2004) Norwalk virus N-terminal nonstructural protein is associated Analysis of the 59 GS of JV The membrane was probed using the anti-V5 monoclonal antibody at the manufacturer’s recommended dilution, followed by an anti-mouse HRP-copnjugated secondary antibody (Santa Cruz) at the recommended diltution. The ECL Western blotting reagents kit (G.E. Healthcare) was used to detect antibody bound protein, which was visualised by exposure to BioMax Light film (Kodak). Degradation analysis CRFK cells were seeded into 6 well trays and transfected with capped JV V5 RNA as described. Following a 24 hr incubation cycloheximide (Sigma) was added to the cells at a final concentration of 50 mg/ml. Cells were harvested at indicated times for the preparation of lysates for V5 Western analysis as described above. Bradford reagent (Sigma) was used to ensure equal loading of lysates according to the manufacturer’s recommendations. Author Contributions Conceived and designed the experiments: IC OS PL. Performed the experiments: OS. Analyzed the data: IC OS PL. Wrote the paper: IC OS PL. with disassembly of the Golgi complex in transfected cells. J Virol 78: 4827–4837. 16. Ettayebi K, Hardy ME (2003) Norwalk virus nonstructural protein p48 forms a complex with the SNARE regulator VAP-A and prevents cell surface expression of vesicular stomatitis virus G protein. J Virol 77: 11790–11797. 17. Seah EL, Marshall JA, Wright PJ (2003) trans Activity of the Norovirus Camberwell Proteinase and Cleavage of the N-Terminal Protein Encoded by ORF1. J Virol 77: 7150–7155. 18. Jiang X, Wang M, Wang K, Estes MK (1993) Sequence and genomic organization of Norwalk virus. Virology 195: 51–61. 19. Gutierrez-Escolano AL, Vazquez-Ochoa M, Escobar-Herrera J, Hernandez- Acosta J (2003) La, PTB, and PAB proteins bind to the 39 untranslated region of Norwalk virus genomic RNA. Biochem Biophys Res Commun 311: 759–766. 20. Blyn LB, Towner JS, Semler BL, Ehrenfeld E (1997) Requirement of Poly(rC) binding protein 2 for translation of poliovirus RNA. J Virol 71: 6243–6246. 21. Haller AA, Semler BL (1995) Stem-Loop Structure Synergy in Binding Cellular Proteins to the 59 Noncoding Region of Poliovirus Rna. Virology 206: 923–934. 22. Kaminski A, Hunt SL, Patton JG, Jackson RJ (1995) Direct evidence that polypyrimidine tract binding protein (PTB) is essential for internal initiation of translation of encephalomyocarditis virus RNA. Rna-A Publication Of The Rna Society 1: 924–938. 23. Meerovitch K, Svitkin YV, Lee HS, Lejbkowicz F, Kenan DJ, Chan EKL, Agol VI, Keene JD, Sonenberg N (1993) La autoantigen enhances and corrects aberrant translation of poliovirus RNA in reticulocyte lysate. J Virol 67: 3798–3807. 24. Gutierrez-Escolano AL, Brito ZU, del Angel RM, Jiang X (2000) Interaction of cellular proteins with the 59 end of Norwalk virus genomic RNA. J Virol 74: 8558–8562. 25. Daughenbaugh KF, Fraser CS, Hershey JWB, Hardy ME (2003) The genome- linked protein VPg of the Norwalk virus binds eIF3, suggesting its role in translation initiation complex recruitment. EMBO J 22: 2852–2859. 26. Daughenbaugh KF, Wobus CE, Hardy ME (2006) VPg of murine norovirus binds translation initiation factors in infected cells. Virology Journal 23. 27. Kozak M (2001) New Ways of Initiating Translation in Eukaryotes? Mol Cell Biol 21: 1899–1907. 28. Liu BL, Clarke IN, Lambden PR (1997) Polyprotein processing in Southampton virus: cleavage in trans by the 3C-like protease. pp 66–71. 29. Southern JA, Young DF, Heaney F, Baumgartner WK, Randall RE (1991) Identification of An Epitope on the P-Proteins and V-Proteins of Simian-Virus 5 That Distinguishes Between 2 Isolates with Different Biological Characteristics. J Gen Virol 72: 1551–1557. 30. Love DN, Sabine M (1975) Electron-Microscopic Observation of Feline Kidney- Cells Infected with A Feline Calicivirus. Arch Virol 48: 213–228. 31. Lopman BA, Reacher MH, van Duijnhoven Y, Hanon FX, Brown D, Koopmans M (2003) Viral gastroenteritis outbreaks in Europe, 1995-2000. Emerg Infect Dis 9: 90–96. PLoS ONE | www.plosone.org 8 May 2008 | Volume 3 | Issue 5 | e2169