Figure 8. Degradation analysis of N-term/V5 following treat- ment of JV/V5 transfected CRFK with cycloheximide (CHX). Whole cell lysate was collected at the following time points following CHX treatment: 0 hr, 1 hr, 3 hr, 6 hr, 12 hr, 24 hr. Bradford analysis was performed on the lysates to ensure equal loading. Following Western analysis the ECL treated membrane was exposed to film for 1 min. Molecular weight marker is represented in lane 1. doi:10.1371/journal.pone.0002169.g008 previously unseen 42 kDa protein in the in vitro transcription and translation profile was unexpected but this protein does represent a translation product for the JV 59GS. To date, it has not been possible to explain the difference in the predicted and observed sizes for the JV N-term, and the addition of the 14 amino acid V5 epitope within JV N-term does not account for this apparent large shift in molecular weight. However, a recent study described a similar anomaly when investigating proteolytic processing in the murine norovirus MNV-1 [12]. The predicted molecular weight for the MNV-1 N-term protein was 38.3 kDa. The authors successfully generated antisera against the MNV-1 N-term and used it to immunoprecipitate the protein from in vitro transcription and translation reactions and observed that the N-term existed as a 45 kDa doublet, in addition to the predicted size of 38 kDa. However, when MNV-1 N-term antisera was used to probe MNV-1-infected cell lysates only the 43–45 kDa doublet and a large 115 kDa precursor could be detected, suggesting that the predicted 38 kDa form of the N-term is not generated in cell culture. Again, it was not possible to conclusively determine the cause of this discrepancy, but it was speculated that the N-term protein may migrate abnormally in SDS-PAGE, or may be proteolytically processed at a previously unknown cleavage site downstream of the protein’s predicted C-terminus. It is also possible that the N-term protein might be modified in some way leading to a shift in observed molecular weight. At this time the same conclusions would seem appropriate for the JV N-term. In addition, it is not known why the JV N-term was previously not detected in in vitro transcription and translation studies prior to the insertion of the V5 epitope. It cannot be ruled out, however, that the wild type JV N-term aberrantly co-migrates with the 39 kDa JV 2C protein in SDS-PAGE. Indeed, the appearance of the V5/ N-term product from transfected cell lysates would appear to be one of a doublet (Figure 8), also analogous to the observed appearance of the MNV N-term protein in infected cells, suggesting the likelihood of a further cleavage site within the JV N-term protein which has yet to be elucidated. Nevertheless, these studies clearly demonstrate that a protein representative of the 59GS of JV is translated both in vitro and in vivo and is proteolytically processed from the ORF1 polyprotein following translation initiation at nucleotide 22. Conclusions Human norovirus infection has been shown to be the leading cause of non-bacterial gastroenteritis [31], however there is currently no cell culture system available to facilitate viral replication and ethical considerations have hindered progress in establishing a permissive human organ culture system. The study of Jena virus offers a potential animal model of enteric noroviral infection. However, until a permissive bovine cell and/or organ culture systems is established analysis of the molecular mechanisms Analysis of the 59 GS of JV underpinning viral replication and pathogenesis rely upon in vitro systems, most notably polyprotein synthesis and processing. Unlike similarly studied human noroviruses JV initially did not appear to express the N-terminal protein, presenting the possibility that the encoding RNA sequence had a regulatory function itself, most likely involved in translation initiation in an IRES-like manner. This was shown not to be the case and, following determination of the site of translation initiation at the predicted nucleotide 22 the N-term protein was detected following the insertion of an epitope tag, both in vitro and in vivo. Although slightly larger than predicted the N-term protein was detected in a processed form in vivo, thus not only demonstrating initial translation of the ORF1 polyprotein but also activity of the viral encoded protease. These important findings indicate that the block to replication of enteric norovirus in cultured cells cannot be attributed to a failure to synthesise and process the non-structural proteins. The detection of processed and active ORF1 proteins in transfected cultured cells, however, highlights the potential for the development of cell and bovine organ based systems to facilitate the replication of Jena virus. Materials and Methods Construction of Bi-cistronic vectors The pEGFP-C1 vector (Clontech) comprises of an EGFP coding sequence under the control of a CMV promoter and a Kozak translation initiation site. Downstream of the EGFP sequence is the multiple cloning site containing unique BglII, SacI, HindIII and ApaI restriction sites. Contruction of pEGFP-C1/JV 59 GS/lacZ was as follows; The JV 59 GS sequence was amplified from the JV full length cDNA clone [7] using Bio-X-Act DNA polymerase (Bioline) with the primers 59 GS F (59-AACTGCA- GATCTTAATAAGTGAATGAAGACTTTGACGAT-39), con- taining the BglII restriction site (bold) and two in-frame translation termination codons (underlined) to ensure that translation of the EGFP sequence did not carry over to the 59 GS, and 59 GS R (59- AACTGCAAGCTTCTGCAGGACACAATGAGG-39), contain- ing theHindIII restriction site. The JV 59 GS amplicon was ligated to the pEGFP-C1 vector, following restriction enzyme digestion of both amplicon and vector with BglII and HindIII restriction enzymes, and the ligated DNA used to transform E.coli Top10 (Invitrogen). This intermediate construct was named pEGFP-C1/ JV 59 GS. The lacZ coding sequence was amplified from the pSV- b-Gal reporter vector (Promega) using Bio-X-Act DNA polymer- ase and the primers lacZ F (59-AACTGCAAGCTTGA- TATGGGGGATCCCGTCGTTTTACAACG-39), containing the HindIII restriction site (bold) and a kozak translation initiation site (underlined), and lacZ R (59-AACTGCGGGCCCTTAT- TATTTTTGACACCAGACCA-39) containing the ApaI restric- tion site (bold) and translation termination codons (underlined). The lacZ amplicon was ligated to the pEGFP-C1/JV 59 GS vector following restriction enzyme digest of both amplicon and vector with HindIII and ApaI restriction enzymes, and the ligated DNA used to transform E.coli Top10. The construct was verified by sequencing. Construction of pEGFP-C1/IRES/lacZ was as follows; The EMCV IRES sequence was amplified from the pIRES2-EGFP vector (Clontech) using Bio-X-Act DNA polymer- ase and the primers IRES Bgl F (59-ACTCGAAGATCTTAA- TAGAGCTTCGAATTCTGCAGTCGA-39), containing the BglII restriction site (bold) and translation termination codons (underlined) to prevent carry over translation as before, and IRES Sac R (59- ACTCGAGAGCTCTGTGGCCATATTATCATC- GTG-39), containing the SacI restriction site (bold). The IRES amplicon was ligated to the pEGFP-C1 vector follwing restriction PLoS ONE | www.plosone.org 6 May 2008 | Volume 3 | Issue 5 | e2169