sequences of various noroviruses indicates little similarity between genogroups within the first 180 residues, however towards the C- terminal end of the protein similarity between the amino acid residues increases. Recent studies investigating the functions of the Norwalk virus N-terminal protein have successfully demonstrated association with the Golgi apparatus in transfected cells [15]. In addition this study also identified a picornaviral 2B like region within the N-terminal protein, suggesting that the protein is involved with host cell membrane interactions, reinforcing other findings that have suggested that the Norwalk virus N-terminal protein disrupts intracellular protein trafficking, including proteins destined for the host cell membrane [16]. A 3C protease-mediated cleavage event within the N-terminal protein (37 kDa) was described for Camberwell virus, a genogroup 2 norovirus, yielding proteins of 22 kDa and 15 kDa [17]. Based on these observations and location within the genome it was hypothesised that the N- terminal protein of noroviruses corresponds to the 2AB region in picornaviruses. Another possibility is that the N-term encoding RNA itself serves to function as a translational enhancer by interacting with cellular proteins involved in translation. Indeed, this phenomenon has been previously reported for Norwalk virus, within which a double stem loop structure has been predicted at the 59 end of the genomic RNA [18]. It was subsequently demonstrated that elements within the 59 end of Norwalk virus bind specifically with cellular proteins such as La, PTB and PCBP2 [19] which have all been implicated in IRES-mediated cap-independent translation in the closely related picornaviruses [20–23]. In this study the role of the JV 59GS was investigated, including its potential to direct cap- independent translation initiation. The precise location of translation initiation in JV was also investigated. Results and Discussion JV ORF1 Polyprotein Processing Previous studies of norovirus polyprotein processing have yielded three major products following in vitro transcription and translation, representing the uncleaved 3ABCD, N-term and 2C proteins. However, initial analysis of JV polyprotein processing indicated that only two major proteins are synthesised initially which, based on molecular weight predictions, are the 3ABCD (110 kDa) and the 2C (39 kDa). The lack of an N-terminal protein encoded by the JV 59GS, predicted to be 35.3 kDa, is unique among the noroviruses that have been studied in this way. The in vitro transcription and translation profile for JV was therefore studied in more detail. As initial experiments had analysed TNTH reactions following a 1 hr incubation, reaction aliquots were harvested at time points before and after the recommended 1 hr incubation. The results in Figure 1 show that there are no major reaction products synthesised prior to the 1 hr time point, at which time the 3ABCD/p110 and 2C/p39 proteins are clearly visible. Extended incubation past the 1 hr point resulted in further proteolytic cleavage of p110 that coincided with the appearance of proteins of the following sizes: 86 kDa, 55 kDa, and 51 kDa. In addition proteins of 29 kDa, 22 kDa and 20 kDa were also visible at the 24 hr time point (Figure 1, lane 7). The only protein that was consistently visible following the 1 hr time point was the 2C/p39 protein. Despite prolonged incubation there was no indication that the N-terminal/p35 protein was synthesised. A comprehensive study of polyprotein processing within the murine norovirus (MNV) suggests likely identities for the equivalent proteins in the similar profile for JV [12]. Using region specific antisera the authors were able to identify p110 as Analysis of the 59 GS of JV Figure 1. TNTH time course for JV displaying the progressive stages of post translational polyprotein processing. Molecular weight marker is represented by lane 1. Lanes 2–7 represent the following time points; 15 min, 30 min, 1 hr, 2 hr, 4 hr and 24 hr respectively. doi:10.1371/journal.pone.0002169.g001 the 3ABCD uncleaved precursor, p90 as the 3BCD, p57.5 as the 3D-like polymerase, p52 as a 3ABC precursor and p40 as the 2C- like NTPase, which was determined by mutagenesis and microsequencing experiments. The 19 kDa protein was identified as the 3C-like protease. The antisera used to detect the MNV N- term protein recognised 3 products; one was the predicted molecular weight at 39 kDa and the other two bands migrated as a 45 kDa doublet. Assessment of translational enhancing potential of the JV 59GS The 59GS region of JV is highly divergent compared to other noroviruses, mainly due to the relatively high cytosine content (32%), which contributes to an overall G/C content of 58%. There are many polypyrimidine tracts within the sequence, potentially yielding a relatively high degree of RNA secondary structure. Previous studies have described potential secondary RNA structure and interaction with proteins involved with IRES- mediated translation within the 59 genomic region of Norwalk virus [18,24]. It was of interest therefore, based on these findings, to ascertain whether or not the 59GS of JV possessed IRES-like properties within the context of a ‘Bi-cistronic’ expression system, independently of other viral proteins, including the VPg which, in other caliciviruses, has been shown to be associated with translation initiation factors [25,26]. Traditionally the bi-cistronic vector system has been used to define potential IRES-like sequences from a variety of viral and cellular mRNAs, and is recognized as being the standard test for this function [27]. A bi-cistronic vector is comprised of a 59 and 39 cistron; translation of the 59 cistron being cap-dependent and translation of the 39 cistron regulated by the putative IRES-like sequence. Thus, if the 39 cistron is translated in addition to the 59 cistron then the sequence of interest is said to have IRES-like properties, as translation is initiating internally. To test for IRES-like function in JV, bi-cistronic constructs were made with a cap dependent 59 EGFP cistron and a 39 lacZ cistron under the translational control of either the JV 59GS (pEGFP-C1/ JV59GS/lacZ) or an authentic EMCV IRES (pEGFP-C1/IRES/ lacZ). CRFK cells were transfected with the bi-cistronic constructs PLoS ONE | www.plosone.org 2 May 2008 | Volume 3 | Issue 5 | e2169