Functional Analysis of the 59 Genomic Sequence of a Bovine Norovirus Omar Salim*, Ian N. Clarke, Paul R. Lambden Molecular Microbiology Group, University of Southampton Medical School, Southampton General Hospital, Southampton, United Kingdom Abstract Background: Jena Virus (JV), a bovine Norovirus, causes enteric disease in cattle and represents a potential model for the study of enteric norovirus infection and pathogenesis. The positive sense RNA genome of JV is organised into ORF1 (non- structural proteins), ORF2 (major capsid protein) and ORF3 (minor capsid protein). The lack of a cell culture system for studying JV replication has meant that work to date has relied upon in vitro systems to study non-structural protein synthesis and processing. Principal Findings: Only two of the three major ORF1 proteins were identified (p110 and 2C) following in vitro translation of JV RNA, the N-term protein was not detected. The N-term encoding genomic sequence (59GS) was tested for IRES-like function in a bi-cistronic system and displayed no evidence of IRES-like activity. The site of translation initiation in JV was determined to be at the predicted nucleotide 22. Following the insertion of an epitope within the 59GS the JV N-term protein was identified in vitro and within RNA transfected cells. Conclusions: The in vitro transcription/translation system is currently the best system for analysing protein synthesis and processing in JV. 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, most likely involved in translation initiation in an IRES-like manner. This was not the case and, following determination of the site of translation initiation the N-term protein was detected using 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 protease. These findings indicate that the block to noroviral replication in cultured cells lies elsewhere. Citation: Salim O, Clarke IN, Lambden PR (2008) Functional Analysis of the 59 Genomic Sequence of a Bovine Norovirus. PLoS ONE 3(5): e2169. doi:10.1371/ journal.pone.0002169 Editor: Lennart Randau, Yale University, United States of America Received February 26, 2008; Accepted April 4, 2008; Published May 14, 2008 Copyright: 2008 Salim et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: OS would like to thank the Medical Research Council for providing a studentship to support this work, which was also supported by Wellcome Trust Grant 069233. Competing Interests: The authors have declared that no competing interests exist. * E-mail: O.Salim@soton.ac.uk Introduction Jena virus, a bovine norovirus, is a member of the Caliciviridae family of positive sense RNA viruses and was first isolated from the diarrhoeic stools of newborn calves [1,2]. JV is a type I genogroup III (GIII) norovirus which is closely related to the type II GIII bovine noroviruses Newbury agent 2 and Dumfries [3,4]. The GIII noroviruses are responsible for causing enteric disease in cattle [2,5] and, thus, likely share a similar tissue tropism to the human-associated enteric noroviruses. Like human noroviruses [6] bovine noroviruses have a high seroprevalence [4]. JV is therefore a potentially useful model for studying the molecular biology of enteric norovirus pathogenesis and replication. The 7.3 kb polyadenylated RNA genome of JV has been characterised previously [7] and, like other noroviruses, is organised into 3 open reading frames (ORFs). ORF1 encodes the non-structural proteins in the form of a large 185 kDa polyprotein, which is subsequently cleaved into functional replication proteins by the viral encoded 3C-like protease. ORF2 encodes the structural capsid protein (56 kDa) and ORF3 encodes a small basic protein, which has been shown to function as a minor capsid component [8]. JV ORF1 is consistent with other caliciviruses in that it encodes a 39 kDa 2C-like nucleoside triphosphatase (NTPase), a 3C-like protease and a 56 kDa 3D-like RNA-dependent RNA polymerase [7,9–13]. However, the genomic sequence within the 59 region of JV ORF1 (59GS) displays a high level of divergence. This divergence is mainly attributed to the presence of several proline-encoding polypyr- imidine tracts within the region predicted to encode a 35 kDa N- terminal protein [7]. The predicted size of N-terminal proteins relative to the size of the respective 2C proteins differs within the norovirus genus. Within the GI noroviruses, such as Southampton virus, the N-terminal protein (44.8 kDa) is larger in size compared to the 2C protein (39.6 kDa). This is in contrast to the GII noroviruses, such as Lordsdale virus and Camberwell virus, in that the N-terminal protein is smaller in size compared to the 2C protein [11,14]. This is also the case for Jena virus in which the predicted JV N-terminal protein (35 kDa) is smaller than the JV 2C protein (39 kDa) [7]. The norovirus N-terminal protein varies in relative size across the genus, and the encoding sequence bears no similarity to other cellular or viral proteins. Alignment of the N-term protein PLoS ONE | www.plosone.org 1 May 2008 | Volume 3 | Issue 5 | e2169