The 3′ End of Norwalk Virus mRNA Contains Determinants That Regulate the Expression and Stability of the Viral Capsid Protein VP1: a Novel Function for the VP2 Protein

Abstract

Norwalk virus (NV) is the prototype strain of a group of noncultivable human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. The capsid protein VP1 is synthesized from a subgenomic RNA that contains two open reading frames (ORFs), ORF2 and ORF3, and the 3 untranslated region (UTR). ORF2 and ORF3 code for the capsid protein (VP1) and a small structural basic protein (VP2), respectively. We discovered that the yields of virus-like particles (VLPs) composed of VP1 are significantly reduced when this protein is expressed from ORF2 alone. To determine how the 3 terminus of the NV subgenomic RNA regulates VP1 expression, we compared VP1 expression levels by using recombinant baculovirus constructs containing different 3 elements. High VP1 levels were detected by using a recombinant baculovirus that contained ORF2, ORF3, and the 3UTR (ORF2؉3؉3UTR). In contrast, expression of VP1 from constructs that lacked the 3UTR (ORF2؉3), ORF3 (ORF2؉3UTR), or both (ORF2 alone) was highly reduced. Elimination of VP2 synthesis from the subgenomic RNA by mutation resulted in VP1 levels similar to those obtained with the ORF2 construct alone, suggesting a cis role for VP2 in upregulation of VP1 expression levels. Comparisons of the kinetics of RNA and capsid protein expression levels by using constructs with or without ORF3 or the 3UTR revealed that the 3UTR increased the levels of VP1 RNA, whereas the presence of VP2 resulted in increased levels of VP1. Furthermore, VP2 increased VP1 stability and protected VP1 from disassembly and protease degradation. The increase in VP1 expression levels caused by the presence of VP2 in cis was also observed in mammalian cells.The Norwalk virus (NV) is a member of the genus Norovirus in the family Caliciviridae. The recent accessibility of molecular diagnostic assays has demonstrated that noroviruses are the leading cause of outbreaks of nonbacterial gastroenteritis worldwide (17). In addition, caliciviruses have been recently classified as category B biodefense pathogens (http://www.niaid .nih.gov/biodefense/). The NV 7.7-kb positive-strand RNA genome is organized into a 5Ј untranslated region (UTR), three open reading frames (ORFs), a 3ЈUTR, and a poly(A) tail (19). The NV predicted subgenomic RNA contains ORF2, ORF3, and the 3ЈUTR. ORF2 and ORF3 code for the major capsid protein (VP1) and a small basic structural protein (VP2), respectively. The ORF3 protein (VP2) has been detected in NV virions and virus-like particles (VLPs) (15). VP2 or a VP2-equivalent protein has been detected in the virions of other members of the Caliciviridae family such as the rabbit hemorrhagic disease virus (RHDV) (34) and feline calicivirus (FCV) (54). VP2 is not essential for the formation of NV VLPs because baculovirus recombinants expressing VP1 alone are able to produce NV VLPs (5, 58). The highly basic VP2 may serve a function similar to the basic N terminus of plant virus capsid proteins, which are involved in RNA binding, since the N terminus of NV VP1 is acidic and is not thought to ...

His-tagged norovirus-like particles: A versatile platform for cellular delivery and surface display

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Human caliciviruses in symptomatic and asymptomatic infections in children in Vellore, South India

“…NoVs that infect humans have been classified into three genetically and antigenically distinct genogroups [Zheng et al, 2006] and SaVs that infect humans into five genogroups . The genome has three open reading frames (ORFs) that encode a polyprotein precursor to nonstructural proteins including the RNA-dependent RNA polymerase (RdRp), and other proteins (ORF 1), the major and minor capsid proteins VP1 and VP2 (ORF 2 and 3) [Jiang et al, 1992[Jiang et al, , 1993Lambden et al, 1993;Bertolotti-Ciarlet et al, 2003]. Knowledge about the role of HuCVs in pediatric gastroenteritis in developing countries has been limited by the lack of sensitive and specific diagnostic techniques.…”

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