Norwalk Virus Minor Capsid Protein VP2 Associates within the VP1 Shell Domain

Abstract

The major capsid protein of norovirus VP1 assembles to form an icosahedral viral particle. Despite evidence that the Norwalk virus (NV) minor structural protein VP2 is present in infectious virions, the available crystallographic and electron cryomicroscopy structures of NV have not revealed the location of VP2. In this study, we determined that VP1 associates with VP2 at the interior surface of the capsid, specifically with the shell (S) domain of VP1. We mapped the interaction site to amino acid 52 of VP1, an isoleucine located within a sequence motif IDPWI in the S domain that is highly conserved across norovirus genogroups. Mutation of this isoleucine abrogated VP2 incorporation into virus-like particles without affecting the ability for VP1 to dimerize and form particles. The highly basic nature of VP2 and its location interior to the viral particle are consistent with its potential role in assisting capsid assembly and genome encapsidation. Noroviruses are nonenveloped viruses with a single-stranded RNA genome of positive polarity and are the leading cause of acute gastroenteritis (1). Infection causes outbreaks of food-borne illness in the community and produces symptoms of vomiting and diarrhea. Noroviruses belong to the Caliciviridae family that is comprised of five genera: Norovirus, Sapovirus, Lagovirus, Vesivirus, and Nebovirus (2). The first two genera contain primarily human viruses, while the other genera represent animal viruses. The noroviruses are genetically diverse since they are divided into six genogroups based on the amino acid sequence of the major structural protein VP1. The noncultivable human noroviruses in genogroups I and II are epidemiologically important (3) and are further subdivided into at least 8 and 21 genotypes, respectively. Norwalk virus (NV) is a prototype member of the Norovirus genus and is designated GI.1, and GII noroviruses such as the GII.4 Houston virus (HoV) are increasingly prevalent in more recent outbreaks (4, 5).The NV genome encodes three open reading frames (ORFs) and ORF1 encodes a large nonstructural polyprotein of 1,789 amino acids. This polyprotein is autocatalytically processed by the viral protease to yield six nonstructural proteins (p48 N-terminal protein, p41 NTPase, p22, VPg, protease, and RNA-dependent RNA polymerase [RdRp]) that are thought to function primarily for viral RNA replication. The structural proteins VP1 and VP2, which form the viral capsid, are translated from a subgenomic mRNA that is 3= coterminal with the viral genome and codes for ORF2 and ORF3 (6-10). In NV, ORF2 overlaps ORF1 and ORF3 by 17 and 1 nucleotides, respectively.The ability to express the norovirus capsid protein to a high levels in insect cells using the baculovirus system and the fact that VP1 proteins will self-assemble into particles that are morphologically and antigenically similar to infectious virions has enabled the structural characterization of the norovirus capsids by cryoelectron microscopy reconstruction and by X-ray crystallography (11-13). These structu...

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