High-Resolution X-Ray Structure and Functional Analysis of the Murine Norovirus 1 Capsid Protein Protruding Domain

Abstract

Murine noroviruses (MNV) are closely related to the human noroviruses (HuNoV), which cause the majority of nonbacterial gastroenteritis. Unlike HuNoV, MNV grow in culture and in a small-animal model that represents a tractable model to study norovirus biology. To begin a detailed investigation of molecular events that occur during norovirus binding to cells, the crystallographic structure of the murine norovirus 1 (MNV-1) capsid protein protruding (P) domain has been determined. Crystallization of the bacterially expressed protein yielded two different crystal forms (Protein Data Bank identifiers [PDB ID], 3LQ6 and 3LQE). Comparison of the structures indicated a large degree of structural mobility in loops on the surface of the P2 subdomain. Specifically, the A-B and E-F loops were found in open and closed conformations. These regions of high mobility include the known escape mutation site for the neutralizing antibody A6.2 and an attenuation mutation site, which arose after serial passaging in culture and led to a loss in lethality in STAT1 ؊/؊ mice, respectively. Modeling of a Fab fragment and crystal structures of the P dimer into the cryoelectron microscopy three-dimensional (3D) image reconstruction of the A6.2/ MNV-1 complex indicated that the closed conformation is most likely bound to the Fab fragment and that the antibody contact is localized to the A-B and E-F loops. Therefore, we hypothesize that these loop regions and the flexibility of the P domains play important roles during MNV-1 binding to the cell surface.Murine noroviruses (MNV) are members of the family Caliciviridae, which contains small icosahedral viruses with positive-sense, single-stranded RNA genomes (18). MNV is related to human noroviruses (HuNoV), which cause most of the sporadic cases and outbreaks of infectious nonbacterial gastroenteritis worldwide in people of all ages (4,15,28,36,38,64). However, noroviruses are an understudied group of viruses due to the previous lack of a tissue culture system and small-animal model. Since its discovery in 2003 (23), MNV has become an increasingly important model to study norovirus biology (66). The availability of a small-animal model, cell culture, and reverse-genetics system, combined with many shared characteristics of human and murine noroviruses, allows detailed studies of norovirus biology (7,23,63,65,66).The norovirus genome is organized into 3 major open reading frames (ORFs), which encode the nonstructural polyprotein (ϳ200 kDa) and the major (VP1; ϳ58-kDa) and minor (VP2; ϳ20-kDa) capsid proteins (18). Recently, a putative ORF-4 was identified in MNV, but the existence of that product and its function remain unknown (60). Norovirus capsids are formed from 180 copies of VP1 arranged with Tϭ3 icosahedral symmetry (9,25,(46)(47)(48). Each capsid protein is divided into an N-terminal arm (N), a shell (S), and a C-terminal protruding (P) domain, with the last two domains connected by a short hinge. VP1 self-assembles into virus-like particles (VLPs) in baculovirus, mammalian, and plant e...

Epitope Insertion at the N-Terminal Molecular Switch of the Rabbit Hemorrhagic Disease Virus T=3 Capsid Protein Leads to Larger T=4 Capsids

“…Reconstructions of the VLP with loop insertions were identical to wt VLP Tϭ3 capsid organization, as the density for the loops was not visible in cryo-EM reconstructions. This indicates that surface insertions were dynamic and suggests great flexibility of the loops at the tip of the P domain; this feature might facilitate the interaction of this tip with its host receptors, in accordance with other studies (27,47). For the C-terminal insertion, the extra density at the center of the hollows at the 6-and 5-fold locations is consistent with the C termini of the subunits facing these hollows.…”

“…X-ray structures of the norovirus Norwalk virus (NV) VLP (37) as well as San Miguel sea lion virus (SMSV) (9) and feline calicivirus (FCV) (34) virions (both vesiviruses) indicate that the virion comprises 90 CP dimers arranged with Tϭ3 symmetry to form 12 pentamers and 20 hexamers. Each monomer has three domains, an N-terminal arm (NTA), a shell (S) composed of an eight-stranded ␤-sandwich, and a flexible protruding domain (P), facilitating virus-host receptor interactions (27,47) at the outermost surface (6). We previously established the mechanism that allows RHDV VP1 (formerly VP60) to switch among quasiequivalent conformational states (5).…”

High-Resolution Functional Profiling of the Norovirus Genome

“…After passage 3, the remaining insertions map largely to flexible loops or unstructured regions that may be able to better accommodate small additional sequences. Interestingly, a small cluster that is stable throughout the passages maps to the C=-D= loop of the P2 domain, which is involved in dimerization interactions (68). This loop may therefore be sufficiently flexible to accommodate insertions while maintaining the interactions, or modification of the interactions does not significantly compromise the overall capsid structure.…”

“…The structure of the P domain of MNV VP1 has recently been determined to a high resolution (68), which allows structural mapping of the insertion sites present in the input and passage 3 profiles (Fig. 5C and D, respectively).…”

“…The P domain is further divided into the P1 domain, which forms the base of the arch and is heavily involved in dimerization interactions, and the P2 domain, which forms the top of the arch, is the least conserved domain, and contains the receptor binding sites and sites for antigenicity (34,38,68). The structure of the P domain of MNV VP1 has recently been determined to a high resolution (68), which allows structural mapping of the insertion sites present in the input and passage 3 profiles (Fig.…”