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

Abstract

Viruses need only one or a few structural capsid proteins to build an infectious particle. This is possible through the extensive use of symmetry and the conformational polymorphism of the structural proteins. Using virus-like particles (VLP) from rabbit hemorrhagic disease virus (RHDV) as a model, we addressed the basis of calicivirus capsid assembly and their application in vaccine design. The RHDV capsid is based on a T=3 lattice containing 180 identical subunits (VP1). We determined the structure of RHDV VLP to 8.0-Å resolution by three-dimensional cryoelectron microscopy; in addition, we used San Miguel sea lion virus (SMSV) and feline calicivirus (FCV) capsid subunit structures to establish the backbone structure of VP1 by homology modeling and flexible docking analysis. Based on the three-domain VP1 model, several insertion mutants were designed to validate the VP1 pseudoatomic model, and foreign epitopes were placed at the N- or C-terminal end, as well as in an exposed loop on the capsid surface. We selected a set of T and B cell epitopes of various lengths derived from viral and eukaryotic origins. Structural analysis of these chimeric capsids further validates the VP1 model to design new chimeras. Whereas most insertions are well tolerated, VP1 with an FCV capsid protein-neutralizing epitope at the N terminus assembled into mixtures of T=3 and larger T=4 capsids. The calicivirus capsid protein, and perhaps that of many other viruses, thus can encode polymorphism modulators that are not anticipated from the plane sequence, with important implications for understanding virus assembly and evolution.

Atomic Model of Rabbit Hemorrhagic Disease Virus by Cryo-Electron Microscopy and Crystallography

“…At each three-fold axis of the virion, three A/B and three C/C dimers pack in alternate fashion via their S domains and clear densities at the interface of each dimer show that each NTA domain folds onto its cognate S domain ( Figure 3A , S5A and B ). The NTA domains of the B and C monomers form a network of interactions with a plug-like density (formed by residues 1–30) surrounding the three-fold axis ( Figure 3A and B ) as was also described previously [16]. Contacts formed by the NTA domains in the inner shell of the virion confirm the importance of this domain for virion assembly, which concurs with previous truncation [27] and insertion studies [16].…”

“…We find that RHDV VP60 has a P2 sub-domain that differs from other caliciviruses. Furthermore, our new model reveals that certain aspects of the P2 and NTA domain structures that were previously reported [16] need reinterpretation. We also examined the putative HBGA binding sites in RHDV by mapping isolate–related sequence variations onto the P domain structure.…”

“…Next, we compared our current structural model of RHDV with the previously reported backbone model derived from the 8.0 Å VLP cryo-reconstruction and homology modeling [16]. Comparison of the three quasi-equivalent monomers (A, B and C) in the two models ( Figure S6 ) showed that the relative positions of the P and S domains correspond closely to each other, but that nevertheless two significant differences are found.…”

“…Previous structural studies of caliciviruses include three-dimensional (3D) cryo-electron microscopic (cryoEM) reconstructions of virus-like particles (VLPs) of Murine Norovirus (MNV, Norovirus ) and Feline calicivirus (FCV, Vesivirus ) at 8- and 16-Å resolution, respectively [10], [11], and determination of the crystal structures of the Norwalk virus (NV, Norovirus ) capsid at 3.4 Å [12], native FCV virions at 3.6 Å [13], and native virions of San Miguel sea lion virus (SMSV, Vesivirus ) at 3.2 Å [14]. CryoEM reconstructions of the RHDV VLP at 8 Å [15] and the native RHDV virion at 11 Å [7] have been computed and a C α homology model of RHDV was built based on the VLP cryo-reconstruction by using the crystal structure models of SMSV and FCV [16]. However, a more complete atomic model of RHDV is still lacking.…”

Cryo-EM Structure of a Novel Calicivirus, Tulane Virus

“…These include the X-ray crystallographic structures of recombinant Norwalk Virus (rNV) [3] from the Norovirus genus, a native San Miguel sea lion virus (SMSV) [4] and a feline calicivirus virion (FCV) [5] from Vesivirus genus, and cryo-electron microscopy (cryo-EM) structures of recombinant Grimsby virus [6], recombinant Parkville virus [6], Rabbit hemorrhagic disease virus (RHDV, both VLPs and virions) [7], [8], [9], [10], murine norovirus (MNV) virion [9], [11] and a genogroup II genotype 10 (GII.10) NoV VLP [12]. All these structures exhibit characteristic similar overall organization: 180 capsid proteins are organized into 90 dimers, which form a T = 3 icosahedral capsid with 32 hollows around the icosahedral 3- and 5-fold axes.…”