84 85 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 1 9 / 2 0 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 1 9 / 2 0 Soft CondensedMatter Group Beamline B21 Howviruses that causewinter-vomiting disease infect host cells Related publication: ConleyM. J., McElweeM., Azmi L., GabrielsenM., Byron O., Goodfellow I. G. & Bhella D. CalicivirusVP2 forms a portal-like assembly following receptor engagement. Nature 565 , 377-381 (2019). DOI: 10.1038/s41586-018-0852-1 Publication keywords: Calicivirus; Portal; VP2; Endosome escape; CryoEM; SAXS T he notorious norovirus that causes winter vomiting disease is part of the calicivirus family. Norovirus is highly contagious and can be very difficult to contain. Other caliciviruses are animal pathogens, including ‘cat flu’ that can cause very high mortality rates in domestic cats. MRC researchers used a combination of cryo-electron microscopy and very high-quality small-angle X-ray scattering (SAXS) (performed on beamline B21) to analyse feline calicivirus (FCV). When viruses infect, they bind to and then enter cells. They often use a process called ‘endocytosis’, which cells use to bring in nutrients from their environment. Viruses trigger endocytosis, causing the cell to enclose the virus particle in a sort of bubble called an endosome. The virus then needs to escape the endosome to infect the cell properly, but how it does this was not understood. The results of this new research show that after they bind to the cell surface, caliciviruses rearrange their protein shell to extrude a funnel- shaped structure. They believe the virus uses this portal-like assembly to inject its RNA into the host cell and begin the infection process. The team were also able to calculate an atomic model of the portal protein - known as VP2. Although VP2 was known to be critical for the production of infectious virus, its exact function was not known. These insights into the early stages of calicivirus infection provide a new target for the development of antiviral drugs. Viruses are intracellular parasites that require a host cell to replicate or multiply. Viruses enter cells by binding to specific receptors on the cell surface. Receptor binding subsequently triggers uptake of the virus into the host cell, folding the host membrane around the virus forming a membranous compartment known as an endosome. The virus must escape the endosome in order to take over the host cell for viral replication. Caliciviruses are small viruses that can infect many species such as humans, cats, sealions and rabbits. The most notable of the caliciviruses is norovirus, the cause of winter vomiting disease outbreaks across the globe, particularly affecting humans in enclosed spaces e.g. cruise ships, hospital wards and classrooms. Until relatively recently, studies on norovirus have been limited by the inability to grow the virus in laboratories and so related veterinary pathogens such as feline calicivirus (FCV) are often used to study calicivirus biology. Caliciviruses have a width of approximately 40 nm (that is 2,500 times smaller than the thickness of a sheet of paper). Viruses are containers for viral genetic information and the calicivirus container is composed of a protein shell mainly composed from 180 copies of a single viral protein (VP1). An additional viral structural protein known asVP2 is also found in the protein shell, although until this study, the function (and structure) of VP2 was unknown. In this study, cryo-electron microscopy (cryoEM) was used to determine the high-resolution structures of FCV, both unbound and bound to its cellular receptor (feline junctional adhesion molecule A (fJAM-A) 1 ). The structure of human and murine JAM-A proteins had been previously determined; however, the structure of feline JAM-A had not. CryoEM studies samples in a close-to- native state and to complement the virus cryoEM studies, small angle X-ray scattering (SAXS) was used to study the portion of fJAM-A that extends into extracellular space where the virus can interact with it. SAXS 2 is a useful technique for studying proteins in solution, as they would be found in a cell. CryoEM and image analysis produced a three-dimensional image reconstruction of the free (Fig. 2) and fJAM-A receptor bound (Fig. 3a) FCV. The structures were of sufficient detail to allow visualisation of structural changes in the virus upon fJAM-A binding. Regions of the VP1 proteins undergo a 15° anti-clockwise rotation whilst some (at the two-fold symmetry axes) also tilt off axis (see below). Interestingly, fJAM-A bound to the virus appeared to adopt different structures than observed by SAXS (Fig. 1). Whilst in solution, fJAM-A proteins appear to couple together to form doublets/dimers consistent with the related human and murine forms. Upon binding to the virus, fJAM-A was found to be present in a head-to-tail arrangement that disrupts/alters the coupling of two fJAM-A proteins.Thus, we concluded that FCV binding appears to disrupt fJAM-A dimeric complexes despite the virus binding site occurring in a region distinct from the fJAM-A dimerisation site suggested by our SAXS data. Due to the highly symmetric (icosahedral) nature of caliciviruses, icosahedral symmetry was imposed during the image reconstruction process, a common practice used in virus structure determination. In some cases, this can lead to a loss of information. Due to this loss of information, a recently developed method of image analysis, known as focussed classification, was adopted. Focussed classification permits the reconstruction of small regions of density without imposing any symmetry 3,4,5 . Whilst performing focussed classification on the three-fold symmetry axes of FCV decorated with fJAM-A, a novel structure was discovered. A portal-like assembly was found at a single three-fold symmetry axis per virus particle (Fig. 3). The portal-like assembly is composed of 12 VP2 proteins in alternating conformations which form a channel-like structure projecting out from the surface of the virus. The distal tips of the VP2 proteins are highly hydrophobic (water fearing) which suggests the portal-like assembly inserts into the membranous endosome during virus entry and acts as a mechanism for delivery of the viral genetic information into the host cell for replication to proceed. This study was the first description of this kind of structure in a mammalian virus. References: 1. Makino A. et al. Junctional adhesion molecule 1 is a functional receptor for feline calicivirus. Journal of Virology , 4482 (2006). DOI: 10.1128/JVI.80.9.4482-4490.2006 2. Franke D. et al. DAMMIF, a program for rapid ab initio shape determination in small-angle scattering. J. Appl. Cryst. 42 , 342 (2009). DOI: 10.1107/S0021889809000338 3. Scheres S. H. W., Processing of structurally heterogeneous cryo-EM data in RELION. Methods in Enzymology 579 , 125 (2016). DOI: 10.1016/bs.mie.2016.04.012 4. Zhou M. et al. Atomic structure of the apoptosome: mechanism of cytochrome c- and dATP-mediated activation of Apaf-1. Genes & Dev. 29 , 2349 (2015). DOI: 10.1101/gad.272278.115 5. McElwee M. et al. Structure of the herpes simplex virus portal-vertex. PLoS Biol. 20 , e2006191 (2018). DOI: 10.1371/journal.pbio.2006191 Funding acknowledgement: We acknowledge Diamond Light Source for time on Beamline B21 under proposal MX11651-24. M. J. Conley was supported by a PhD studentship from the UK Biotechnology and Biological Sciences Research Council (BBSRC WestBIO DTP: BB/J013854/1). D. Bhella was supported by the UK Medical Research Council (MC_UU_12014/7). Corresponding author: Dr Michaela Jayne Conley, MRC-University of Glasgow Centre for Virus Research,
[email protected] Figure 1: Dummy atommodel of feline junctional adhesion molecule A (fJAM-A) computed with the program DAMMIF from size-exclusion chromatography small angle X-ray scattering (SEC-SAXS) data. Figure 2: Structure of feline calicivirus at 3 Å resolution as determined by cryo-electron microscopy and three-dimensional image analysis. . Figure 3: Interaction of feline calicivirus with fJAM-A results in the formation of a portal-like assembly. (a) Full virus structure (coloured by radius) showing the portal-like assembly (orange) and surrounding fJAM-A proteins (blue). (b) Atomic model of the portal-like assembly (coloured by VP2 conformer). (c) Atomic model of the two conformers of VP2 present in the portal-like assembly.