The role of cellular adhesion molecules in virus attachment and entry

As obligate intracellular parasites, viruses must traverse the host-cell plasma membrane to initiate infection. This presents a formidable barrier, which they have evolved diverse strategies to overcome. Common to all entry pathways, however, is a mechanism of specific attachment to cell-surface macromolecules or ‘receptors’. Receptor usage frequently defines viral tropism, and consequently, the evolutionary changes in receptor specificity can lead to emergence of new strains exhibiting altered pathogenicity or host range. Several classes of molecules are exploited as receptors by diverse groups of viruses, including, for example, sialic acid moieties and integrins. In particular, many cell-adhesion molecules that belong to the immunoglobulin-like superfamily of proteins (IgSF CAMs) have been identified as viral receptors. Structural analysis of the interactions between viruses and IgSF CAM receptors has not shown binding to specific features, implying that the Ig-like fold may not be key. Both proteinaceous and enveloped viruses exploit these proteins, however, suggesting convergent evolution of this trait. Their use is surprising given the usually occluded position of CAMs on the cell surface, such as at tight junctions. Nonetheless, the reason for their widespread involvement in virus entry most probably originates in their functional rather than structural characteristics

Cryo-electron microscopy: an introduction to the technique, and considerations when working to establish a national facility

No abstract available

Structure of the herpes-simplex virus portal-vertex

Herpesviruses include many important human pathogens such as herpes simplex virus, cytomegalovirus, varicella-zoster virus, and the oncogenic Epstein–Barr virus and Kaposi sarcoma–associated herpesvirus. Herpes virions contain a large icosahedral capsid that has a portal at a unique 5-fold vertex, similar to that seen in the tailed bacteriophages. The portal is a molecular motor through which the viral genome enters the capsid during virion morphogenesis. The genome also exits the capsid through the portal-vertex when it is injected through the nuclear pore into the nucleus of a new host cell to initiate infection. Structural investigations of the herpesvirus portal-vertex have proven challenging, owing to the small size of the tail-like portal-vertex–associated tegument (PVAT) and the presence of the tegument layer that lays between the nucleocapsid and the viral envelope, obscuring the view of the portal-vertex. Here, we show the structure of the herpes simplex virus portal-vertex at subnanometer resolution, solved by electron cryomicroscopy (cryoEM) and single-particle 3D reconstruction. This led to a number of new discoveries, including the presence of two previously unknown portal-associated structures that occupy the sites normally taken by the penton and the Ta triplex. Our data revealed that the PVAT is composed of 10 copies of the C-terminal domain of pUL25, which are uniquely arranged as two tiers of star-shaped density. Our 3D reconstruction of the portal-vertex also shows that one end of the viral genome extends outside the portal in the manner described for some bacteriophages but not previously seen in any eukaryote viruses. Finally, we show that the viral genome is consistently packed in a highly ordered left-handed spool to form concentric shells of DNA. Our data provide new insights into the structure of a molecular machine critical to the biology of an important class of human pathogens

Strategies for maximizing ATP supply in the microsporidian Encephalitozoon cuniculi: direct binding of mitochondria to the parasitophorous vacuole and clustering of the mitochondrial porin VDAC

Microsporidia are obligate intracellular parasites with extremely reduced genomes and a dependence on host-derived ATP. The microsporidium Encephalitozoon cuniculi proliferates within a membranous vacuole and we investigated how the ATP supply is optimized at the vacuole–host interface. Using spatial EM quantification (stereology), we found a single layer of mitochondria coating substantial proportions of the parasitophorous vacuole. Mitochondrial binding occurred preferentially over the vegetative ‘meront’ stages of the parasite, which bulged into the cytoplasm, thereby increasing the membrane surface available for mitochondrial interaction. In a broken cell system mitochondrial binding was maintained and was typified by electron dense structures (<10 nm long) bridging between outer mitochondrial and vacuole membranes. In broken cells mitochondrial binding was sensitive to a range of protease treatments. The function of directly bound mitochondria, as measured by the membrane potential sensitive dye JC-1, was indistinguishable from other mitochondria in the cell although there was a generalized depression of the membrane potential in infected cells. Finally, quantitative immuno-EM revealed that the ATP-delivering mitochondrial porin, VDAC, was concentrated atthe mitochondria-vacuole interaction site. Thus E. cuniculi appears to maximize ATP supply by direct binding of mitochondria to the parasitophorous vacuole bringing this organelle within 0.020 microns of the growing vegetative form of the parasite. ATP-delivery is further enhanced by clustering of ATP transporting porins in those regions of the outer mitochondrial membrane lying closest to the parasite

The respiratory syncytial virus nucleoprotein–RNA complex forms a left-handed helical nucleocapsid

Respiratory Syncytial Virus (RSV) is an important human pathogen. Its nucleocapsid (NC), which comprises the negative sense RNA viral genome coated by the viral nucleoprotein N, is a critical assembly that serves as template for both mRNA synthesis and genome replication. We have previously described the X-ray structure of a nucleocapsid-like structure: a decameric ring formed of N-RNA that mimics one turn of the helical NC. In the absence of experimental data we had hypothesized that the NC helix would be right-handed, as the N-N contacts in the ring appeared to more easily adapt to that conformation. We now unambiguously show that the RSV NC is a left-handed helix. We further show that the contacts in the ring can be distorted to maintain key N-N protein interactions in a left-handed helix, and discuss the implications of the resulting atomic model of the helical NC for viral replication and transcription

Vesivirus 2117 capsids more closely resemble sapovirus and lagovirus particles than other known vesivirus structures

Vesivirus 2117 is an adventitious agent that in 2009, was identified as a contaminant of CHO cells propagated in bioreactors at a pharmaceutical manufacturing plant belonging to Genzyme. The consequent interruption in supply of Fabrazyme and Cerezyme (drugs used to treat Fabry and Gaucher disease respectively), caused significant economic losses. Vesivirus 2117 is a member of the Caliciviridae; a family of small icosahedral viruses encoding a positive sense RNA genome. We have used cryo-electron microscopy and three dimensional image reconstruction to calculate a structure of vesivirus 2117 virus like particles as well as feline calicivirus and a chimeric sapovirus. We present a structural comparison of several members of the Caliciviridae, showing that the distal P domain of vesivirus 2117 is morphologically distinct from that seen in other known vesivirus structures. Furthermore, at intermediate resolutions we found a high level of structural similarity between vesivirus 2117 and Caliciviridae from other genera, such as sapovirus and rabbit haemorrhagic disease virus. Phylogenetic analysis confirms vesivirus 2117 as a vesivirus closely related to canine vesiviruses. We postulate that morphological differences in virion structure seen between vesivirus clades may reflect differences in receptor usage

In situ structure of virus capsids within cell nuclei by correlative light and cryo-electron tomography

Cryo electron microscopy (cryo-EM), a key method for structure determination involves imaging purified material embedded in vitreous ice. Images are then computationally processed to obtain three-dimensional structures approaching atomic resolution. There is increasing interest in extending structural studies by cryo-EM into the cell, where biological structures and processes may be imaged in context. The limited penetrating power of electrons prevents imaging of thick specimens (> 500 nm) however. Cryo-sectioning methods employed to overcome this are technically challenging, subject to artefacts or involve specialised and costly equipment. Here we describe the first structure of herpesvirus capsids determined by sub-tomogram averaging from nuclei of eukaryotic cells, achieved by cryo-electron tomography (cryo-ET) of re-vitrified cell sections prepared using the Tokuyasu method. Our reconstructions confirm that the capsid associated tegument complex is present on capsids prior to nuclear egress. We demonstrate that this method is suited to both 3D structure determination and correlative light/electron microscopy, thus expanding the scope of cryogenic cellular imaging

A tail-like assembly at the portal vertex in intact herpes simplex type-1 virions

Herpes viruses are prevalent and well characterized human pathogens. Despite extensive study, much remains to be learned about the structure of the genome packaging and release machinery in the capsids of these large and complex double-stranded DNA viruses. However, such machinery is well characterized in tailed bacteriophage, which share a common evolutionary origin with herpesvirus. In tailed bacteriophage, the genome exits from the virus particle through a portal and is transferred into the host cell by a complex apparatus (i.e. the tail) located at the portal vertex. Here we use electron cryo-tomography of human herpes simplex type-1 (HSV-1) virions to reveal a previously unsuspected feature at the portal vertex, which extends across the HSV-1 tegument layer to form a connection between the capsid and the viral membrane. The location of this assembly suggests that it plays a role in genome release into the nucleus and is also important for virion architecture

Calicivirus VP2 forms a portal-like assembly following receptor engagement

To initiate infection, many viruses enter their host cells by triggering endocytosis following receptor engagement. However, the mechanisms by which non-enveloped viruses escape the endosome are poorly understood. Here we present near-atomic-resolution cryo-electron microscopy structures for feline calicivirus both undecorated and labelled with a soluble fragment of its cellular receptor, feline junctional adhesion molecule A. We show that VP2, a minor capsid protein encoded by all caliciviruses1,2, forms a large portal-like assembly at a unique three-fold axis of symmetry, following receptor engagement. This assembly—which was not detected in undecorated virions—is formed of twelve copies of VP2, arranged with their hydrophobic N termini pointing away from the virion surface. Local rearrangement at the portal site leads to the opening of a pore in the capsid shell. We hypothesize that the portal-like assembly functions as a channel for the delivery of the calicivirus genome, through the endosomal membrane, into the cytoplasm of a host cell, thereby initiating infection. VP2 was previously known to be critical for the production of infectious virus3; our findings provide insights into its structure and function that advance our understanding of the Caliciviridae

An N-terminal extension to the hepatitis B virus core protein forms a poorly ordered trimeric spike in assembled virus-like particles

Virus-like particles composed of the core antigen of hepatitis B virus (HBcAg) have been shown to be an effective platform for the display of foreign epitopes in vaccine development. Heterologous sequences have been successfully inserted at both amino and carboxy termini as well as internally at the major immunodominant epitope. We used cryogenic electron microscopy (CryoEM) and three-dimensional image reconstruction to investigate the structure of VLPs assembled from an N-terminal extended HBcAg that contained a polyhistidine tag. The insert was seen to form a trimeric spike on the capsid surface that was poorly resolved, most likely owing to it being flexible. We hypothesise that the capacity of N-terminal inserts to form trimers may have application in the development of multivalent vaccines to trimeric antigens. Our analysis also highlights the value of tools for local resolution assessment in studies of partially disordered macromolecular assemblies by cryoEM