A.Richard Bellamy

Molecular interactions in rotavirus assembly and uncoating seen by high-resolution cryo-EM

Rotaviruses, major causes of childhood gastroenteritis, are nonenveloped, icosahedral particles with double-strand RNA genomes. By the use of electron cryomicroscopy and single-particle reconstruction, we have visualized a rotavirus particle comprising the inner capsid coated with the trimeric outer-layer protein, VP7, at a resolution (4 Å) comparable with that of X-ray crystallography. We have traced the VP7 polypeptide chain, including parts not seen in its X-ray crystal structure. The 3 well-ordered, 30-residue, N-terminal “arms” of each VP7 trimer grip the underlying trimer of VP6, an inner-capsid protein. Structural differences between free and particle-bound VP7 and between free and VP7-coated inner capsids may regulate mRNA transcription and release. The Ca2+-stabilized VP7 intratrimer contact region, which presents important neutralizing epitopes, is unaltered upon capsid binding.

Immobilization of the early secretory pathway by a virus glycoprotein that binds to microtubules

Membrane trafficking from the endoplasmic reticulum (ER) to the Golgi complex is mediated by pleiomorphic carrier vesicles that are driven along microtubule tracks by the action of motor proteins. Here we describe how NSP4, a rotavirus membrane glycoprotein, binds to microtubules and blocks ER‐to‐Golgi trafficking in vivo. NSP4 accumulates in a post‐ER, microtubule‐associated membrane compartment and prevents targeting of vesicular stomatitis virus glycoprotein (VSV‐G) at a pre‐Golgi step. NSP4 also redistributes β‐COP and ERGIC53, markers of a vesicular compartment that dynamically cycles between the ER and Golgi, to structures aligned along linear tracks radiating throughout the cytoplasm. This block in membrane trafficking is released when microtubules are depolymerized with nocodazole, indicating that vesicles containing NSP4 are tethered to the microtubule cytoskeleton. Disruption of microtubule‐mediated membrane transport by a viral glycoprotein may represent a novel pathogenic mechanism and provides a new experimental tool for the dissection of early steps in exocytic transport.

Interaction of rotavirus cores with the nonstructural glycoprotein NS28

The nonstructural rotavirus receptor glycoprotein NS28 is 175 amino acids long and oriented in the RER membrane with the NH2 terminus on the luminal side and approximately 131 amino acids accessible from the cytoplasmic side. Au et al. (1988) have demonstrated that NS28 is able to interact with rotavirus single-shelled particles (cores) in a receptor:ligand interaction in which NS28 appears to act as the receptor and the rotavirus core as the ligand. This interaction appears to model the events that occur in the infected cell in which virus maturation involves budding of the core into the lumen of the RER. We have investigated the nature of the interaction between cores and NS28 in vitro using membranes derived from SA11 rotavirus-infected MA104 cells and membranes from cells where NS28 and other rotavirus proteins have been expressed using a series of recombinant vaccinia viruses that incorporate appropriate cloned rotavirus genes. The interaction between the core and the receptor is enhanced by the presence of Ca2+ and Mg2+ and Scatchard analysis yields a dissociation constant (Kd) of 5 x 10(-11) M. The major core protein VP6 is the ligand involved because (i) a monoclonal antibody specific for VP6 blocks the reaction, (ii) membranes prepared from cells infected with a double recombinant vaccinia virus which expresses both NS28 and VP6 exhibit a reduced capacity to bind cores, and (iii) VP6 prepared from virus blocks the ability of membranes to bind cores. When VP6, VP7, VP4, and NS28 are expressed singly as the sole viral proteins present in the cell, only membranes from cells expressing NS28 mediate receptor function, indicating that the presence of NS28 is sufficient to mediate the interaction between cores and the membrane and that other viral proteins probably are not involved in the initial receptor:ligand interaction.

Membrane-destabilizing activity of rotavirus NSP4 is mediated by a membrane-proximal amphipathic domain

Expression of the rotavirus non-structural glycoprotein NSP4 in E. coli leads to a decrease in optical density of the culture and release of [(3)H]uridine into the medium, effects attributable to the ability of NSP4 to perturb the bacterial membrane. To identify a domain of NSP4 responsible, different regions of the polypeptide were expressed in E. coli. Membrane destabilization is associated with a region of the protein located within residues 48-91, which includes a potential cationic amphipathic helix. A second region of NSP4 that contains a coiled-coil oligomerization domain and a sequence reported to function as a viral enterotoxin enhances the membrane-destabilizing activity of residues 48-91, but has no direct effect on the membrane stability. These studies suggest that the membrane-destabilizing and enterotoxic properties of NSP4 may be mediated by different regions of the polypeptide and suggest a possible basis for the cytotoxicity of NSP4 in mammalian cells.

The potential of plant viral vectors and transgenic plants for subunit vaccine production.

Publisher Summary This chapter presents the case that plants can be used for the production of subunit vaccines and outlines the systems that are available for their production. The limits of plant biology are explored along with the limits of the differing expression systems that are available. Vaccination has led to a significant improvement in the health of the worlds population. The use of vaccines has reduced the spread and infection of a number of major human diseases, including measles, mumps, rubella, and tetanus. With the advent of recombinant technologies, subunit vaccines based on proteins expressed in bacteria and yeast have grown in popularity. Because subunit vaccines do not contain an infectious agent that can revert to a more virulent form or survive the inactivation process, they offer advantages over live vaccines, because they are incapable of causing disease. Interest in vaccine production in plants has also expanded rapidly. There are considerable advantages in expressing antigenic proteins in plants. Plants can be grown locally and cheaply using standard methods, thus reducing problems with distribution, transport, and storage. Several considerations must be addressed when expressing proteins in plants for the purpose of vaccination; the protein must retain the immunogenic characteristics of the original protein and be capable of inducing a protective response to the disease.

Vaccinia—rotavirus VP7 recombinants protect mice against rotavirus-induced diarrhoea

Recombinant vaccinia viruses expressing wild type intracellular VP7 (VP7wt) from rotavirus SA11 or VP7sc, a cell surface-anchored variant, boosted antibody titres in SA11-immune mice. Pups born to these mice were protected from diarrhoea following challenge with SA11. In rotavirus-naive mice, two immunizations with recombinant vaccinia virus expressing VP7sc stimulated protective immunity that could be transferred to pups, whereas viruses expressing VP7wt did not stimulate protective immunity. Recombinant vaccinia viruses expressing intracellular or cell surface-anchored VP6, the rotavirus group-reactive antigen from the inner capsid, did not stimulate protective immunity. These experiments demonstrate that a live viral vector expressing cell surface anchored VP7 may represent a strategy for the development of safe, effective vaccines against rotavirus-induced diarrhoea.

Rotavirus VP6 modified for expression on the plasma membrane forms arrays and exhibits enhanced immunogenicity

The major inner capsid protein of rotavirus is VP6, a 42-kDa polypeptide that forms the icosahedral surface of the rotavirus single-shelled particle. A chimeric form of VP6 (VP6sc) was constructed containing an upstream leader sequence derived from the influenza virus hemagglutinin and a downstream membrane-spanning (anchor) domain from a mouse immunoglobulin gene. When VP6sc was expressed in cells using a recombinant vaccinia virus, the protein was transported, glycosylated, and anchored in the plasma membrane as a trimer with the major domains of the protein orientated externally. Immunofluorescence and immunolabeling with colloidal gold indicated that VP6sc also localized in patches on the cell surface; electron microscopy revealed that the protein assembled into two-dimensional arrays which exhibited the same periodicity as the paracrystalline arrays formed by purified (viral) VP6. Mice inoculated with a recombinant vaccinia virus that expressed VP6sc produced rotavirus-specific antibodies at a titer 10 times higher than that achieved when wild-type, intracellular VP6 was delivered in the same way. Presentation at the cell surface therefore may represent a general method for enhancing the immunogenicity of rotavirus proteins.

II, 7. Interaction of the rotavirus nonstructural glycoprotein NSP4 with viral and cellular components

Publisher Summary The assembly and pathogenesis of rotavirus (RV) exhibit a number of unique features. Following the discovery of the enterotoxic properties of NSP4, attention has been focused on the role of this non-structural RV glycoprotein in the pathogenesis of viral infection. This chapter discusses the structure and function of NSP4, the intracellular interactions that occur between NSP4 and viral and host components, and the potential consequences of these interactions for the RV-infected cell. The chapter provides some preliminary insights not only into the potential pathogenic roles played by NSP4 in RV replication but also into the effect(s) of NSP4 expression on animal cells and the protein–protein and protein–lipid interactions that involve this protein. High-resolution structural analysis of the cytoplasmic region is required to reveal molecular details of its interaction with viral proteins 4 (VP4), the double-layered particle (DLP), and microtubules.