Localization of membrane permeabilization and receptor binding sites on the VP4 hemagglutinin of rotavirus: implications for cell entry.

The surface of rotavirus is decorated with 60 spike-like projections, each composed of a dimer of VP4, the viral hemagglutinin. Trypsin cleavage of VP4 generates two fragments, VP8*, which binds sialic acid (SA), and VP5*, containing an integrin binding motif and a hydrophobic region that permeabilizes membranes and is homologous to fusion domains. Although the mechanism for cell entry by this non-enveloped virus is unclear, it is known that trypsin cleavage enhances viral infectivity and facilitates viral entry. We used electron cryo-microscopy and difference map analysis to localize the binding sites for two neutralizing monoclonal antibodies, 7A12 and 2G4, which are directed against the SA-binding site within VP8* and the membrane permeabilization domain within VP5*, respectively. Fab 7A12 binds at the tips of the dimeric heads of VP4, and 2G4 binds in the cleft between the two heads of the spike. When these binding results are combined with secondary structure analysis, we predict that the VP4 heads are composed primarily of beta-sheets in VP8* and that VP5* forms the body and base primarily in beta-structure and alpha-helical conformations, respectively. Based on these results and those of others, a model is proposed for cell entry in which VP8* and VP5* mediate receptor binding and membrane permeabilization, and uncoating occurs during transfer across the lipid bilayer, thereby generating the transcriptionally active particle.

Structure of native and expanded sobemoviruses by electron cryo-microscopy and image reconstruction.

Rice yellow mottle virus (RYMV) and southern bean mosaic virus, cowpea strain (SCPMV) are members of the Sobemovirus genus of RNA-containing viruses. We used electron cryo-microscopy (cryo-EM) and icosahedral image analysis to examine the native structures of these two viruses at 25 A resolution. Both viruses have a single tightly packed capsid layer with 180 subunits assembled on a T=3 icosahedral lattice. Distinctive crown-like pentamers emanate from the 12 5-fold axes of symmetry. The exterior face of SCPMV displays deep valleys along the 2-fold axes and protrusions at the quasi-3-fold axes. While having a similar topography, the surface of RYMV is comparatively smooth. Two concentric shells of density reside beneath the capsid layer of RYMV and SCPMV, which we interpret as ordered regions of genomic RNA. In the presence of divalent cations, SCPMV particles swell and fracture, whereas the expanded form of RYMV is stable. We previously proposed that the cell-to-cell movement of RYMV in xylem involves chelation of Ca(2+) from pit membranes of infected cells, thereby stabilizing the capsid shells and allowing a pathway for spread of RYMV through destabilized membranes. In the context of this model, we propose that the expanded form of RYMV is an intermediate in the in vivo assembly of virions.

Large conformational changes in the maturation of a simple RNA virus, nudaurelia capensis omega virus (NomegaV).

An assembly intermediate of a small, non-enveloped RNA virus has been discovered that exhibits striking differences from the mature virion. Virus-like particles (VLPs) of Nudaurelia capensis omega virus (NomegaV), a T=4 icosahedral virus infecting Lepidoptera insects, were produced in insect cells using a baculovirus vector expressing the coat protein. A procapsid form was discovered when NomegaV VLPs were purified at neutral pH conditions. These VLPs were fragile and did not undergo the autoproteolytic maturation that occurs in the infectious virus. Electron cryo-microscopy (cryoEM) and image analysis showed that, compared with the native virion, the VLPs were 16% larger in diameter, more rounded, porous, and contained an additional internal domain. Upon lowering the pH to 5.0, the VLP capsids became structurally indistinguishable from the authentic virion and the subunits autoproteolyzed. The NomegaV protein subunit coordinates, which were previously determined crystallographically, were modelled into the 28 A resolution cryoEM map of the procapsid. The resulting pseudo-atomic model of the NomegaV procapsid demonstrated the large rearrangements in quaternary and tertiary structure needed for the maturation of the VLPs and presumably of the virus. Based on this model, we propose that electrostatically driven rearrangements of interior helical regions are responsible for the large conformational change. These results are surprising because large structural rearrangements have not been found in the maturation of any other small RNA viruses. However, similarities of this conformational change to the maturational processes of more complex DNA viruses (e.g. bacteriophages and herpesvirus) and to the swelling of simple plant viruses suggest that structural changes in icosahedral viruses, which are integral to their function, have similar strategies and perhaps mechanisms.

Particle polymorphism caused by deletion of a peptide molecular switch in a quasiequivalent icosahedral virus.

The capsid of flock house virus is composed of 180 copies of a single type of coat protein which forms a T=3 icosahedral shell. High-resolution structural analysis has shown that the protein subunits, although chemically identical, form different contacts across the twofold axes of the virus particle. Subunits that are related by icosahedral twofold symmetry form flat contacts, whereas subunits that are related by quasi-twofold symmetry form bent contacts. The flat contacts are due to the presence of ordered genomic RNA and an ordered peptide arm which is inserted in the groove between the subunits and prevents them from forming the dihedral angle observed at the bent quasi-twofold contacts. We hypothesized that by deleting the residues that constitute the ordered peptide arm, formation of flat contacts should be impossible and therefore result in assembly of particles with only bent contacts. Such particles would have T=1 symmetry. To test this hypothesis we generated two deletion mutants in which either 50 or 31 residues were eliminated from the N terminus of the coat protein. We found that in the absence of residues 1 to 50, assembly was completely inhibited, presumably because the mutation removed a cluster of positively charged amino acids required for neutralization of encapsidated RNA. When the deletion was restricted to residues 1 to 31, assembly occurred, but the products were highly heterogeneous. Small bacilliform-like structures and irregular structures as well as wild-type-like T=3 particles were detected. The anticipated T=1 particles, on the other hand, were not observed. We conclude that residues 20 to 30 are not critical for formation of flat protein contacts and formation of T=3 particles. However, the N terminus of the coat protein appears to play an essential role in regulating assembly such that only one product, T=3 particles, is synthesized.

Cytochrome c oxidase in proteoliposomes visualised by platinum-carbon and by tungsten-tantalum shadowing: image analysis.

Beef heart cytochrome c oxidase complexes incorporated into phospholipid liposomes were examined by freeze-fracture electron microscopy. Enzyme molecules are inserted into the membrane asymmetrically, with larger projections on the 'C' side, where cytochrome c binding occurs, than on the 'M' (matrix-facing) side. Visualisation of the complexes was improved by: (i) image analysis, to determine details of size and shape, and (ii) tungsten-tantalum (W/Ta) rather than platinum-carbon (Pt/C) shadowing, which permits examination of smaller entities. Enzyme molecules are incorporated as dimers in the proteoliposomes. Some surface structural details of the embedded molecules can be discerned. Around each complex is seen a small area of modified lipid, the frozen annulus whose existence has been predicted with other methods.

Electron microscopy of cytochrome c oxidase-containing proteoliposomes: imaging analysis of protein orientation and monomer-dimer behaviour.

1. Cytochrome c oxidase-containing vesicles were prepared by cholate dialysis using bovine heart cytochrome c oxidase with egg and dioleoylphosphatidylcholine/dioleoylphosphatidylethanolamines (1:1, w/w) at two ratios of phospholipid to protein (25 mg/mg and 10 mg/mg). With each mixture, one or two (FII, FIII) fractions with mostly outward-facing cytochrome aa3 were separated from a fraction (FI) containing mostly inward-facing enzyme and protein-free liposomes by DEAE-Sephacel chromatography. 2. FII and FIII fractions from egg phospholipid mixtures had 60-80% outward-facing enzyme; FII and FIII fractions from dioleoyl phospholipids showed 50-70% outward-facing enzyme. Egg and dioleoyl phospholipid mixtures maintained good respiratory control ratios (8-13) only at the higher lipid/protein ratios. 3. Platinum/carbon replicas of freeze-fractured vesicle surfaces were subjected to image analysis. The results showed two types of membrane projection with average heights of 7.5 nm and 3.5 nm from the fracture plane. The former were more numerous on the convex faces. Calculated areas of the projections indicated the probable presence of both enzyme dimers and higher aggregates. Oxidase dimers may have membrane areas of 70-80 nm2 at the high (7.5 nm) side and 40-50 nm2 on the low (3.5 nm) side. 4. Proteoliposomes prepared with enzyme depleted of subunit III contained predominantly much smaller projecting areas. These probably represent monomers with high side areas of 35-40 nm2 and low side areas of 20-25 nm2. Electron microscopy thus directly confirms the predicted change of aggregation state resulting from subunit depletion. 5. The results are compared with those from two-dimensional crystals. Assuming that the high and low projections are two sides of one family of transmembrane molecules, a total length of 11 nm matches 11-12 nm lengths obtained by crystallography. Our membrane areas match the areas obtained in earlier 'crystal' studies better than the small areas obtained recently by electron cryomicroscopy.

A probability concept about size distributions of sonicated lipid vesicles.

The sonication procedure of preparation of small unilamellar vesicles is modelled as a process of uniform random fragmentation of the lipid aggregates. The vesicle size distribution evolving in this process is shown to be identical with the Weibull extremal probability distribution. Size histograms of sonicated small vesicles of various phospholipid composition were obtained by using electron microscopy (negative staining). Their successful simulation with Weibull curves shows that theory agrees with experiment. A similarly good agreement is found also with size histograms obtained by freeze-fracture of phosphatidylcholine-cholesterol vesicles (Van Venetië, R., Leunissen-Bijvelt, J., Verkleij, A.J. and Ververgaert, P.H.J.T. (1980) J. Microsc. 118, 401-408). This analysis allows a refinement of some earlier conclusions about the effect of cholesterol on the size of the sonicated vesicles. It follows from the theoretical model that the only intrinsic characteristic of the sonicated vesicles is the lower limit of their size. The other characteristics of the size distribution such as expectancy, dispersion, position and height of the maximum depend on the intensity of fragmentation. It is concluded that the size distribution of sonicated small vesicles is completely determined by the procedure of their preparation and, therefore, the condition of thermodynamic equilibrium between aggregated and monomeric lipid is irrelevant in this case.