Structural investigations of influenza B virus

Purified preparations of influenza B/Hong Kong/5/72 have been characterized by hydrodynamical measurements, electron microscopy and small-angle neutron scattering. As judged by these techniques the preparations are highly monodisperse, the virus particles being spherical and of molecular weight about 200 × 10⁶. The lipid bilayer is located at a radius of 425 Å and its molecular weight is estimated to be 60 × 10⁶, constituting about 30% of the total virus mass. The external radius is about 580 Å.     

Structure of clathrin-coated vesicles from small-angle scattering experiments

Previously published small-angle neutron and X-ray scattering data from coated vesicles, reassembled coats, and stripped vesicles have been analyzed in terms of one common model. The neutron data sets include contrast variation measurements at three different D20 solvent concentrations. The model used for interpreting the data has spherical symmetry and explicitly takes into account polydispersity, which is described by a Gaussian distribution. Å constant thickness of the clathrin coats is assumed. The fitting of the model shows that the coated vesicles consist of a low-density outer protein shell (clathrin) and a central protein shell (accessory polypeptides and receptors) of approximately six times higher density. For the X-ray scattering and neutron contrast variation data, the polydispersity of the samples is of the order of 90 Å (full-width-at-half-maximum value) and the average outer radius is approximately 400 Å. The inner high-density shell has inner and outer radii of 115 and 190 Å, respectively. Å simultaneous fit to the three neutron contrast variation data sets identifies the lipid membrane with a thickness of 40 Å and an outer radius of 196 Å. Thus, the membrane and the high-density protein shell overlap in space, which shows that the lipid membrane contains protein. The molecular mass of the average particle is 27 × 106 Da. The coated vesicles consist, on average, of approximately 85 % protein and 15 lipids. About 40% of the protein mass is situated in the central high-density shell, which gives a large amount of protein in the lipid membrane. The densities of the central shell and the lipid membrane show that the hydration is small in the central region. Å comparison of the total mass, the mass distribution, and the structure of the average-size particles with the barrel structure shows that the accessory polypeptides are incorporated in the lipid membrane. The results from the neutron data for the reassembled coats show that the structure of these particles is very similar to the structure of the native coats. The main difference is a higher density of the central protein shell, which shows that the membrane is replaced by protein in the reassembled coats.     

Thermal Fluctuations in Amphipol A8-35 Particles: A Neutron Scattering and Molecular Dynamics Study

Amphipols are a class of polymeric surfactants that can stabilize membrane proteins in aqueous solutions as compared to detergents. A8-35, the best-characterized amphipol to date, is composed of a polyacrylate backbone with ~35 % of the carboxylates free, ~25 % grafted with octyl side-chains, and ~40 % with isopropyl ones. In aqueous solutions, A8-35 self-organizes into globular particles with a molecular mass of ~40 kDa. The thermal dynamics of A8-35 particles was measured by neutron scattering in the 10-picosecond, 18-picosecond, and 1-nanosecond time-scales on natural abundance and deuterium-labeled molecules, which permitted to separate backbone and side-chain motions. A parallel analysis was performed on molecular dynamics trajectories (Perlmutter et al., Langmuir 27:10523–10537, 2011). Experimental results and simulations converge, from their respective time-scales, to show that A8-35 particles feature a more fluid hydrophobic core, predominantly containing the octyl chains, and a more rigid solvent-exposed surface, made up predominantly of the hydrophilic polymer backbone. The fluidity of the core is comparable to that of the lipid environment around proteins in the center of biological membranes, as also measured by neutron scattering. The biological activity of proteins depends sensitively on molecular dynamics, which itself is strongly dependent on the immediate macromolecular environment. In this context, the characterization of A8-35 particle dynamics constitutes a step toward understanding the effect of amphipols on membrane protein stability and function.     

Biochemical and morphological properties of hepatitis C virus particles and determination of their lipidome

A hallmark of hepatitis C virus (HCV) particles is their association with host cell lipids, most notably lipoprotein components. It is thought that this property accounts for the low density of virus particles and their large heterogeneity. However, the composition of infectious virions and their biochemical and morphological properties are largely unknown. We developed a system in which the envelope glycoprotein E2 was N-terminally tagged with a FLAG epitope. This virus, designated Jc1E2(FLAG), produced infectivity titers to wild type levels and allowed affinity purification of virus particles that were analyzed for their protein and lipid composition. By using mass spectrometry, we found the lipid composition of Jc1E2(FLAG) particles to resemble the one very low- and low density-lipoprotein with cholesteryl esters accounting for almost half of the total HCV lipids. Thus, HCV particles possess a unique lipid composition that is very distinct from all other viruses analyzed so far and from the human liver cells in which HCV was produced. By electron microscopy (EM), we found purified Jc1E2(FLAG) particles to be heterogeneous, mostly spherical structures, with an average diameter of about 73 nm. Importantly, the majority of E2-containing particles also contained apoE on their surface as assessed by immuno-EM. Taken together, we describe a rapid and efficient system for the production of large quantities of affinity-purified HCV allowing a comprehensive analysis of the infectious virion, including the determination of its lipid composition.     

Molecular modeling of the domain structure of C9 of human complement by neutron and X-ray solution scattering.

C9 is the most abundant component of the membrane attack complex of the complement system of immune defense. This is a typical mosaic protein with thrombospondin (TSR) and low density lipoprotein receptor (LDLr) domains at its N-terminus and an epidermal growth factor-like (EGF) domain at its C-terminus. Between these lies a perforin-like sequence. In order to define the arrangement in solution of these four moieties in C9, high-flux neutron and synchrotron X-ray solution scattering studies were carried out. The neutron radius of gyration RG at infinite contrast is 3.33 nm, and its cross-sectional RG (RXS) is 1.66 nm. Similar values were obtained by synchrotron X-ray scattering after allowance for radiation effects. Stuhrmann analyses showed that the neutron radial inhomogeneity of scattering density alpha is 35 X 10(-5) from the RG data and 16 X 10(-5) from the RXS data. These values are typical for soluble glycoproteins and show no evidence for the existence of any large hydrophobic surface patches on free C9 that might form contacts with lipids. Indirect transformation of the neutron and X-ray scattering curves into real space showed that C9 had a maximum dimension estimated at 12 +/- 2 nm, and this suggests that the lengths of 7-8 nm deduced from previous electron microscopy studies in vacuo are underestimated. Molecular modeling of the C9 scattering curves utilized small spheres in the Debye equation, in which the analyses were constrained by the known volumes of the four moieties of C9 and the known sizes of the TSR and EGF-like domains. The most likely models for C9 suggest that these four regions of C9 are arranged in a V-shaped structure, with an angle of 10 degrees between the two arms, each of length 11.1 nm. This structure has a more hydrophobic character between the two arms. The scattering model is fully consistent with hydrodynamic sedimentation data on C9. Similar V-shaped hydrodynamic models could be developed for C6, C7, C8, and C9 of complement. Such a compact structure is atypical of other multidomain complement proteins so far studied by solution scattering and is fully compatible with mechanisms in which C9 is postulated, on activation, to undergo a drastic unfolding of its domain structure and to expose a more hydrophobic surface which can be embedded into lipid bilayers.     

Two concentric protein shell structure with spikes of silkworm Bombyx mori cytoplasmic polyhedrosis virus revealed by small-angle neutron scattering using the contrast variation method

The overall and internal structures of the silkworm Bombyx mori cytoplasmic polyhedrosis virus was investigated by small-angle neutron scattering using the contrast variation method. Data were collected in aqueous buffer solutions containing 0, 50, 75, and 100% D2O in the q range of 0.002 to 0.0774 A-1 at 5 degrees C. The radius of gyration at infinite contrast was estimated to be 336 A. The contrast matching point of the virus was determined to correspond to about 50% D2O level, evidence that the virus is composed of protein and nucleic acid. The virus was basically spherical and had a diameter of about 700 A. The main feature of its structure is the clustering of protein into two concentric shells separated by about 100 A. Most of the RNA moieties are located in the central core and between these two protein shells. However, the distance distribution function P(r) showed a minor distribution beyond a distance of r = 700 A, with a maximum particle distance of the virus of 1350 A. This is indicative of an external structure region with very low scattering density, in addition to the basic spherical structure. This external region is thought to correspond to twelve pyramidal protruding spikes shown by electron microscopic studies.     

Characterization of a glycoprotein fusogen isolated from Sendai virus

After isolation from Sendai virus, the glycoproteins HN and F retained their ability to induce hemagglutination and both heterologous and homologous cell-cell fusion. Both methods for demonstrating cell fusion indicated that the isolated HN and F glycoproteins compared favorably with whole Sendai virus as a fusogen. Conditions affecting the degree of fusion were examined and optimized. Whole virus and isolated glycoprotein preparations were characterized by electron microscopy and by SDS-polyacrylamide gel electrophoresis. Lipid analysis of the glycoprotein preparations by thin layer chromatography and gas chromatography/mass spectrometry indicated that they were partially lipid-depleted during the isolation protocol and the ratio of cholesterol to phospholipid was higher than in the whole virus. A complete fatty acid analysis was performed on lipid extracts from whole virus and from glycoprotein preparations. Detergent was removed from the glycoproteins by dialysis and by incubation with Amberlite XAD-2 resin. The detergent content of the glycoprotein preparations was monitored by gas chromatography and with [3H]Triton X-100. Both methods showed that virtually all (greater than or equal to 99.8%) of the originally added detergent was removed. Electron microscopy of the negatively-stained HN and F preparations showed primarily spherical particles 120 +/- 20 A in diameter (range 80-250 A). Since no organization reminiscent of envelopes could be demonstrated, we conclude that the fusogenic activity of Sendai virus resides in the glycoproteins per se rather than in bilayer integrated lipid-protein complexes.     

Negative-sense RNA viruses: An underexplored platform for examining virus–host lipid interactions

Viruses are pathogenic agents that can infect all varieties of organisms, including plants, animals, and humans. These microscopic particles are genetically simple as they encode a limited number of proteins that undertake a wide range of functions. While structurally distinct, viruses often share common characteristics that have evolved to aid in their infectious life cycles. A commonly underappreciated characteristic of many deadly viruses is a lipid envelope that surrounds their protein and genetic contents. Notably, the lipid envelope is formed from the host cell the virus infects. Lipid-enveloped viruses comprise a diverse range of pathogenic viruses, which often lead to high fatality rates and many lack effective therapeutics and/or vaccines. This perspective primarily focuses on the negative-sense RNA viruses from the order Mononegavirales, which obtain their lipid envelope from the host plasma membrane. Specifically, the perspective highlights the common themes of host cell lipid and membrane biology necessary for virus replication, assembly, and budding.     

Physical, chemical and morphologic dimensions of human hepatitis A virus strain CR326 (38578).

CR326 human hepatitis A virus purified by isopycnic banding from infected marmoset sera was shown to consist of 27 nm spherical particles on electron microscopic examination. The particles were identified as hepatitis A virus by tests for infectivity and by specific neutralization of infectivity with convalescent human hepatitis A serum. Also, indentical 27 nm viruses in liver extracts gave specific reactions with hepatitis A antisera when tested by immune electron microscopy. The bouyant density of the virus in CsCl was 1.34 and it was heat (60 degrees), ether and acid stable but was destroyed by heat (100 degrees), formalin (1:4000) and ultraviolet irradiation. Electron microscopic studies of sections of infected marmoset liver showed intracytoplasmic virus particles, usually in vesicles. Presumptive findings for RNA, together with the intracytoplasmic location of the virus, indicated the virus to be of RNA-type. The attributes of the virus indicate it is closely related to the enterovirus family and not to hepatitis B virus.     

Architecture of bacterial lipid A in solution. A neutron small-angle scattering study

The phase structure of isolated bacterial lipid A, the lipid anchor of the lipopolysaccharides of the outer membrane of Gram-negative bacteria, has been investigated by neutron small-angle scattering. The shape of the scattering curves obtained at different H2O/2H2O ratios revealed a lamellar organisation of the lipid A at neutral pH both above and below its main phase temperature (approximately 40-45 degrees C). Analysis of the scattering curves and interpretation of the corresponding thickness distance distribution functions of the lamellar aggregates led to a model in which the lipid A molecules form a bilayer of about 5 nm in thickness. This value for the thickness of the bilayer, as well as the neutron-scattering density profile across the bilayer, can be explained by a molecular model which shows interdigitation of the fatty acid chains of the lipid A.     

Lipid fingerprints of intact viruses by MALDI-TOF/mass spectrometry

A number of viruses contain lipid membranes, which are in close contact with capsid proteins and/or nucleic acids and have an important role in the viral infection process. In this study membrane lipids of intact viruses have been analysed by MALDI-TOF/MS with a novel methodology avoiding lipid extraction and separation steps. To validate the novel method, a wide screening of viral lipids has been performed analysing highly purified intact bacterial and archaeal viruses displaying different virion architectures. Lipid profiles reported here contain all lipids previously detected by mass spectrometry analyses of virus lipid extracts. Novel details on the membrane lipid composition of selected viruses have also been obtained. In addition we show that this technique allows the study of lipid distribution easily in subviral particles during virus fractionation. The possibility to reliably analyse minute amounts of intact viruses by mass spectrometry opens new perspectives in analytical and functional lipid studies on a wider range of viruses including pathogenic human ones, which are difficult to purify in large amounts.     

Site-specific maturation of enveloped viruses in L cells treated with cytochalasin B

Treatment of infected L cells with 10 micrograms/ml cytochalasin B (CB) was found to promote a rapid relocalization of viral glycoproteins on the cell surface. Whereas the vesicular stomatitis virus G protein and the influenza virus hemagglutinin were uniformly distributed on the surface of untreated cells, in CB-treated cells, they were strikingly concentrated at cell extremities in the regions of clustered blebs. Glycoprotein concentration at cell extremities was accompanied by preferential maturation of virus particles from the same sites; both vesicular stomatitis and influenza viruses budded predominantly from the vicinity of clustered blebs. This effect of CB was completely reversible. Removal of CB from the cell growth medium resulted in a return of viral glycoproteins to the uniform distribution characteristic of untreated cells and to uniform virus budding. The results of this study are interpreted in terms of a model that suggests that preferential budding of viruses from the regions of bleb clusters is due to the concentration of viral glycoproteins at these sites.     

Intracellular trafficking of Gag and Env proteins and their interactions modulate pseudotyping of retroviruses

Glycoproteins derived from most retroviruses and from several families of enveloped viruses can form infectious pseudotypes with murine leukemia virus (MLV) and lentiviral core particles, like the MLV envelope glycoproteins (Env) that are incorporated on either virus type. However, coexpression of a given glycoprotein with heterologous core proteins does not always give rise to highly infectious viral particles, and restrictions on pseudotype formation have been reported. To understand the mechanisms that control the recruitment of viral surface glycoproteins on lentiviral and retroviral cores, we exploited the fact that the feline endogenous retrovirus RD114 glycoprotein does not efficiently pseudotype lentiviral cores derived from simian immunodeficiency virus, whereas it is readily incorporated onto MLV particles. Our results indicate that recruitment of glycoproteins by the MLV and lentiviral core proteins occurs in intracellular compartments and not at the cell surface. We found that Env and core protein colocalization in intracytoplasmic vesicles is required for pseudotype formation. By investigating MLV/RD114 Env chimeras, we show that signals in the cytoplasmic tail of either glycoprotein differentially influenced their intracellular localization; that of MLV allows endosomal localization and hence recruitment by both lentiviral and MLV cores. Furthermore, we found that upon membrane binding, MLV core proteins could relocalize Env glycoproteins in late endosomes and allow their incorporation on viral particles. Thus, intracellular colocalization, as well as interactions between Env and core proteins, may influence the recruitment of the glycoprotein onto viral particles and generate infectious pseudotyped viruses.     

Use of lipid solvents for viral inactivation in factor VIII concentrates

Model virus inactivation studies with lipid solvents were carried our in antihemophilic factor concentrates. The procoagulant activity obtained was >/=80% recovery with 20% amyl acetate-0.1% deoxycholate. A concurrent reduction of four logs of virus titer was obtained for model viruses provided the viral mass contained significant amounts (>/=20%) of lipid. From this preliminary study it appears that further investigations in animal models may be warranted to demonstrate the inactivation of hepatitis B virus, non-A-non-B virus, and AIDS virus with 20% amyl acetate-0.1% deoxycholate in antihemophilic factor concentrates.     

The purification of alphavirus virions and subviral particles using ultrafiltration and gel exclusion chromatography

Conventional methods of virus purification using ultracentrifugation frequently result in distorted particles with low levels of biological activity, and are thus unsuitable for preparing samples for high-resolution techniques such as neutron scattering, X-ray scattering in solution, and X-ray crystallography. Moreover, in the event of an instrument failure, ultracentrifugation can also pose a significant hazard when preparing pathogenic viruses or subunits derived from them. By employing exclusively ultrafiltration and gel exclusion chromatography, a method has been developed to prepare highly purified, intact alphavirus particles retaining high levels of biological activity. These procedures have also been adapted to prepare aggregates of viral envelope protein with a defined immunogenic content.     

Mini-plasmin found in the epithelial cells of bronchioles triggers infection by broad-spectrum influenza A viruses and Sendai virus.

Extracellular cleavage of virus envelope fusion glycoproteins by host cellular proteases is a prerequisite for the infectivity of mammalian and nonpathogenic avian influenza viruses, and Sendai virus. Here we report a protease present in the airway that, like tryptase Clara, can process influenza A virus haemagglutinin and Sendai virus envelope fusion glycoprotein. This protease was extracted from the membrane fraction of rat lungs, purified and then identified as a mini-plasmin. Mini-plasmin was distributed predominantly in the epithelial cells of the upward divisions of bronchioles and potentiated the replication of broad-spectrum influenza A viruses and Sendai virus, even that of the plasmin-insensitive influenza A virus strain. In comparison with plasmin, its increased hydrophobicity, leading to its higher local concentrations on membranes, and decreased molecular mass may enable mini-plasmin to gain ready access to the cleavage sites of various haemagglutinins and fusion glycoproteins after expression of these viral proteins on the cell surface. These findings suggest that mini-plasmin in the airway may play a pivotal role in the spread of viruses and their pathogenicity.     

Divalent ion-dependent swelling of tomato bushy stunt virus: a multi-approach study

Time-resolved small-angle X-ray and neutron scattering (SAXS and SANS) in solution were used to study the swelling reaction of TBSV upon chelation of its constituent calcium at mildly basic pH. SAXS intensities comprise contribution from the protein capsid and the RNA moiety, while neutron scattering, recorded in 72% D2O, is essentially due to the protein capsid. Cryo-electron micrographs of compact and swollen virus were used to produce 3D reconstructions of the initial and final conformations of the virus at a resolution of 13 A and 19 A, respectively. While compact particles appear to be very homogeneous in size, solutions of swollen particles exhibit some size heterogeneity. A procedure has been developed to compute the SAXS pattern from the 3D reconstruction for comparison with experimental data. Cryo-electron microscopy thereby provides an invaluable starting (and ending) point for the analysis of the time-resolved swelling process using the scattering data.     

Silencing the morphogenesis of rotavirus

The morphogenesis of rotaviruses follows a unique pathway in which immature double-layered particles (DLPs) assembled in the cytoplasm bud across the membrane of the endoplasmic reticulum (ER), acquiring during this process a transient lipid membrane which is modified with the ER resident viral glycoproteins NSP4 and VP7; these enveloped particles also contain VP4. As the particles move towards the interior of the ER cisternae, the transient lipid membrane and the nonstructural protein NSP4 are lost, while the virus surface proteins VP4 and VP7 rearrange to form the outermost virus protein layer, yielding mature infectious triple-layered particles (TLPs). In this work, we have characterized the role of NSP4 and VP7 in rotavirus morphogenesis by silencing the expression of both glycoproteins through RNA interference. Silencing the expression of either NSP4 or VP7 reduced the yield of viral progeny by 75 to 80%, although the underlying mechanism of this reduction was different in each case. Blocking the synthesis of NSP4 affected the intracellular accumulation and the cellular distribution of several viral proteins, and little or no virus particles (neither DLPs nor TLPs) were assembled. VP7 silencing, in contrast, did not affect the expression or distribution of other viral proteins, but in its absence, enveloped particles accumulated within the lumen of the ER, and no mature infectious virus was produced. Altogether, these results indicate that during a viral infection, NSP4 serves as a receptor for DLPs on the ER membrane and drives the budding of these particles into the ER lumen, while VP7 is required for removing the lipid envelope during the final step of virus morphogenesis.     

Influenza virus hemagglutinin and neuraminidase cytoplasmic tails control particle shape.

The cytoplasmic tails of the influenza virus glycoproteins hemagglutinin (HA) and neuraminidase (NA) are highly conserved in sequence for all virus subtypes and it is believed that assembly of this enveloped virus depends on interactions of these domains with cytoplasmic viral components. However, it is possible to rescue altered influenza viruses lacking either the HA or NA cytoplasmic tails. We have obtained an influenza virus that lacks both the cytoplasmic tail of HA and NA. Particle production is reduced approximately 10-fold but these particles, although having a fairly normal protein composition, are greatly elongated and of extended irregular shape. We propose a model in which the interactions of the cytoplasmic tails of HA and NA with an internal viral component are so important for spherical virion shape that there is dual redundancy in the interactions.