Stability and immunogenicity of empty particles of foot-and-mouth disease virus

Summary Three strains of foot-and-mouth disease virus were shown to contain significant amounts of naturally occurring 75 S, empty particles as well as the infectious, 140 S full particles. One of these strains — A Pando (1970) — was studied in detail.The empty particles from this virus strain were shown to have an observed sedimentation coefficient of 67S in 0.04m phosphate buffer; they were labile in SDS, non-infectious and probably RNA-free and, on heating, they broke down to 12 S subunits as did the 140 S particles. The empty particles differed from the full particles in their polypeptide composition since they contained VP₀, but there was no evidence for a diminished content of VP₄.The 75 S particles were shown to be present in significant amounts and to be stable to AEI inactivation. At 4° C they were stable for at least two years. In guinea pigs they were as immunogenic as the 140 S particles. The antisera raised against the 75 S particles had the same serological specificity in neutralization tests as sera prepared against the 140 S particle. It was concluded that the 75 S particles from the A Pando (1970) strain of FMD virus may provide as important a contribution as 140 S particles to the immunogenicity of inactivated vaccines prepared from this virus strain.With 5 Figures     

Characterization of three antigenic particles of swine vesicular disease virus.

Three distinct particles were isolated from cell culture harvests of swine vesicular disease virus (SVDV) by sucrose and CsCl gradient centrifugation. Virions (148S), RNA-free empty capsids (81S), and a third particle (49S) also free of RNA showed immune reactivity with SVDV antiserum. The 81S and 49S particles had polypeptides typical of naturally occurring empty capsids. Injection of purified antigens into guinea pigs produced antisera which distinguished empty capsids from virions on immunodiffusion; the 49S antigen appeared similar to virions. Antisera produced to freshly prepared virus antigen grown in brains of baby mice distinguished SVDV from the serologically related Coxsackie B-5 virus but did not distinguish the individual S particle antigens. Partly purified virus preparations degraded to empty capsids when incubated in guinea pig serum. The possible origin of empty capsids and 49S particles and their relationship to antigenicity of virus preparations are discussed.     

Identification and partial characterization of a new (50 S) subviral particle in Mengo virus-infected L cells.

A previously undetected subviral particle has been found in Mengo virus-infected L cells by sucrose density gradient centrifugal analysis of cytoplasmic supernatants (S₂₀) prepared from cells after labeling with [³H]amino acids during the early to mid-log phase of virus production. The particle (designated the “50 S particle” from its position between the ribosomal subunits in the gradient), together with mature virions (150 S) and previously described 14 S particles (McGregor et al., 1975), can be recovered from the S₂₀ fraction by high-speed centrifugation. It contains no RNA and is composed of equimolar amounts of the polypeptides ?, α, and γ. The results of conventional pulse-chase experiments suggest that it may be a precursor in the assembly of Mengo virions, but more convincing evidence that this is the case was obtained from experiments in which the chase was carried out in the presence of cordycepin (3′-deoxyadenosine). In the presence of this inhibitor of viral RNA synthesis, there is a significant accumulation of 50 S particles, and when the inhibition in reversed, a quantitative transfer of radiolabel from 50 S particles to mature virions ensues. The recovery of 50 S particles (and of mature virions) from cell homogenates is strongly dependent upon the concentration of KCl in the suspending buffer; only trace amounts are recovered at concentrations of less than 60 mM, while maximum recovery is achieved at a concentration of 100 mM.     

Barley stripe mosaic virus in sperm and vegetative cells of barley pollen

We have developed a method that allows us to isolate with high yield RNP particles containing intact RNA from BHK21 cells infected with Semliki Forest virus. Two types of virus-specific RNA sedimenting with about 42 S and 26 S on sucrose gradients are found in the gradient fractions containing the large and intermediate-sized polyribosomes. That the 26 S RNA and at least 50% of the 42 S RNA are presumably part of the polyribosomes has been shown by CsCl density gradient analysis of the fixed ribonucleoprotein particles. We suggest therefore that both molecules are virus specific mRNA. From the quantitative aspects of our results, we conclude that they are the main species of virus specific polyribosome associated RNA in the infected cells. The total virus-specific RNA synthesized during different 2 hr intervals, which is incorporated into polyribosomes, is greater than 20% during the 0 to 2 hr p.i. interval and decreases later. We suggest therefore that especially during the first hours after infection, a major part of the total RNA synthetic capacity of the virus is directed toward mRNA production. Both polyribosome-associated molecules do not hybridize to RNA extracted from the SFV particle.     

Purification and characterization of retrovirus vector particles by rate zonal ultracentrifugation

Sucrose equilibrium density ultracentrifugation remains the most widely used technique for retrovirus purification. However, purified virus preparations obtained by this routine method usually contain considerable amounts of contaminating cell membrane vesicles. In addition, sucrose solutions are highly viscous and hyperosmotic which jeopardizes the integrity and functionality of the retrovirus particle. In order to overcome these limitations, an alternative purification technique using rate zonal ultracentrifugation and iodixanol as gradient medium was developed. Recombinant retrovirus particles were produced by 293-GPG packaging cells grown in suspension in the presence of 10% FBS. Concentrated supernatants were purified by rate zonal sedimentation on a 10-30% continuous iodixanol gradient. Virus particles were recovered intact and active from the central fractions of the gradient. By using this strategy, high levels of purification were achieved, with no evident contamination with cell membrane vesicles as indicated by subtilisin treatment studies. The level of purity of the retrovirus preparation is over 95% as shown by SDS-PAGE analysis and size-exclusion chromatography. Purified particles appear homogenous in size and morphology according to negative stain electron microscopy. In addition, large amounts of defective retrovirus particles produced by 293-GPG packaging cells can be separated from functional retrovirus particles using this purification strategy.     

Characterization of the minor polypeptides in the foot-and-mouth disease particle.

In addition to the four major polypeptides VP1 and VP4, foot-and-mouth disease virus particles contain two minor polypeptides, mol. wt. 40 X 10(3) (P40) and 52 X 10(3) (P52). Extensive purification procedures failed to remove these minor polypeptides from the virus particles. Polypeptide P40 co-electrophoresed in SDS-polyacrylamide gels with VP0, the probable precursor of VP2 and VP4 and was inaccessible to iodination in situ. The second minor polypeptide, P52, co-electrophoresed with the virus infection associated (VIA) antigen found in large amounts in harvests of the virus grown in BHK 21 cells. Polypeptide P52 was shown to be located near the surface of the virus particle by iodination experiments and by its removal on incubating the particles with trypsin or chymotrypsin. Pactamycin mapping showed that this polypeptide was not a precursor of the structural polypeptides. About one copy of P52 and 4 copies of P40 were found in the virus particles sedimenting at 146S. However a larger number of copies was found in those virus particles sedimenting faster than the 146S peak.     

Binding of cowpea chlorotic mottle virus to cowpea protoplasts and relation of binding to virus entry and infection

Cowpea chlorotic mottle virus (CCMV) and cowpea protoplasts were used to study initial interactions between virus and protoplast. Protoplasts and virus were incubated under varying conditions of temperature, pH, ionic strength, and the presence of added compounds. Both the amount of 35S-labeled virus bound to protoplasts and the percentage of infected cells were determined. At 0 and 25 degrees the amount of virus associated with protoplasts increased with the amount of virus added. With inoculum of 25 x 10(6) virus particles per protoplast, 4 x 10(3) and 14 x 10(3) particles per protoplast were bound at 0 and 25 degrees, respectively. In the presence of polyethylene glycol, 85 x 10(3) associated particles per protoplast were bound at both temperatures and ca. 50% of the protoplasts became infected. No infection occurred in the absence of PEG. Variation of pH or ionic strength in the absence of PEG caused little to no change in binding and no infection. In the presence of PEG, increase of pH resulted in lower binding, but infectivity was not affected. Increasing ionic strength, however, increased both binding and infectivity. The presence of unlabeled CCMV, tobacco mosaic virus coat protein, bovine serum albumin, and polycations during inoculation in the absence of PEG decreased the amount of bound CCMV. In contrast, CCMV coat protein, which has a positively charged N-terminal arm, increased binding. In the presence of PEG the effects were similar, although larger amounts of virus were bound. The percentage of infection was reduced by all additives to 5-25%. Addition of ammonium chloride, which inhibits endocytotic virus uptake in animal cells, during inoculation as well as in culture media, did not reduce infectivity. These data do not support a specific receptor-mediated endocytotic uptake of virus but favor a nonspecific mechanism of entry, possibly through membrane lesions. Observations in the electron microscope support the latter mechanism.     

In vitro assembly of polioviruses. 3. Assembly of 14 S particles into empty capsids by poliovirus-infected HeLa cell membranes

Rough membranes obtained from poliovirus-infected HeLa cells have the capacity to assemble 14 S particles into 73 S empty capsids in vitro. A corresponding fraction from uninfected cells did not possess this activity. When labeled 14 S particles were incubated with smooth membranes obtained from infected cells, a significant amount of radioactivity sedimented as heterogeneous material in the 30–70 S region of the gradient.Sucrose gradient analysis of ¹⁴C-labeled rough and smooth membrane fractions lysed with deoxycholate (DOC) demonstrated the presence of 14 S particles, 73 S empty capsids, and a 110 S structure in the rough membrane fraction. These structures were found only in trace amounts in the smooth membrane fraction. In the absence of DOC treatment, no 14 S particles were found in any of the membrane fractions. The 73 S empty capsids, on the other hand, were detected in the presence or absence of DOC treatment in the rough-membrane fraction. Therefore, it appears that 14 S particles are associated with the rough membranes where they are assembled into 73 S empty capsids and/or complete virions.     

Identification and protein analysis of polyomavirus assembly intermediates from infected primary mouse embryo cells

A method is described for the isolation of polyoma virus assembly intermediates from infected mouse embryo cells. Sucrose gradient profiles revealed the presence of 90 S, 200 S, and 240 S intermediates. These intermediates were shown to be sensitive to a number of factors: ionic condition of the isolation buffer, presence of chelating agents and nonionic detergents during isolation, and sonication of nuclei during extraction of intermediates. Pulse-chase experiments demonstrated that the order of formation of the intermediates to be 90 S----240 S, with the 200 S particles as a possible intermediate form linking the 90 S and 240 S particles. Viral structural proteins VP1, VP2, and VP3 were shown to be present on all three intermediates, but the ratio of each protein varied on each intermediate species. Two-dimensional gel electrophoresis demonstrated that the distribution of the VP1 isoelectric focusing species were different among the three intermediates. Histone H1 was found exclusively with the 90 S species.     

Purification and electron microscopy of lactic dehydrogenase virus of mice.

Lactic dehydrogenase virus (LDV) was purified from infectious ascites fluid of mice bearing Ehrlich tumours using Sepharose gel filtration and rate zonal and isopycnic sedimentation. In glycerol gradients, a sedimentation coefficient of about 200S and a buoyant density of 1-14 g/ml was determined for the virus particle. Spherical particles with diam. between 62 and 80 nm, depending on the method of fixation and staining, have been identified electron microscopically. The virus particle consists of a spherical nucleocapsid wrapped into a double-layered envelope. The nucleocapsids, isolated by treatment with NP40 and purified by centrifuging on sucrose gradients had a sedimentation coefficient of 176S. Electron micrographs show spherical particles with a diam of 35 plus or minus nm. Classification of LDV as a member of the togaviridae family is discussed.     

RNA contents of abnormally long particles of certain strains of alfalfa mosaic virus

A nucleoprotein particle longer than bottom component occurring in small amounts in preparations of the alfalfa mosaic virus (AMV) strain $\text{15}\text{64}$ has been partially purified by gradient centrifugation and characterized. The particle has the same shape, width, and RNA percentage as the major components of the virus. Its length and sedimentation coefficient are 87 nm and 113 S, respectively. It does not contain a single abnormally long RNA molecule, but two molecules of middle component RNA. Electrophoresis in polyacrylamide gel revealed that in addition to this type of particle other nucleoproteins containing different sets of RNA molecules occur in trace amounts in preparations of strain $\text{15}\text{64}$.Particles with a length of up to about 400 nm of strain VRU have also been studied after they were freed from normal length particles by gradient centrifugation. In accordance with the results of others it has been found that the abnormally long particles did not contain any RNA molecules longer than bottom component RNA.     

Further characterization of Mengo subviral particles: a new hypothesis for picornavirus assembly.

The “50S particle” found in Mengo virus-infected L cells (P. W. K. Lee, E. Paucha, and J. S. Colter, 1978, Virology 85, 286–295) has been further characterized. Its sedimentation coefficient has been estimated to be 53 S from centrifugal analysis in sucrose density gradients. When, during the isolation of 53 S particles, the KCI concentration in the suspending buffer is increased to 150 mM or higher, some of the particles are converted to structures having a significantly larger sedimentation coefficient. A similar conversion of 53 S to more rapidly sedimenting particles occurs when the former are centrifuged to equilibrium in a CsCI density gradient. The sedimentation coefficient of this new particle has been estimated to be 75 S. Molecular weight determinations of the previously described 14 S particles and of the 53 and 75 S particles by means of Sepharose 4B exclusion chromatography suggest that the molecular compositions of these particles are (?αγ)₅, (?αγ)₂₅, and (?αγ)₅₀, respectively. Based on this information and the previously reported evidence suggesting a precursor role for the 53 S particles, a new hypothesis regarding the mechanism of Mengo virus assembly has been proposed. In this model, the viral RNA interacts with either a 75 S particle or two 53 S particles to form a complex represented by RNA[(?αγ)₅]₁₀, before assembly is completed by the addition of two 14 S subunits.     

Isolation and characterization of mitochondrial ribosomes and ribosomal subunits from Leishmania tarentolae

We have analyzed Leishmania tarentolae mitochondrial ribonucleoprotein (RNP) complexes using the 9S small subunit (SSU) rRNA and the 12S large subunit (LSU) rRNA as markers, and have identified a 50S RNP particle as the putative mitochondrial monosome, a 40S particle as the putative LSU and a 30S particle as the putative SSU. These assignments are supported by morphological analysis by cryo-electron microscopy and proteomics analyses by mass spectrometry. The presence of additional rRNA-containing particles complicated the analysis and most likely was the basis for previous difficulties in identification of these ribosomes; thus, in addition to the monosomes and their subunits, there are abundant stable 45S particles (SSU(*)) containing only 9S rRNA, which may represent homodimers of the SSU or SSU associated with additional proteins, and variable minor amounts of 65S and 70S particles, which represent homodimers of the LSU and SSU(*), respectively. These additional rRNA particles might be due to the lengthy mitochondrial isolation and ribosome isolation procedures or may be present in vivo and play yet undetermined roles.     

Hydrophilic and cationic latex particles for the specific extraction of nucleic acids

The adsorption of BSA and RNA onto hydrophilic and thermosensitive poly(N-isopropyl-acrylamide) (NIPAM) latex particles was described as a function of pH, ionic strength and temperature. The hydrogel poly(NIPAM) latex was synthesized by precipitation polymerization in the presence of a cationic amino-containing monomer. The latex obtained was characterized in terms of particle size, and electrophoretic mobility as a function of pertinent variables: pH, temperature and ionic strength. The adsorption of BSA onto the latex was investigated to identify the conditions at which the adsorbed amount of BSA was negligible. The adsorption of RNA was studied to establish the conditions which give rise to maximal adsorption of RNA. In order to favor the desorption of RNA, desorption was investigated by changing the pH, ionic strength, and temperature. The adsorption of BSA was found to be lower at 20 than at 40 degrees C. However, the adsorption of RNA is drastically affected by the pH and the ionic strength of the medium. Maximal adsorbed amounts were obtained at acidic pH, 20 degrees C, and low ionic strength. The adsorption is shown to decrease when the pH, temperature and ionic strength increase, implying that the adsorption was mainly governed by electrostatic interactions. Maximal release of RNA molecules was obtained at high ionic strength and basic pH.     

Biophysical studies of reovirus type 3. II. Properties of the hydrated particle

The hydrodynamic radii of reovirus, top component, sub-viral particles, and core particles have been determined by dynamic light scattering under various conditions of pH, temperature, and ionic strength. The hydrodynamic radius of virions and top component increased when the ionic strength of the suspending medium was decreased. This process was found to be reversible. In contrast the hydrodynamic radius of viral cores was not influenced by the ionic strength of the suspending medium. The frictional coefficient of core particles has been determined by direct experimental measurement using macroscopic models and the size of hydrated core particles determined from the measured frictional coefficient. Core particles were found to be dense rigid particles of low water content having the same size under all conditions investigated. Intact virus particles were found to be much larger when hydrated than when dehydrated having a large water content and low average density. The measurements lead to an outline of the structure of hydrated reovirus.     

Aggregation states of Brome mosaic virus protein

The self-assembly of Brome mosaic virus protein has been studied as a function of pH and ionic strength at 22°. At ionic strengths above 0.25, the dissociated protein (dimers of the subunit) polymerized quantitatively below pH 5 into empty capsids or PTC (pseudo top component) in a fully reversible manner. The threshold of reassociation was displaced to higher pH values with increasing ionic strength. Once formed, PTC particles could as well be dissociated into ribbons of protein at low pH, but at low ionic strength only; in the latter configuration, BMV protein was unable to form shells upon reverting to initial conditions. Filamentous structures also formed from dissociated protein when reassociation was performed at low ionic strength; these ribbons too could hardly, if ever, give rise to PTC particles upon shifting to conditions promoting shell formation. Here again, an increase in ionic strength at constant pH resulted in a higher degree of polymerization, suggesting a major role for hydrophobic forces in the formation of organized structures from BMV protein subunits.     

Purification and properties of virus particles, infectious subviral particles, and cores of bluetongue virus serotypes 1 and 4

Effective purification methods have been developed for virus particles, infectious subviral particles (ISVP), and virus cores of bluetongue virus (BTV) serotypes 1 and 4. The purified particles were analysed by indirect ELISA or PAGE using either silver staining, or fluorography of [35S]methionine-labelled preparations. No significant contamination with host cell proteins, or with the majority of BTV nonstructural proteins was detectable in any of the particle preparations. In addition to the two major outer capsid and five core proteins previously described, the purified virus particles of both serotypes were consistently found to contain small amounts of BTV protein NS2, previously regarded as exclusively nonstructural. This protein could be removed from the particle surface by treatment with a combination of chymotrypsin and sodium N-lauroyl sarcosinate, which also resulted in the cleavage of the larger of the two major outer capsid components (protein VP2). Two of the cleavage products of VP2 and the whole of the other major outer capsid component (protein VP5) formed a modified outer capsid layer in the resultant ISVP. These subviral particles were as or more infectious than the intact virus particles but had lost haemagglutinating activity. The core-associated RNA polymerase remained inactive in ISVP.     

In vitro assembly of poliovirus. II. Evidence for the self-assembly of 14 S particles into empty capsids

A small (14S) virus-specific particle, isolated from poliovirus-infected HeLa cells, is able to self-assemble into a 73S particle. This 73S particle, when negatively stained and examined in the electron microscope, resembles the empty capsids found in infected cells. The kinetics of the self-assembly reaction was determined and found to be similar, but not identical, to the extract-mediated assembly of 14S particles into 73S particles. The self-assembly reaction, unlike the extract-mediated reaction, exexhibits a marked dependence on the initial concentration of 14S particles. The polypeptide content of 14S particles has been determined.     

Virus-ribosome complexes from cell-free translation systems supplemented with cowpea chlorotic mottle virus particles

When particles of cowpea chlorotic mottle virus (CCMV) were added to cell-free extracts from wheat germ, the encapsidated viral genome was translated into polypeptides similar to the translation products specified by unencapsidated viral RNA (as shown before by M.J. Brisco, R. Hull, and T.M.A. Wilson, 1986, Virology 148, 210-217). The rate of protein synthesis observed upon addition of virus particles was much slower than that of extracted RNA and the quantity of protein formed was only 10% of that of extracted RNA. Using sucrose and cesium-chloride gradient analysis, virus-ribosome complexes, containing up to four ribosomes per virus particle, were isolated from translation mixtures supplemented with CCMV particles. These complexes, with densities intermediate of those of virus (1.36 g cm-3) and ribosomes (1.58 g cm-3), were analyzed and quantified in the electron microscope. Less than 5% of the particles was found in association with ribosomes. To verify whether these complexes were involved in the process of cotranslational disassembly, tobacco mosaic virus was analyzed with the same techniques and methods. The results found for TMV were similar to those found for CCMV except that virus-ribosome complexes with up to 20 ribosomes per virus particle were observed. The implications of the process of virion-directed translation for the structure of the particle as well as the role of this process in vivo are discussed.     

In vitro assembly of poliovirus: V. Evidence that the self-assembly activity of 14 S particles is independent of extract assembly factor(s) and host proteins

Experiments were performed to determine whether or not the self-assembly of 14 S precursor particles into empty capsids was caused by the contamination of certain 14 S isolates with assembly factor(s) present in poliovirus-infected cell extracts. The self-assembly capacity of 14 S particle preparations was found to be directly related to the amount of viral-specific protein present. Dilution of 14 S particles markedly inhibited their self-assembly activity, whereas using ultrafiltration methods to concentrate 14 S particles increased their self-assembly activity. No consistent relationship was found for the presence or absence of particular viral or host proteins and the ability of 14 S particle preparations to self-assemble. The self-assembly capacity of 14 S particles was more sensitive to uv-inactivation than their ability to assemble into empty capsids in the presence of infec cted cell extracts. On the basis of these data and a detailed reanalysis of the effect of relative initial 14 S particle concentration on the rate of formation of empty capsids in extracts, we propose that the assembly of 14 S particles into empty capsids occurs in two steps, an initiation event and subsequent polymerization, and that extracts act by promoting the initiation event.