The study of amorphous aggregation of tobacco mosaic virus coat protein by dynamic light scattering

The kinetics of heat-induced and cetyltrimethylammonium bromide induced amorphous aggregation of tobacco mosaic virus coat protein in Na(+)/Na(+) phosphate buffer, pH 8.0, have been studied using dynamic light scattering. In the case of thermal aggregation (52 degrees C) the character of the dependence of the hydrodynamic radius (R(h)) on time indicates that at certain instant the population of aggregates is split into two components. The size of the aggregates of one kind remains practically constant in time, whereas the size of aggregates of other kind increases monotonously in time reaching the values characteristic of aggregates prone to precipitation (R(h)=900-1500 nm). The construction of the light scattering intensity versus R(h) plots shows that the large aggregates (the start aggregates) exist in the system at the instant the initial increase in the light scattering intensity is observed. For thermal aggregation the R(h) value for the start aggregates is independent of the protein concentration and equal to 21.6 nm. In the case of the surfactant-induced aggregation (at 25 degrees C) no splitting of the aggregates into two components is observed and the size of the start aggregates turns out to be much larger (107 nm) than on the thermal aggregation. The dependence of R(h) on time for both heat-induced aggregation and surfactant-induced aggregation after a lapse of time follows the power law indicating that the aggregation process proceeds in the kinetic regime of diffusion-limited cluster-cluster aggregation. Fractal dimension is close to 1.8. The molecular chaperone alpha-crystallin does not affect the kinetics of tobacco mosaic virus coat protein thermal aggregation.     

Alterations of the conformation of the RNAs of alfalfa mosaic virus upon binding of a few coat protein molecules

Structural changes in the single-stranded genome RNAs (RNAs 1, 2 and 3) and the subgenomic coat protein messenger (RNA 4) of alfalfa mosaic virus upon addition of a few coat protein molecules of the virus were investigated by measuring the fluorescent intensity of bound ethidium bromide and by circular dichroism. No effect could be observed in the case of the genome RNAs. However, in RNA 4, which is of much less complexity than the genome RNAs, a reduction of the ethidium bromide binding by 30% was found, whereas the positive molar ellipticity at 265 nm was reduced by 9% upon binding of the coat protein. Both changes point to a reduction of the ordered structure of the RNA. Since the protein is known to bind first at the 3′-terminus of RNA 4 and probably also of the genome RNAs, the conformational changes observed could be those thought to be necessary for replicase recognition in this positive-stranded RNA virus which needs the coat protein for starting an infection cycle.     

Low sodium dodecyl sulfate concentrations inhibit tobacco mosaic virus coat protein amorphous aggregation and change the protein stability

Effects of low SDS concentrations on amorphous aggregation of tobacco mosaic virus (TMV) coat protein (CP) at 52 degrees C and on the protein structure were studied. It was found that SDS completely inhibits the TMV CP (11.5 microM) unordered aggregation at the detergent/CP molar ratio of 15 : 1 (0.005% SDS). As judged by fluorescence spectroscopy, these SDS concentrations did not prevent heating-induced disordering of the large-distance part of the TMV CP subunit, including the so-called "hydrophobic girdle". At somewhat higher SDS/protein ratio (40 : 1) the detergent completely disrupted the TMV CP hydrophobic girdle structure even at room temperature. At the same time, these low SDS concentrations (15 : 1, 40 : 1) strongly stabilized the structure of the small-distance part of the TMV CP molecule (the four alpha-helix bundle) against thermal disordering as judged by the far-UV (200-250 nm) CD spectra. Possible mechanisms of TMV CP heating-induced unordered aggregation initiation are discussed.     

Comparative structural stability of subunits of the potato virus X coat protein in solution and in virus particles

Several optical methods and differential scanning calorimetry were used to study the structure and stability of free coat protein (CP) molecules and CP molecules in the virion of the potato virus X (PVX), a filamentous plant virus. All criteria suggest that PVX CP (hereinafter, CP) subunits in solution at room temperature display a certain preserved tertiary structure; however, this structure is very unstable and already denatures at 35°C. Very low concentrations of sodium dodecylsulfate or cetyltrimethylammonium bromide also disrupt the CP tertiary structure, three-five molecules of these detergents per one protein molecule being sufficient. However, the secondary structure of CP molecules does not change under the same conditions. Once included into the virion, CP subunits become considerably more stable towards increased temperature and detergents. This combination of a highly labile tertiary structure and a fairly stable secondary structure of free CP can be a structural basis for the recently discovered ability of PVX CP to assume two distinct functional states within the virion.     

Modification of Tobacco Mosaic Virus by Polyornithine and Lecithin

Combined action of polyornithine and lecithin modified tobacco mosaic virus (TMV) virions making them sensitive to ribonuclease (RNase), pronase or Triton X-100. Sedimentational analysis and examination of the fluorescence spectrum revealed that the reaction product obtained after RNase treatment of modified TMV was a three-component complex made of coat protein, polyornithine and lecithin. The minimum requirement for the modification was completely fulfilled by cetyltrimethylammonium bromide, suggesting that a positively charged nitrogen group and an alkyl group of moderate size, C_10–18, are necessary components. These components react with the surface region of TMV which is considered to have an important role in connecting coat protein subunits in TMV virions.     

A mechanism of macroscopic (amorphous) aggregation of the tobacco mosaic virus coat protein

To gain more insight into the mechanisms of heating-induced irreversible macroscopic aggregation of the tobacco mosaic virus (TMV) coat protein (CP), the effects of pH and ionic strength on this process were studied using turbidimetry, CD spectroscopy, and fluorescence spectroscopy. At 42 degrees C, the TMV CP passed very rapidly (in less than 15s) into a slightly unfolded conformation, presumably because heating disordered a segment of the subunit where the so-called hydrophobic girdle of the molecule resides. We suppose that the amino acid residues of this girdle are responsible for the aberrant hydrophobic interactions between subunits that initiate macroscopic protein aggregation. Its rate increased by several thousands of times as the phosphate buffer molarity was varied from 20 to 70 mM, suggesting that neutralization of strong repulsive electrostatic interactions of TMV CP molecules at high ionic strengths is a prerequisite for amorphous aggregation of this protein.     

Subcellular localization of the coat protein in tobacco cells infected by cucumber mosaic virus isolated from Catharanthus roseus

Electron microscopy and immunolabelling with antiserum specific to cucumber mosaic virus coat protein were used to examine tobacco leaf cells infected by cucumber mosaic virus isolated from Catharanthus roseus (CMV-Cr). Crystalline and amorphous inclusions in the vacuoles were the most obvious cytological modifications seen. Immunogold labelling indicated that the crystalline inclusion was made up of virus particles and amorphous inclusions contained coat protein. Rows of CMV-Cr particles were found between membranes of dictyosomes, but membranous bodies and tonoplast-associated vesicles were not evident. Virus particles and/or free coat protein were easily detected in the cytoplasm by immunolabelling. No gold labelling was found within nuclei, chloroplasts and mitochondria.     

Oligonucleotide binding to the coat protein disk of tobacco mosaic virus. Possible steps in the mechanism of assembly

Binding of the oligoribonucleotides AAG, AAGAAG and AAGAAGUUG to the disk aggregate of tobacco mosaic virus coat protein has been studied in solution under conditions favourable for virus assembly. The two longer oligomers bind strongly with Kd around 1 microM, approach complete saturation of binding sites and cause the formation of long, nicked helical rods resembling the virus. It is suggested that the binding of these oligomers, with sequences chosen from the assembly origin of the viral RNA, simulates the tobacco mosaic virus assembly process. No binding could be detected for AAG, indicating that chain length is a crucial determinant in the interaction. The binding of AAGAAG to coat protein crystals is very much weaker than that observed in solution, and the crystals crack at high oligomer concentrations. The corresponding oligodeoxyribonucleotide, d(AAGAAG), shows no binding to the protein in solution; the interaction is extremely specific for RNA.     

Biological, Serological and Coat Protein Properties of a Strain of Turnip Mosaic Virus Causing a Mosaic Disease of Brassica campestris and B. juncea in India

Biological, serological and coat protein properties of a potyvirus (Poty-Rape) causing a mosaic disease of Brassica campestris and B. juncea in India were investigated. The virus readily infected 4 of the 5 plant species in the family Brassicaceae in which it induced severe systemic mosaic symptoms; it also induced chlorotic and necrotic local lesions in Chenopodium amaranticolor , but failed to infect 4 other species of Chenopodiaceae or 20 species of Amaranthaceae, Apiaceae, Canabinaceae, Compositae, Cucurbitaceae, Euphorbiaceae, Leguminosae and Solanaceae. The virus was transmitted in a non-persistant manner by Myzus persicae, Brevicoryne brassicae and Aphis gossypii. The Average size, of the virus particles in a purified preparation was 740 nm × 12 nm. SDS-PAGE analysis of the viral coat protein showed two major bands of approximately 37 kDa and 31 kDa, a pattern very similar to that of a reference isolate of turnip mosaic virus (TuMV) from the U.S. In Western-blot immunoassay, an antiserum to TuMV reacted with both the coat protein bands of the Poty-Rape islate and the reference TuMV, but not with the coat proteins of four other potyviruses. The high performance liquid chromatographic profile of tryptic peptides from the coat protein of Poty-Rape was found to be very similar to that of the reference TuMV, but differed substantially from those of four other potyviruses. The Poty-Rape isolate is considered to be a distinct strain of, TuMV.     

A barley lectin that binds free amino sugars I. Purification and characterization

A lectin was isolated from barley seed which bound the coat glycoprotein of barley stripe mosaic virus (Type strain) and precipitated the virus from solution. Purification of the barley lectin was achieved by fractionation with ammonium sulfate and successive column chromatography on DEAE cellulose and cellulose phosphate. The barley lectin was homogeneous as ascertained by polyacrylamide gel electrophoresis, isoelectric focusing, and from immunochemical tests. No isolectins were detected. The lectin has a molecular weight of 31 000 daltons and is not a glycoprotein. Each virion can accomodate between 200 to 300 molecules of lectin. Barley lectin was shown to be specific for D-glucosamine, D-galactosamine and D-mannosamine with little distinction among the epimeric configurations at carbons 2 and 4. Free amino groups of D-glucosamine and D-galactosamine were detected on the coat glycoprotein of Type strain barley stripe mosaic virus and these sugars appear to serve as receptors for the barley lectin.     

Thiopental enhances human platelet aggregation by increasing arachidonic acid release.

Conflicting results have been reported regarding the effect of thiopental on aggregation and cytosolic calcium levels in platelets. The present study attempted to clarify these phenomena. Using platelet-rich plasma or washed suspensions, platelet aggregation, thromboxane (TX) B2 formation, arachidonic acid (AA) release, and cytosolic free calcium concentrations ([Ca2+]i) were measured in the presence or absence of thiopental (30-300 microM). Platelet activation was induced by adenosine diphosphate (ADP, 0.5-15 microM), epinephrine (0.1-20 microM) arachidonic acid (0.5-1.5 mM), or (+)-9,11-epithia-11,12-methano-TXA2 (STA2, 30-500 nM). Measurements of primary aggregation were performed in the presence of indomethacin (10 microM). Low concentrations of ADP and epinephrine, which did not induce secondary aggregation in a control study, induced strong secondary aggregation in the presence of thiopental (> or = 100 microM). Thiopental (> or = 100 microM) also increased the TXB2 formation induced by ADP and epinephrine. Thiopental (300 microM) increased ADP- and epinephrine-induced 3H-AA release. Thiopental (300 microM) also augmented the ADP- and epinephrine-induced increases in [Ca2+]i in the presence of indomethacin. Thiopental appears to enhance ADP- and epinephrine-induced secondary platelet aggregation by increasing AA release during primary aggregation, possibly by the activation of phospholipase A2.     

Physical regulation of the self-assembly of tobacco mosaic virus coat protein

We present a statistical mechanical model based on the principle of mass action that explains the main features of the in vitro aggregation behavior of the coat protein of tobacco mosaic virus (TMV). By comparing our model to experimentally obtained stability diagrams, titration experiments, and calorimetric data, we pin down three competing factors that regulate the transitions between the different kinds of aggregated state of the coat protein. These are hydrophobic interactions, electrostatic interactions, and the formation of so-called "Caspar" carboxylate pairs. We suggest that these factors could be universal and relevant to a large class of virus coat proteins.     

Translation of the RNAs of brome mosaic virus: The monocistronic nature of RNA1 and RNA2

Each of the two largest brome mosaic virus RNAs, RNA1 and RNA2, directs the synthesis of a large protein in cell-free extracts derived from wheat embryo. The size of each protein represents the translation of almost the entire length of the corresponding RNA. It was shown previously that brome mosaic virus RNA4 directs the synthesis of the coat protein and that brome mosaic virus RNA3, although it also contains the coat protein cistron, is translated mostly into a single product unrelated to the coat protein (Shih & Kaesberg, 1973). Thus, the brome mosaic virus genome encodes a total of four proteins.     

Spectroscopic studies on the comparative interaction of cationic single-chain and gemini surfactants with human serum albumin

To gain insights into the comparative effect of single-chain/gemini surfactants on proteins and the possible implications, the interaction of human serum albumin (HSA) with cationic single-chain surfactant cetyltrimethylammonium bromide (CTAB) and its gemini counterpart bis(cetyldimethylammonium)butane dibromide with spacer -(CH(2))(4)- (designated as G4) using turbidity measurements, far-UV and near-UV circular dichroism (CD), intrinsic fluorescence and extrinsic fluorescence spectroscopy at pH 7.0 are reported in this contribution. A decrease of 33.5% alpha-helical content at 22.5 microM G4 was monitored compared to a 15% decrease at 2,250 microM CTAB. Against a 3.5% increase at 11,250 microM CTAB, a rise of 21.1% in the alpha-helical content was observed 375 microM G4. The result is related to the stronger electrostatic and hydrophobic forces in G4, owing to the presence of two charged headgroups and two hydrophobic hydrocarbon tails that make it to bind strongly to the protein compared to its single chain counterpart, CTAB, resulting in larger unfolding. The stabilization at higher concentrations is attributed to the highly hydrophobic microdomain of the G4 aggregates formed at such concentrations. The results of the multi-technique approach are consistent with the fact that the gemini surfactants are more efficient than their conventional single-chain counterparts and hence may be used more effectively in the renaturation of proteins produced in the genetically engineered cells via the artificial chaperone protocol, as solubilizing agents to recover proteins from insoluble inclusion bodies and in drug delivery.     

A Recombinant Rotavirus Antigen Based on the Coat Protein of Alternanthera Mosaic Virus

Thanks to their strong immunostimulating properties and safety for humans, plant viruses represent an appropriate basis for the design of novel vaccines. The coat protein of Alternanthera mosaic virus can form virus-like particles that are stable under physiological conditions and have adjuvant properties. This work presents a recombinant human rotavirus A antigen based on the epitope of rotavirus structural protein VP6, using Alternanthera mosaic virus coat protein as a carrier. An expression vector containing the gene of Alternanthera mosaic virus (MU strain) coat protein fused to the epitope of rotavirus protein VP6 was designed. Immunoblot analysis showed that the chimeric protein was effectively recognized by commercial polyclonal antibodies to rotavirus and therefore is a suitable candidate for development of a vaccine prototype. Interaction of the chimeric recombinant protein with the native coat protein of Alternanthera mosaic virus and its RNA resulted in the formation of ribonucleoprotein complexes that were recognized by anti-rotavirus antibodies.     

The interaction of sodium dodecyl sulfate with cucumber green mottle mosaic virus protein and tobacco mosaic virus protein

The binding of sodium dodecyl sulfate to coat protein subunits of cucumber green mottle mosaic virus and tobacco mosaic virus was studied by equilibrium dialysis. The amount of dodecyl sulfate bound to the cucumber virus protein in 0.1 m phosphate buffer (pH 7.2) was found to be 1.55 g/g, which was the same value as that obtained with the tobacco virus protein. The presence of 8 m urea markedly decreased the degree of binding of dodecyl sulfate to the proteins. The amount of binding to the cucumber virus protein was reduced to 0.56 g/g, and that to the tobacco virus protein decreased to 0.8 g/g. The net charges of both proteins were negative at neutral pH and the amount of negative charge of the cucumber virus protein, obtained from the potentiometric titration curves, was larger than that of the tobacco virus protein, either in the native state or in the denatured state. In dodecyl sulfate/polyacrylamide gel electrophoresis the cucumber virus protein migrated faster than the tobacco virus protein. On the other hand, in the presence of 8 m urea, the electrophoretic migration rate of the cucumber virus protein was equal to that of the tobacco virus protein. Sedimentation equilibrium experiments in 6 m guanidinium chloride gave molecular weights of 17,700 and 17,200 for the tobacco mosaic virus and the cucumber virus proteins, respectively. These results suggest that the effective negative charge density of the cucumber virus protein-dodecyl sulfate complex is higher than that of the tobacco virus proteindodecyl sulfate complex in 0.1% dodecyl sulfate solution. The conformation of both proteins was investigated by circular dichroism measurements. Both proteins have a slightly higher degree of α-helix content in dodecyl sulfate solution than in the native state. The addition of 8 m urea to both proteins while in this solution induced a change in conformation to one having a much smaller degree of ordered structure, although the change in the cucumber virus protein was more intense than that in the tobacco virus protein.     

Protection against virus infection in tobacco plants expressing the coat protein of grapevine fanleaf nepovirus

Summary Grapevine fanleaf nepovirus (GFLV) is responsible for the economically significant court-noué disease in vineyards. Its genome is made up of two single-stranded RNA molecules (RNA1 and RNA2) which direct the synthesis of polyproteins P1 and P2 respectively. A chimeric coat protein gene derived from the C-terminal part of P2 was constructed and subsequently introduced into a binary transformation vector. Transgenic Nicotiana benthamiana plants expressing the coat protein under the control of the CaMV 35S promoter were engineered by Agrobacterium tumefaciens-mediated transformation. Protection against infection with virions or viral RNA was tested in coat protein-expressing plants. A significant delay of systemic invasion was observed in transgenic plants inoculated with virus compared to control plants. This effect was also observed when plants were inoculated with viral RNA. No coat protein-mediated cross-protection was observed when transgenic plants were infected with arabis mosaic virus (ArMV), a closely related nepovirus also responsible for a court-noué disease.Abbreviations GFLV-F13 grapevine fanleaf virus F13 isolate - ArMV arabis mosaic virus - CP coat protein - MS Murashige and Skoog - NPTII neomycin phosphotransferase II - CaMV cauliflower mosaic virus - ELISA enzyme linked immunosorbent assay - VPg genome linked viral protein - TMV tobacco mosaic virus - PVX potato virus X - PVY potato virus Y - TRV tobacco rattle virus - +CP CP expressing - -CP control plant, not expressing CP - CPMP coat protein-mediated protection - CPMCP coat crotein-mediated cross protection     

Translational stability of plant viral RNAs microinjected into living cells. Influence of a 3'-poly(A) segment

Three different alternative structural features have been shown to be present at the 3' terminus of plant viral RNAs: (a) a poly(A) track, (b) a tRNA-like structure, (c) no special structural or sequence characteristic. We have compared the translational stability after injection into frog oocytes of a representative of each type: (a) the small genomic RNA (M-RNA) of cowpea mosaic virus (CPMV), (b) the subgenomic mRNA for coat protein (RNA 4) of brome mosaic virus (BMV), (c) the subgenomic mRNA for coat protein (RNA 4) of alfalfa mosaic virus (AIMV). It has been shown that CPMV M-RNA exhibits the highest translational stability. However, the stability of AIMV RNA 4 is remarkably high and moreover significantly higher than that of BMV RNA 4. We demonstrate that, for all three viral RNA species considered, the presence of a poly(A) segment at the 3' end of the molecules improves the translational stability. From a comparative investigation in which AIMV RNA 4 was also injected into HeLa cells, it is concluded that the stability of a given non-adenylylated mRNA depends on the nature of the cytoplastic environment.     

Physical and functional heterogeneity in TYMV RNA: evidence for the existence of an independent messenger coding for coat protein.

Turnip yellow mosaic virus RNA can be separated into two distinct components of 2 times 10(6) and 300 000 daltons molecular weight after moderate heat treatment in the presence of SDS or EDTA. The two species cannot have arisen by accidental in vitro degradation of a larger RNA, as they both possess capped 5' ends. Analysis of the newly synthesized proteins resulting from translation of each RNA by a wheat germ extract shows that the 300 000 molecular weight RNA can be translated very efficiently into coat protein. When translated in vitro the longer RNA gave a series of high molecular weight polypeptides but only very small amounts of a polypeptide having about the same mass as the coat protein. Thus our results suggest that the small RNA is the functional messenger for coat protein synthesis in infected cells.