Identification of a peptide of the membrane protein of tobacco mosaic virus, cross-linked with intraviral RNA under the effect of UV-radiation

As reported previously, UV-irradiation induces crosslinking between tobacco mosaic virus (TMV) coat protein molecules and intraviral RNA nucleotides. We have irradiated [3H]-uridine labeled TMV and isolated TMV coat protein subunits with the attached nucleotide label. These TMV protein subunits were hydrolyzed with trypsin. The tryptic peptides were separated by high-performance liquid chromatography and [3H]-labeled peptides were identified. The UV-irradiation of TMV was found to result in crosslinking to intraviral RNA of the T8 tryptic peptide (residues 93-112) of TMV coat protein.     

Possibility to study cell membrane topography using tritium planigraphy

The method of tritium planigraphy was adopted for the investigation of intact cells. Conditions for the incorporation of thermally activated tritium atoms in the erythrocytes are described. The accessibility of erythrocytes hemoglobin for tritium was compared to that of free hemoglobin. By comparing specific radioactivities of amino acids it was shown that the incorporation of the label into free hemoglobin was over 100 times higher than into that in erythrocytes. The cell membrane was highly tritiated. Thus the plasma membrane protects the cell inner regions from penetration of the hot tritium atoms. Tritium planigraphy can be used for studying the cell surface topography.     

In situ spatial organization of Potato virus A coat protein subunits as assessed by tritium bombardment

Potato virus A (PVA) particles were bombarded with thermally activated tritium atoms, and the intramolecular distribution of the label in the amino acids of the coat protein was determined to assess their in situ steric accessibility. This method revealed that the N-terminal 15 amino acids of the PVA coat protein and a region comprising amino acids 27 to 50 are the most accessible at the particle surface to labeling with tritium atoms. A model of the spatial arrangement of the PVA coat protein polypeptide chain within the virus particle was derived from the experimental data obtained by tritium bombardment combined with predictions of secondary-structure elements and the principles of packing alpha-helices and beta-structures in proteins. The model predicts three regions of tertiary structure: (i) the surface-exposed N-terminal region, comprising an unstructured N terminus of 8 amino acids and two beta-strands, (ii) a C-terminal region including two alpha-helices, as well as three beta-strands that form a two-layer structure called an abCd unit, and (iii) a central region comprising a bundle of four alpha-helices in a fold similar to that found in tobacco mosaic virus coat protein. This is the first model of the three-dimensional structure of a potyvirus coat protein.     

Tobacco mosaic virus RNA directs the synthesis of a coat protein peptide in a cell-free system from wheat

Tobacco mosaic virus (TMV) RNA stimulates amino acid incorporation into protein in cell-free extracts from wheat germ, rye embryo and Escherichia coli. The properties of the wheat germ system are examined and the nature of the viral RNA-induced products studied with the aid of a virus mutant carrying a threonine → methionine replacement in its coat protein. A peptide containing this methionine residue is present in tryptic digests of mutant RNA-directed cell-free products, and is absent from digests of wild type RNA-directed products. The undigested cell-free product contains a very large number of polypeptides with molecular weights from 10,000 to 140,000, but little or no synthesis of correct sized coat protein is observed.     

Model System for the Adsorption of Tobacco Mosaic Virus

Materials which can adsorb tobacco mosaic virus (TMV) were isolated from tobacco leaves and studied for applicability as a model system for TMV adsorption. Leaves were homogenized and fractionated by sucrose density gradient centrifugation. One fraction adsorbed TMV in the presence of polyornithine. Deduced from its sensitivity to trypsin and detergent as well as from its manner of isolation, the material responsible for adsorption of TMV seemed to be cytoplasmic membrane. Membrane derived from light particulate, as well as cytoplasmic membrane, seemed to be capable of adsorbing TMV. Shorter rods obtained by sodium dodecyl sulfate or sonic treatment of TMV could adsorb to membrane as efficiently as TMV. Viral protein subunit could not adsorb whereas helical rods made of viral protein aggregates could. A two-step nature of the adsorption of TMV was suggested: a salt-sensitive and a subsequent salt-resistant steps. In the first step, ionic bonding plays a main role in the combination between TMV and membrane. Adsorption of ¹⁴C-labeled TMV was inhibited by an excess amount of non-labeled TMV or cucumber green mottle mosaic virus but not by potato virus X or rice dwarf virus, suggesting the specific nature of adsorption. In contrast to the observed specificity on the part of virus, a membrane fraction isolated from various plants, including non-hosts for TMV, could adsorb TMV. This may imply that adsorption and injection are not the determinant of host specificity in plant viral infection.     

Quantitation of the glycoprotein spike area on the surface of enveloped viruses

The density of glycoprotein (GP) distribution on the virion surface substantially influences the virus infectivity and pathogenicity. A method to quantitatively determine the area occupied by surface GP spikes was proposed for influenza virus (Flu) strain A/PR/8/34 on the basis of data of tritium bombardment and dynamic light scattering. The latter was used to measure the diameter of intact virions and subviral particles (Flu virions lacking GP spikes after bromelain digestion). Intact virions and subviral particles were bombarded with a hot tritium atom flux, and the specific radioactivity of the matrix M1 protein was analyzed. The tritium label was incorporated into the amino acid residues of a thin exposed protein layer and partly penetrated through the lipid bilayer of the viral envelope, labeling M1, located under the lipid bilayer. The tritium label distribution among different amino acid residues was the same in M1 isolated from subviral particles and M1 isolated from intact virions, demonstrating that the M1 spatial structure remained unchanged during proteolysis of GP spikes. The difference in specific radioactivity between the M1 proteins isolated from intact virions and subviral particles was used to calculate the GP-free portion of the viral surface. Approximating the Flu virion as a sphere, the GP-covered area was estimated at 1.4 × 10⁴ nm², about 40% of the total virion surface. This was consistent with the cryoelectron tomography data published for Flu strain A/X-31. The approach can be applied for other enveloped high pathogenic viruses, such as HIV and the Ebola virus.     

A proteinless mutant of tobacco mosaic virus: Evidence against the role of a viral coat protein for interference

Summary A coat-protein-free mutant of tobacco mosaic virus as well as mutants with a non-functional coat protein were found to interfere with the establishment and spread of challenging strains of TMV. The results do not support an earlier concept, according to which the genome of a related challenging virus could be captured by the coat protein of the virus introduced in advance. The presence of a viral coat protein is obviously not essential and a competition among the viral genomes for some specific site seems to be a more likely mechanism of cross protection.A part of the data was presented at the 5th International Congress for Virology, Strassburg, August 2–7, 1981     

外壳蛋白羧端序列缺失对烟草花叶病毒侵染性的影响

对野生型烟草花叶病毒(TMV-U1)的外壳蛋白羧端序列进行系列缺失突变,观察到TMV-U1株系的外壳蛋白羧端序列缺失6个氨基酸(保留152个氨基酸),仍能较强系统侵染烟草并高水平表达外壳蛋白,且能在新生叶里复制大量完整的病毒粒子。该研究结果表明：外壳蛋白羧端6个氨基酸序列并非烟草花叶病毒感染和复制所必需,并对利用外壳蛋白羧端缺失型病毒载体表达外源多肽具有一定的启示性。     

Structures of Helical Plant Virus Ribonucleoproteins as Assessed by Tritium Planigraphy and Theoretical Modeling

This review considers the results of probing the structure of ribonucleoprotein particles of helical plant viruses by tritium planigraphy (TP). This method works by exposing macromolecular targets to a beam of tritium atoms and analyzing the tritium label distribution along the macromolecule length. The TP data combined with theoretical predictions made it possible to propose a structural model of the coat protein for the virions of potato viruses X (the type representative of potexviruses) and A (a potyvirus), which eluded X-ray diffraction analysis so far. TP revealed fine structural differences between the wild-type tobacco mosaic virus (strain U1) and its temperature-sensitive mutant with an altered coat protein and host specificity. The possibilities of using TP for studying the RNA–protein interactions in helical virus particles are discussed.     

The antigenicity of tobacco mosaic virus

The antigenic properties of the tobacco mosaic virus (TMV) have been studied extensively for more than 50 years. Distinct antigenic determinants called neotopes and cryptotopes have been identified at the surface of intact virions and dissociated coat protein subunits, respectively, indicating that the quaternary structure of the virus influences the antigenic properties. A correlation has been found to exist between the location of seven to ten residue-long continuous epitopes in the TMV coat protein and the degree of segmental mobility along the polypeptide chain. Immunoelectron microscopy, using antibodies specific for the bottom surface of the protein subunit, showed that these antibodies reacted with both ends of the stacked-disk aggregates of viral protein. This finding indicates that the stacked disks are bipolar and cannot be converted directly into helical viral rods as has been previously assumed. TMV epitopes have been mapped at the surface of coat protein subunits using biosensor technology. The ability of certain monoclonal antibodies to block the cotranslational disassembly of virions during the infection process was found to be linked to the precise location of their complementary epitopes and not to their binding affinity. Such blocking antibodies, which act by sterically preventing the interaction between virions and ribosomes may, when expressed in plants, be useful for controlling virus infection.     

Spatial structure peculiarities of influenza A virus matrix M1 protein in an acidic solution that simulates the internal lysosomal medium

The structure of the C-terminal domain of the influenza virus A matrix M1 protein, for which X-ray diffraction data were still missing, was studied in acidic solution. Matrix M1 protein was bombarded with thermally-activated tritium atoms, and the resulting intramolecular distribution of the tritium label was analyzed to assess the steric accessibility of the amino acid residues in this protein. This technique revealed that interdomain loops and the C-terminal domain of the protein are the most accessible to labeling with tritium atoms. A model of the spatial arrangement of the C-terminal domain of matrix M1 protein was generated using rosetta software adjusted to the data obtained by tritium planigraphy experiments. This model suggests that the C-terminal domain is an almost flat layer with a three-α-helical structure. To explain the high level of tritium label incorporation into the C-terminal domain of the M1 protein in an acidic solution, we also used independent experimental approaches (CD spectroscopy, limited proteolysis and MALDI-TOF MS analysis of the proteolysis products, dynamic light scattering and analytical ultracentrifugation), as well as multiple computational algorithms, to analyse the intrinsic protein disorder. Taken together, the results obtained in the present study indicate that the C-terminal domain is weakly structured. We hypothesize that the specific 3D structural peculiarities of the M1 protein revealed in acidic pH solution allow the protein greater structural flexibility and enable it to interact effectively with the components of the host cell.     

Identification of a new viral protein containing CAp30 and NCp10 sequences in murine and feline leukemia retroviruses.

Because Pr65gag is in part located in the nucleus and contains a putative bipartite nuclear targeting signal, we investigated the cellular location and structure of P55gag, a gag-encoded polyprotein known to lack the nucleocapsid (NC) protein NCp10. P55gag was found to be restricted to the cytoplasm of Moloney murine leukemia virus-infected cells. Of interest, P55gag was produced in cells infected by a viral protease deletion mutant and by a recombinant murine sarcoma virus known to lack the protease gene. Surprisingly, our structural and immunological studies indicated that P55gag also lacks carboxy-terminal residues of CAp30. During the course of studying P55gag, we detected a new viral protein within purified virus particles that contained NCp10 tryptic peptide sequences and a CAp30 tryptic peptide lacking in P55gag. This viral protein, which we have named nucleocapsid-related protein (NCRP), also contained antigenic epitopes present in CAp30 and NCp10. P55gag- and NCRP-like proteins were also observed in AKV murine leukemia virus and feline leukemia virus systems. The precise site of cleavage within Pr65gag that produces P55gag and NCRP is unknown but lies upstream of the CAp30-NCp10 junction within the carboxy-terminal domain of CAp30. The existence of a form of NCp10 containing carboxy-terminal CAp30 sequences raises interesting possibilities about its functional role in genomic RNA packaging and/or viral RNA dimerization.     

Simian virus 40 T-antigen: identification of tryptic peptides in the C-terminal region and definition of the reading frame

T-antigen (the simian virus 40 A cistron protein) was purified by immunoprecipitation and electrophoresis on polyacrylamide gels from monkey kidney CV-1 cells infected with simian virus S (SV-S), dl1263, or dl1265 and digested with trypsin. The tryptic peptides, labeled with [35S]methionine, [35S]cysteine, or [3H]proline, were fractionated either by chromatography on Chromobead-P resin or by two-dimensional electrophoresis and chromatography on cellulose thin layers. The T-antigen of SV-S was shown to give rise to a proline-rich (approximately 6 mol of proline) tryptic peptide which was absent in dl1265 T-antigen and hence, on the basis of DNA sequence data, must originate from the C-terminus of the SV-S protein. T-antigen from dl1265, but not SV-S, yielded a cysteine-rich terminal tryptic peptide. The presence of these cysteines caused the protein to be retarded during electrophoresis under the usual conditions in polyacrylamide gels. The T-antigen of dl1263 possessed the proline-rich tryptic peptide; the data are consistent with there being only one peptide altered by the deletion. Both deletion mutants produced a T-antigen that had a higher electrophoretic mobility than SV-S T-antigen but still a larger apparent molecular weight than was predicted by the DNA sequence. The major form of T-antigen found in several lines of 3T3 cells transformed by these mutants was indistinguishable from the T-antigen found in infected cells, and in addition seemed to associate normally with the host-coded 53,000-dalton protein. Except for a minor form of T-antigen with a slightly lower mobility in gels but the same C-terminus, no other polypeptides were detected among the extracted and immunoprecipitated proteins whose electrophoretic mobility was affected by either deletion.     

Expression and purification of a neuropeptide nocistatin using two related plant viral vectors

Both odontoglossum ringspot virus (ORSV) and tobacco mosaic virus (TMV) were investigated as expression viral vectors for the expression of a neuropeptide nocistatin. Chimeras of ORSV and TMV were constructed by fusion of 17 amino acids of mouse nocistatin (mNST) to the C-terminal of the coat protein (CP) gene via a Factor Xa cleavage linker to yield ORSV-mNST and TMV-mNST. Expression of the mNST peptide was demonstrated by immuno-transmission electron microscopy, western blot, mass spectrometry and radioimmunoassay. Serial passaging of the chimeric viruses revealed loss of mNST from TMV-mNST by the fifth passage. The mNST was maintained in ORSV-mNST throughout six passages. The mNST peptide could be effectively cleaved and purified from chimeric ORSV CP. To our knowledge, this is the first successful attempt in obtaining a complete peptide with no additional amino acid sequence after expression and purification through the use of either ORSV or TMV as vectors.     

Tobacco mosaic virus particle structure and the initiation of disassembly

The structure of an intact tobacco mosaic virus (TMV) particle was determined at 2.9 A resolution using fibre diffraction methods. All residues of the coat protein and the three nucleotides of RNA that are bound to each protein subunit were visible in the electron density map. Examination of the structures of TMV, cucumber green mottle mosaic virus and ribgrass mosaic virus, and site-directed mutagenesis experiments in which carboxylate groups were changed to the corresponding amides, showed that initial stages of disassembly are driven by complex electrostatic interactions involving at least seven carboxylate side-chains and a phosphate group. The locations of these interactions can drift during evolution, allowing the viruses to evade plant defensive responses that depend on recognition of the viral coat protein surface.     

Multiple serine phosphorylation sites on the 30 kDa TMV cell-to-cell movement protein synthesized in tobacco protoplasts

p30, the protein required for cell-to-cell movement of tobacco mosaic virus (TMV), has a slightly reduced mobility on SDS-polyacrylamide gels when isolated by immunoprecipitation from TMV-infected protoplasts compared with that of p30 translated from viral RNA in vitro . Further investigation established a probable cause for the difference in mobility between the two: protoplasts incorporate [³²P]orthophosphate into p30 at multiple sites, predominantly as phosphoserine. Tryptic peptide mapping reveals at least five internal phosphopeptides in p30, besides the C-terminal tryptic phosphopeptide already reported, involving at least two distinct domains of the protein (at residues 61–114 and residues 212–231), which may be substrates for different protein kinases. These structural results are consistent with a three-domain model for the TMV movement protein with two regulatory domains similar to that recently proposed on genetic grounds for dianthovirus movement proteins.     

Propagation of plant viruses in hairy root cultures: a potential method for in vitro production of epitope vaccines and foreign proteins

Hairy roots were used as an in vitro culture system for the propagation of wild-type and transgenic plant viruses. Tobacco mosaic virus (TMV) was added to the liquid culture medium at the same time as root inoculation. Hairy root growth was unaffected by viral infection. Maximum concentrations of TMV in Nicotiana benthamiana hairy roots were 1-2 orders of magnitude greater than in suspended N. benthamiana cells and reached levels of 1-2 mg g(-1) dry weight or 20-28% total soluble protein. Virus accumulated in the roots initially with a constant doubling time of about 1.0 day; subsequent reductions in viral growth rate were correlated with a significant decline in infectivity relative to the inoculum virus. The morphological integrity of the viral particles was maintained during propagation in hairy roots. The contribution to the overall viral titer of passive association of virus with the biomass, for example, by surface adsorption, was negligible compared with active viral replication. N. benthamiana hairy roots were also infected with a TMV-based viral vector developed to express green fluorescent protein (GFP). This vector was about 260-fold less infectious than wild-type TMV and accumulated much more slowly in the roots. Maximum levels of TMV-GFP in the biomass were about 65-fold lower than for TMV. This work demonstrates that hairy root cultures are a feasible means for in vitro propagation of wild-type and transgenic plant viruses under conditions that allow a high degree of environmental containment and control.     

Studies of coat protein-mediated resistance to tobacco mosaic tobamovirus: correlation between assembly of mutant coat proteins and resistance.

Coat protein-mediated resistance (CP-MR) has been widely used to protect transgenic plants against virus diseases. To characterize the mechanisms of CP-MR to tobacco mosaic tobamovirus (TMV) we developed mutants of the coat protein that affected subunit-subunit interactions. Mutant CPs were expressed during TMV replication as well as in transgenic Nicotiana tabacum plants. The mutation T42-->W increased protein aggregation and T28-->W abolished aggregation and assembly, while the mutations T28-->W plus T42-->W and T89-->W altered normal CP subunit-subunit interactions. The mutant T28W was unable to assemble virus-like particles (VLPs) during infection and in transgenic plants failed to aggregate; this protein conferred no protection against challenge of transgenic plants by TMV. The mutant T42W had strong CP subunit-subunit interactions and formed VLPs but not infectious virions. Transgenic lines with this protein exhibited stronger protection against TMV infection than transgenic plants that contained the wild-type (wt) CP. It is proposed that increased resistance conferred by the T42W mutant results from strong interaction between transgenic CP subunits and challenge virus CP subunits. CP carrying the mutation T89-->W formed flexuous and unstable VLPs whereas the double mutant T28W:T42W formed open helical structures that accumulated as paracrystalline arrays. In transgenic plants, T89W and the double mutant CPs showed reduced ability to aggregate and provided lower protection against TMV infection than wt CP. A strong correlation between normal CP subunit-subunit interactions and CP-MR is observed, and a model for CP-MR involving interactions between the transgenic CP and the CP of the challenge virus as well as interference with virus movement is discussed.     

Activation of ATPase activity of simian virus 40 large T antigen by the covalent affinity analog of ATP, fluorosulfonylbenzoyl 5''-adenosine.

Fluorosulfonylbenzoyl 5'-adenosine (FSBA) bound to one site in simian virus 40 large T antigen (T) and covalently modified greater than 95% of the molecules in a complete reaction. This analog for ATP specifically cross-links to the Mg-phosphate pocket in ATP-binding sites. Cyanogen bromide cleavage and tryptic digestion of [14C]FSBA-labeled protein, paired with T-specific monoclonal antibody analyses, were used to map the site in T to a tryptic peptide just C terminal to the PAb204 epitope. The location of the FSBA linkage was consistent with the predicted tertiary structure of the ATP-binding region in T described previously (M. K. Bradley, T. F. Smith, R. H. Lathrop, D. M. Livingston, and T. A. Webster, Proc. Natl. Acad. Sci. USA 84:4026-4030, 1987). Binding of FSBA to T was cooperative, implying an interaction between two binding sites. This could occur if the protein formed a dimer, and it is known that the ATPase activity is associated with a dimeric T. Most interesting was the activation of the ATPase when up to 50% of T was bound by the analog. The effect was also produced by preincubation with millimolar concentrations of ATP or the nonhydrolyzable analog gamma beta-methylene 5'-adenosine diphosphate at elevated temperatures. When greater than 50% of T was modified by FSBA, the ATPase was inhibited as the analog cross-linked to the second, previously activated, binding site. These data support a dual function for the one ATP-binding site in T as both regulatory and catalytic.