John G. Shaw

Potyviral proteins share amino acid sequence homology with picorna-, como-, and caulimoviral proteins.

The predicted amino acid sequences of the polyproteins of two potyviruses, tobacco vein mottling virus (TVMV) and tobacco etch virus (TEV), were compared to each other, to proteins of other viruses, and to the National Biomedical Research Foundation protein sequence bank. Three potyviral proteins, the cylindrical inclusion and the two nuclear inclusion proteins, were found to be homologous to proteins considered to be involved in the replication and expression of picorna- and comovirus RNA. A lower, but also significant, level of homology was observed between a putative N-terminal 28-kDa protein of TVMV, the 30-kDa protein of tobacco mosaic virus, and the 29-kDa protein of tobacco rattle virus, the latter two of which are thought to be involved in cell-to-cell movement of virus or viral RNA. The aphid transmission helper component of TVMV was homologous to the aphid transmission factors of two strains of cauliflower mosaic virus. A region of the putative TEV polyprotein, located between the helper component and the cylindrical inclusion protein, contained a sequence that was homologous to the conserved region of the 2A protease of picornaviruses. These results suggest functions for all of the potyviral proteins and also indicate that poty-, como-, and picornaviruses may share a similar replication strategy and genome organization.

The VPg of tobacco etch virus RNA is the 49-kDa proteinase or the N-terminal 24-kDa part of the proteinase.

Preparations of tobacco etch virus (TEV) RNA which were purified by sucrose gradient centrifugation, digested with RNase, and analyzed by SDS-polyacrylamide gel electrophoresis contained proteins of 49, 32, and 24 kDa. The 49- and 24-kDa proteins reacted with polyclonal antiserum to the TEV 49-kDa proteinase while the 32-kDa protein reacted with anti-TEV serum. Further purification of the RNA by centrifugation through CsCl removed the coat protein (32 kDa), but not the 49- and 24-kDa proteins. The 49- and 24-kDa proteins did not migrate into a polyacrylamdie gel when the RNA was not digested with RNase. These results indicate that the VPg of TEV is either the 49-kDa proteinase or the 24 kDa that represents the amino-terminal half thereof.

Two newly detected nonstructural viral proteins in potyvirus-infected cells

The existence of two viral RNA-encoded proteins in cells infected with tobacco vein mottling potyvirus (TVMV) has been demonstrated. One of the proteins (named 34K) maps at the N-terminus of the TVMV polyprotein and the other (42K) between the helper component and cylindrical inclusion proteins; both had previously been predicted in the consensus potyviral genetic map. The 34K and 42K coding regions of TVMV were cloned separately in a bacterial expression vector and the proteins were isolated from transformed Escherichia coli. These were used to raise polyclonal antibodies which reacted specifically with proteins of the expected size in immunoblots of extracts of TVMV-infected tobacco leaves and protoplasts. In addition to 42K, the anti-42K serum detected similar amounts of a second protein of apparent size 37 kDa that was absent in 42K-expressing bacteria. Both 34K and 42K were present predominantly in membrane-enriched fractions of extracts of TVMV-infected tobacco leaves. Computer analysis of the deduced amino acid sequence of 42K suggests that this viral protein may be an integral transmembrane protein.

Cell-free translation of tobacco vein mottling virus RNA. II. Immunoprecipitation of products by antisera to cylindrical inclusion, nuclear inclusion, and helper component proteins.

The genomic RNA of tobacco vein mottling virus (TVMV) was translated in a cell-free system derived from rabbit reticulocytes. Antisera against TVMV coat protein, TVMV cylindrical inclusions, the helper component required for aphid transmission of TVMV, and the 49- and 54-kd nuclear inclusion proteins of tobacco etch virus (TEV) were used to characterize the translational products. Each of the five antisera precipitated a distinctive pattern of polypeptides. Specificity of immunoprecipitation was shown by competing with the various proteins to which antisera were raised and by sequentially precipitating with two different antisera. These experiments showed that the five antisera define five different nonoverlapping regions of the TVMV genome.

Association of the non-structural P3 viral protein with cylindrical inclusions in potyvirus-infected cells

Antibodies raised against the 42K non-structural P3 protein encoded by the RNA genome of a potyvirus (tobacco vein mottling virus, TVMV) have been used with immunogold labelling procedures to determine the subcellular location of P3 in infected Nicotiana tabacum cells. P3-specific gold label was found almost exclusively associated with the cylindrical inclusions typically formed in the cytoplasm of potyvirus-infected cells by another non-structural viral protein, the cylindrical inclusion protein. The P3 antibodies reacted with inclusions in both the transverse (pinwheel-like) and longitudinal (bundle-like) orientations of these structures. Immunocytological examination of TVMV-infected protoplasts showed the association of P3 with cylindrical inclusions during the early stages of formation of these structures and suggests that the P3 may be involved in the replication of potyviral RNA.

Mapping of the tobacco vein mottling virus VPg cistron

The location of the cistron encoding the genome-linked protein (VPg) in the potyvirus tobacco vein mottling virus (TVMV) was investigated. Precipitation of 125I-labeled VPg with anti-tobacco etch virus 49K nuclear inclusion protein antiserum (which reacts with the NIa nuclear inclusion protein of TVMV) indicated that the TVMV VPg is immunologically related to NIa. Lysyl residues were found to be present at positions 2, 11, and 16 of the amino-terminal region of the VPg. A search of the TVMV polyprotein sequence for this distribution of lysyl residues revealed a unique location beginning at amino acid residue 1801, the proposed amino-terminus of the NIa protein.

Evidence that tobacco mosaic virus particles disassemble contranslationally in vivo

The possibility that a cotranslational disassembly mechanism, similar to that observed when pH 8-treated tobacco mosaic virus (TMV) particles are incubated in an in vitro translation system [T. M. A. Wilson. (1984), Virology 137, 255-265], may be involved in the early stages of virus infection was investigated. Extracts of tobacco leaf epidermal cells, collected between 10 and 70 min after inoculation with 32P- and [3H]leucine-labeled TMV, contained material which had higher buoyant densities in Cs2SO4 gradients and higher 32P:3H ratios than did virus particles. The material sedimented to positions similar to those of in vitro-prepared complexes of partially stripped virus particles and ribosomes and to those of the in vivo-produced complexes of TMV rodlets and in vivo-labeled, nascent polypeptides that formed after inoculation with unlabeled, untreated TMV. In the electron microscope, some of this material resembled the complexes observed in the in vitro translation system. Experiments in which TMV or partially stripped TMV was mixed with epidermal cells from mock-inoculated leaves indicated that the material did not arise by dissociation of virus particles. nor by binding of subcellular components to partially uncoated TMV, during extraction and analysis. These observations provide evidence of the occurrence of a cotranslational disassembly mechanism during the early stages of infection with TMV.

Cell-free translation of tobacco vein mottling virus RNA.

Tobacco vein mottling virus (TVMV), a member of the potyvirus group, was translated in a rabbit reticulocyte cell-free system. The RNA was approximately 5% as active as rabbit globin mRNA in directing protein synthesis. Translation was stimulated approximately 15% by the cap analog pppm7G, a phenomenon which has also been observed with uncapped viral RNAs. Treatment with NaIO4 and NaB(3H]4 under conditions which label the caps of globin and ovalbumin mRNA failed to produce labeled cap structures in TVMV RNA. Approximately 20 polypeptides, ranging from 20,000 to 100,000 daltons, were produced in the reticulocyte system programmed with TVMV RNA. The predominant species (P75) had a molecular weight of 75,000. The same major polypeptides were produced in the wheat germ system, but there was relatively greater synthesis of the smaller polypeptides. Incubation of the reticulocyte system in the presence of cycloheximide following an initial period of synthesis failed to cause breakdown of larger polypeptides into smaller polypeptides. Preincubation of TVMV RNA with the reticulocyte system before addition of labeled amino acids caused greater synthesis of the smaller polypeptides, suggesting that they are derived from fragments of the RNA produced during the incubation. Translation of TVMV RNA which had been bound to oligo(dT)-cellulose yielded nearly the same spectrum of polypeptides, but synthesis of polypeptides smaller than 52,000 daltons was reduced. At low ionic strength, only polypeptides of 52,000 daltons and smaller were synthesized. At high ionic strength, P75 was essentially the only product synthesized. Antibody to whole virus precipitated many of the polypeptides, including P75, suggesting that they contain the coat protein sequence. No polypeptide with the electrophoretic mobility of authentic coat protein, however, was precipitated. Comparison of partial proteolytic digests of P75 and authentic coat protein provided further evidence for sequence homology.

In vivo removal of protein from tobacco mosaic virus after inoculation of tobacco leaves

Abstract Tobacco leaves were inoculated with reconstituted tobacco mosaic virus labeled with 14C in its protein moiety only. After various intervals, extracts of the leaves were prepared and analyzed by density gradient centrifugation. In extracts from leaves infected for 7–8 minutes, about 25% of the virus protein was found in a top zone dissociated from the main virus zone. The amount of radioactive protein in the top zone gradually increased with longer infection periods until 5–9 hours, when about half the 14C was removed from the virus. Analysis of extracts of leaves infected with labeled native virus for 8 minutes yielded almost the same results obtained with reconstituted virus. Other control experiments indicated that the dissociation was not due to a breakdown of virus particles during extraction and analysis. There was some evidence that most of the virus particles retained by the leaves were at least partially uncoated.

A hypersensitive response-like mechanism is involved in resistance of potato plants bearing the Ry(sto) gene to the potyviruses potato virus Y and tobacco etch virus.

Potato plants carrying the Ry(sto) gene from Solanum stoloniferum are extremely resistant to a number of potyviruses, but it is not known at what stage of infection the resistance is expressed. The resistance may be due to Ry(sto) or to a closely linked gene. In this investigation, we used potato virus Y (PVY) and a tobacco etch virus construct that encodes beta-glucuronidase (TEV-GUS) to monitor virus infections of potato plants. Systemic spread of either virus in resistant potato plants was not detectable by serology, RT-PCR, GUS assay or bioassay although each replicated in the initially infected cells of leaves from resistant potato cultivars and was transported into neighbouring cells. However, 3 days post-inoculation (p.i.) a necrotic reaction set in that stopped movement and accumulation of both viruses by 7 days p.i. The resistance reaction (probably a hypersensitive reaction) became visible as necrotic streaks on veins on the lower leaflet surfaces of some potato cultivars carrying the Ry(sto) gene and may be elicited by a common potyviral gene product.