Ernest Hiebert

Translation of potyvirus RNA in a rabbit reticulocyte lysate: identification of nuclear inclusion proteins as products of tobacco etch virus RNA translation and cylindrical inclusion protein as a product of the potyvirus genome.

The translations of tobacco etch virus (TEV) RNA and pepper mottle virus (PeMV) RNA in the mRNA-dependent rabbit reticulocyte lysate produced products which comigrated with potyviral inclusion proteins on polyacrylamide gels. The TEV RNA translation products comigrating with the 49,000 and 54,000 molecular weight TEV nuclear inclusion proteins were also immunoprecipitated by antisera specific to the nuclear inclusion proteins. The proteolytic peptide map of the comigrating in vitro translation products for TEV RNA was similar to that generated by the nuclear inclusion proteins. The PeMV RNA translation product comigrating with the cylindrical (pinwheel) inclusion protein was immunoprecipitated by antiserum to PeMV cylindrical inclusion protein. These results provided direct evidence that the inclusions associated with potyviruses consist of virus-coded, nonstructural proteins.

Location of Geminiviruses in the Whitefly Bemisia tabaci (Homoptera: Aleyrodidae)

The location of tomato mottle virus (ToMoV) and cabbage leaf curl virus (CabLCV) (Geminiviridae, genus Begomovirus) in the whitefly vector Bemisia tabaci B-biotype (Homoptera: Aleyrodidae) was elucidated using a novel technique incorporating indirect immunofluorescent labeling in freshly dissected whiteflies. Begomoviruses were visualized in the whitefly by indirect-fluorescent-microscopy. Polyclonal and monoclonal primary antibodies were used to successfully detect both ToMoV and CabLCV. Both begomoviruses were located in the anterior region of the midgut and filter-chamber of adult whiteflies, with ToMoV detected in the salivary glands. CabLCV was detected at a greater frequency than ToMoV, with a positive detection of 16% (89 out of 560) for CabLCV and 3% (25 out of 840) for ToMoV. Possible sites involved in geminivirus transport from the gut lumen of whiteflies into the hemocoel were located in the filter-chamber and anterior portion of the midgut. The location of these begomoviruses suggests a possible scenario of virus movement through the whitefly, which is discussed.

Characterization of some proteins associated with viruses in the potato Y group

Abstract Cytoplasmic inclusions induced by tobacco etch virus (TEV), potato virus Y (PVY), pepper mottle virus (PMV), bidens mottle virus (BiMV), and turnip mosaic virus (TuMV) as well as the respective viruses were purified from infected tissue and their constituent proteins investigated. A procedure is described for the simultaneous purification of virus and its associated inclusions. Electron microscopy of the purified inclusion preparations revealed structures characteristic of those seen in situ for each virus and are described as laminated aggregates shaped as triangles for TEV and as rectangles for BiMV, as scrolls for PVY and PMV, and as scrolls and laminated aggregates shaped as triangles for TuMV. Polyacrylamide electrophoresis of the cytoplasmic inclusions in 0.1% sodium dodecyl sulfate (SDS) revealed one protein component with an estimated molecular weight of 67,000 for PVY; 67,400 for PMV; 69,300 for BiMV; 69,600 for TEV; and 70,300 for TuMV. SDS electrophoresis of the viral coat proteins revealed anomalies, dependent in part on the history of the purified virus. Molecular weights of viral coat protein were estimated to be 26,000 and 32,000 for TEV; 28,000 and 34,000 for PVY; 28,000 and 34,000 for PMV; 28,000 and 33,000 for BiMV; and 27,000 and 36,000 for TuMV. Electrophoresis using different acrylamide gel concentrations and cellulose acetate support medium suggested that the larger of the two molecular weights estimated for each viral coat protein was a charge isomer.

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.

Phenotypic Variation in Transgenic Tobacco Expressing Mutated Geminivirus Movement/Pathogenicity (BC1) Proteins

Tobacco plants were transformed with the movement protein (pathogenicity) gene (BC1) from tomato mottle geminivirus (TMoV), using Agrobacterium-mediated transformation. Different transgenic tobacco lines that expressed high levels of the BC1 protein had phenotypes ranging from plants with severe stunting and leaf mottling (resembling geminivirus symptoms) to plants with no visible symptoms. The sequence data for the BC1 transgene from the transgenic plants with the different phenotypes indicated an association of spontaneously mutated forms of the BC1 gene in the transformed tobacco with phenotype variations. One mutated transgene associated with an asymptomatic phenotype had a major deletion at the C terminus of 119 amino acid residues with a recombination resulting in the addition of 26 amino acid residues of unidentified origin. This asymptomatic, mutated BC1 attenuated the phenotypic expression of the symptomatic BC1 in a tobacco line containing both copies of the BC1 gene. Another mutated form of the BC1 gene amplified from an asymptomatic, multicopy transgenic tobacco plant did not induce symptoms when transiently expressed in tobacco via a virus vector. The symptom attenuation in the transgenic tobacco by the asymptomatic BC1 may involve trans-dominant negative interference.

Partial purification and some properties of tobacco etch virus induced intranuclear inclusions

Abstract Tobacco etch virus (TEV) induced intranuclear inclusions (NI) were isolated from tissue of Datura stramonium L. by homogenization in buffer followed by Triton X-100 detergent treatment and by a combination of successive low speed and sucrose density-gradient centrifugations. The isolated NI, which appeared similar to those seen in situ, were rectangular with a striated surface. The NI were readily dissociated in sodium dodecyl sulfate (SDS), 6 M urea and 67% formic or acetic acids. Spectral analysis of dissociated NI revealed a typical protein spectrum with a maximum at 277 nm and a minimum at 250 nm. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of NI revealed two major zones and two minor zones all of which migrated at different rates than viral or cytoplasmic inclusion protein. Molecular weights were estimated to be 49,800 and 54,500 for protein in the major zones and 95,600 and 101,400 for protein in the minor zones. An antiserum produced to purified NI reacted specifically with NI antigens, but not with TEV coat protein, TEV-induced cytoplasmic inclusions, or extracts from uninoculated D. stramonium L. No reaction was detected with purified NI tested against antisera to virus or to cytoplasmic inclusions.

Immunochemical specificity of cytoplasmic inclusions induced by viruses in the potato Y group

Antisera were produced to the cytoplasmic inclusions induced by Bidens mottle, pepper mottle, potato Y, tobacco etch, and turnip mosaic viruses. Immunodiffusion tests with sodium dodecyl sulfate-denatured antigens showed that the inclusion proteins induced by each virus were immunochemically unique, although related in some cases. No changes in the antigenic specificities of the inclusions were detected by propagating the viruses in different plant species. These findings are taken as new evidence that the inclusions are coded by the viral genome, and it is proposed that immunochemical properties of the inclusions may be useful in classifying and diagnosing viruses in the potato Y group.

The nucleotide sequence of tomato mottle virus, a new geminivirus isolated from tomatoes in Florida

A new geminivirus, tomato mottle virus (TMoV), affecting tomato production in Florida has been cloned and sequenced. Sequence analysis of the cloned replicative forms of TMoV revealed four potential coding regions for the A component [2601 nucleotides (nt)] and two for the B component (2541 nt). Comparisons of the nucleotide sequence of the TMoV genome with those of other whitefly-transmitted geminiviruses indicate that TMoV is a typical bipartite geminivirus of the New World and is closely related to but distinct from abutilon mosaic virus.

Geminivirus Resistance in Transgenic Tobacco Expressing Mutated BC1 Protein

Tobacco explants were transformed by Agrobacterium-mediated transformation with sense and antisense constructs of the movement protein genes (BC1 and BV1) of tomato mottle geminivirus (TMoV). Transgenic plants were tested for virus resistance either by exposure to viruliferous whiteflies carrying TMoV or cabbage leaf curl geminivirus (CabLCV) for a 72-h inoculation period or by continuous exposure to TMoV during the life of the plants. The transgenic lines were scored for disease symptoms, and virus replication and distribution were determined by enzyme-linked immunosorbent assay and dot blot hybridizations. Transgenic plants which expressed a mutated form (identified in a previous study) of the BC1 gene showed TMoV and CabLCV resistance. Three resistant phenotypes were observed: a delay in symptom development, a recovery from early symptoms, and an absence of virus symptoms at all stages. Geminivirus was detected in inoculated leaves but was not readily detected in leaves beyond the inoculation sites in ...

Immunoprecipitation analysis of potyviral in vitro translation products using antisera to helper component of tobacco vein mottling virus and potato virus Y

The serological relationships of the products of in vitro translation of the RNA of various potyviruses were analyzed by using antisera to helper component (HC) from tobacco plants infected with either tobacco vein mottling virus (TVMV) or potato virus Y (PVY). The PVY-HC antiserum immunoprecipitated a specific PVY-RNA translation product; this product was not reactive with antisera to PVY-induced cylindrical inclusion protein or capsid protein or to the two tobacco etch virus nuclear inclusion proteins. The antiserum to PVY-HC did not immunoprecipitate significant amounts of any translation products of 16 other potyviruses including TVMV. In contrast the antiserum to TVMV-HC efficiently immunoprecipitated a specific product(s) of four different potyviruses, some isolates of which are poorly transmitted or nontransmissible by aphids, and less efficiently a product(s) from 12 other potyviruses, including PVY. Distinct serotypes were resolved among the major in vitro translation products of 17 different potyviral RNAs by the antisera to TVMV-HC and PVY-HC. There appears not to be a correlation between the serological reactivities of HC-related polypeptides and the ability of different HC-virus combinations to effect aphid transmission of the virus.