Reusing nylon membranes for radioactive hybridizations.

We describe a procedure for recycling nylon hybridization membranes, enabling their repeated use for radioactive Southern hybridization analysis of different DNA samples. Following hybridization and probe removal, nylon membranes containing covalently linked DNAs were treated with 0.55% sodium hypochlorite. This destroyed the DNA, thereby preventing it from participating in further hybridization and enabling the membranes to be used subsequently for binding new DNA samples. With this procedure, we were able to reuse a single membrane as many as 13 times, with no detectable loss in signal. This method was shown to be effective for membranes supplied by three different manufacturers.

A specific interaction between coat protein and helper component correlates with aphid transmission of a potyvirus.

Specific binding between the coat protein (CP) and the helper component (HC) of the tobacco vein mottling potyvirus (TVMV) was characterized using a protein blotting-overlay protocol. In this in vitro assay, HC interacted with either virions or CP monomers originating from the aphid-transmissible TVMV-AT but not from the non-aphid-transmissible TVMV-NAT. There was a strong correlation between the aphid transmissibility of a series of TVMV variants having mutations in the DAG motif of the CP and their ability to bind HC. Expression of TVMV CP derivatives in bacteria allowed a precise determination of the minimum domain mediating HC binding. This domain is composed of seven amino acids, including the DAG motif (DTVDAGK), located in the N-terminus of the TVMV CP at amino acid positions 2 to 8.

Loss of potyvirus transmissibility and helper-component activity correlate with non-retention of virions in aphid stylets.

The hypothesis that loss of aphid transmissibility of potyvirus mutants is due to non-retention of virions in the mouthparts was tested by feeding aphids through membranes on purified virions of aphid transmissible (AT or HAT) and non-aphid-transmissible (NAT) tobacco vein mottling virus (TVMV) or tobacco etch virus (TEV), in the presence of functional [potato virus Y (PVY) HC or TVMV HC] or non-functional (PVC HC) helper component (HC). TVMV virions were detected, by electron microscopic examination of immunogold-labelled thin sections, in the food canal or cibarium of 57% of 28 aphids fed on the transmissible combination of TVMV-AT and functional HC, while no virions were found in these structures in 25 aphids fed on the non-transmissible combinations: TVMV-NAT and PVY HC, or TVMV-AT and PVC HC. Autoradiography of intact stylets allowed the examination of much larger numbers of aphids, fed on 125I-labelled TEV; 48% of 523 aphids fed on the TEV-HAT and PVY HC combination retained label in the stylets: this correlated well with the percentage transmission in bioassays. In contrast, in non-transmissible combinations, label was found in the stylets of 0.77% of 389 aphids fed on TEV-NAT and PVY HC, and 1.35% of 223 aphids fed on TEV-HAT and PVC HC. No differences were found in the overall amount of label in the bodies of aphids fed on the transmissible and non-transmissible combinations. There was a strong tendency for virions to be retained in the distal third of the stylets; 56% of aphids positive for TVMV, and 82% of those positive for TEV, had label in this area. These data support the concept that virions retained within the stylets are those that are primarily involved in potyvirus transmission.

Expression of potyvirus proteins in insect cells infected with a recombinant baculovirus.

The N-terminal portion (P1-HC-Pro-P3) of the tobacco vein mottling virus (TVMV) polyprotein was expressed in insect cells and larvae by a recombinant baculovirus. The proteases necessary to process this TVMV polyprotein fragment were active in insect cells, since mature P1, HC-Pro and P3 proteins were detected by specific antisera in Western blots. Antisera to P1, HC-Pro and P3 also recognized polypeptides with apparent M(r) values predicted for the intermediate processing products of the polyprotein fragment. The results of this study indicate that the autocatalytic processing of TVMV HC-Pro from the polyprotein is supported by insect cells. Helper component activity in extracts of cells infected with recombinant baculovirus was not detected by aphid transmission assay.

A simple and efficient procedure for the oral inoculation of Trichoplusia ni larvae with polyhedrin-negative recombinant baculovirus.

Current procedures for inoculating lepidopteran larvae with polyhedrin-negative recombinant baculovirus, i.e. intracoelomic injection or coinfection with wild type virus, are laborious and can compromise final yields of recombinant protein. Herein is described a simple and efficient method for oral inoculation. Up to 100% infection was obtained when individual early fifth instar Trichoplusia ni larvae were fed a small piece of a formaldehyde-free insect diet to which 4.2 x 10(5) PFU of a polyhedrin-negative recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) containing the gene for beta-galactosidase was applied. Infected larvae were identified by assaying hemolymph for beta-galactosidase activity. The maximum levels of beta-galactosidase detected in these hemolymph samples were identical to those obtained for larvae infected by intracoelomic injection. The dose of polyhedrin-negative recombinant virus recommended for intracoelomic injection of T. ni was efficacious for the oral route of inoculation.

Site-directed mutations in the potyvirus HC-Pro gene affect helper component activity, virus accumulation, and symptom expression in infected tobacco plants.

Helper component (HC-Pro) is a virus-encoded nonstructural protein required for aphid transmission of potyviruses. In the tobacco vein mottling virus (TVMV) polyprotein, HC-Pro represents a 457 residue polypeptide from amino acid position 257 to 713. Previous sequence comparison studies have suggested that mutations of one or two specific amino acid residues in the HC-Pro protein might result in loss of aphid transmission activity. To test this hypothesis, the initial targets were the residues corresponding to these specific amino acids, a lys to glu change and an ile to val change at amino acid positions 307 and 482, respectively, of the TVMV polyprotein, as well as the combination of the two. Two additional mutations within the HC-Pro representing dipeptide changes thr-ser to ile-asp and thr-ala to leu-glu at amino acid positions (283/284) and (368/369), respectively, were also tested to further define the effects of mutations in this region on helper component activity. The mutations at positions 482 and (368/369) had no effect on aphid transmission activity, while mutation at position 307 completely abolished the activity. Except for the 482 mutation, all the mutations also affected symptomatology and virus accumulation in infected plants. Due to the very low concentrations of HC-Pro in plants infected with the (283/284) mutant, the effect of this dipeptide change on aphid transmission activity could not be assessed. The majority of the tested mutations fall within a putative zinc-finger motif postulated in the cysteine-rich N-terminus of HC-Pro. The possible role of this motif in the potyviruses is further discussed in the light of our present results with TVMV.

Comparative sequence of the helper component (HC) region of potato virus Y and a HC-defective strain, potato virus C.

Potato virus C (PVC), a non-aphid transmissible strain of potato virus Y (PVY), was found to code for a protein (PVC-HC) which is similar in molecular weight and immunological reactivity to the helper component protein of PVY (PVY-HC). PVC-HC, however, was inactive with respect to its ability to effect aphid transmission of either PVC or PVY. The 5'-terminal 2.7-kb regions of PVC and PVY were sequenced. Within the HC region there was 92% nucleotide homology between the two strains; comparison of the derived amino acid sequences revealed 24 amino acid differences. Comparison of the PVC-HC sequence with that of five potyviruses revealed 2 amino acid changes which were specific to PVC-HC. These amino acids are prime targets for mutational analysis of HC activity.

Expression in transgenic plants of a viral gene product that mediates insect transmission of potyviruses.

Helper component (HC) is a virus-encoded nonstructural protein that is required for transmission of potyviruses by their aphid vectors. As a prelude to studies on the molecular basis of HC activity, a cDNA clone (pPB-3) was constructed that contained the first three cistrons (34 kDa-HC-42 kDa) of the RNA genome of the potyvirus tobacco vein mottling virus, the first six nucleotides of the adjacent cylindrical inclusion body protein cistron, and a synthetic translation termination codon. This construction was introduced into tobacco cells via a Ti plasmid-based vector. Northern blot analysis of transgenic plants demonstrated the presence of an RNA of the size expected from the construction of pPB-3, and Western blot analysis revealed the presence of a protein that comigrated with authentic HC, indicating that the proteolytic activity necessary to produce mature-sized HC was encoded by pPB-3. The HC produced in the transgenic plants was demonstrated to be active in a virus transmission bioassay with aphids.

Detection of picogram quantities of potyviruses using a dot blot immunobinding assay.

Highly sensitive direct visual detection of potyviruses was achieved using a dot blot immunobinding assay (DBIA). The small sample volumes required permit the detection of as little as 0.5 pg virus in purified preparations. The binding of rabbit antibodies could be visualized using goat anti-rabbit IgG (GAR) conjugated to alkaline phosphatase, beta-D-galactosidase, glucose oxidase, or horseradish peroxidase and histochemical substrates. The avidin-biotin system was also useful, but somewhat less sensitive than GAR-enzyme conjugates. Detection of potyviruses in an aphid vector was also attempted, but without success due to endogenous aphid enzymes.

Purification and characterization of potyvirus helper component.

Helper component (HC) was purified from tobacco vein mottling (TVMV)- and potato virus Y (PVY)- infected tobacco plants by sucrose gradient fractionation followed by affinity chromatography on oligo(dT)-cellulose and by gel electrophoresis. The subunit apparent molecular weights (M(r)) of the purified HCs were 53,000 (53K) and 58K for TVMV and PVY, respectively. Antisera to these purified polypeptides specifically blocked the activity of the homologous HC, as determined by aphid transmission assays, and specifically precipitated 75K products of the cell-free translation of the homologous RNA. The molecular weight of undissociated, biologically active TVMV or PVY HC, as determined by high-pressure liquid chromatography (HPLC)-gel permeation chromatography was found to be between 100K and 150K, suggesting that the active molecule is a dimer.

Identification of potyviral amorphous inclusion protein as a nonstructural, virus-specific protein related to helper component.

Antisera to amorphous inclusion (AI) proteins associated with infections by pepper mottle virus (PeMV) and the watermelon mosaic virus-1 strain of papaya ringspot virus (PRSV-W) were used to probe in vitro translation products of the viral RNAs. The major translation product of PeMV RNA in the rabbit reticulocyte lysate (RRL) system was a previously reported polypeptide of apparent molecular weight 78,000 (Mr 78K). It reacted with anti-AI serum, whereas the major translation product in the wheat germ (WG) system was a 30K polypeptide that did not react with the antiserum. These results, the Mr values, and analyses of peptides generated by partial digestion with proteinase indicate that the amino acid sequences of the 30K polypeptide and the (Mr) 51K AI protein are distinct subsets of the 78K polypeptide amino acid sequence. Similar results were obtained with PRSV-W except that the Mr values of the corresponding translation products are 110K (RRL) and 60K (WG). Thus the 5'-most region of the PeMV and PRSV-W RNAs (corresponding to 78K and 110K, respectively) appears to encode two proteins rather than one as previously supposed on the basis of RRL translation products. Reciprocal serological tests revealed that the tobacco vein mottling virus aphid transmission helper component protein was related to AI protein. There is direct evidence that the AI represent another potyviral-coded nonstructural protein and the first evidence that a biologically functional protein is related to a component of a potyviral inclusion.

The involvement of a helper component in nonpersistent transmission of plant viruses by aphids.

Certain plant viruses require a 'helper component' in order to be transmitted by aphids. The helper component for potyviruses is a virus-encoded protein with a subunit molecular weight of between 50,000 and 60,000. Aphid transmission of caulimoviruses is associated with the presence of an 18,000 molecular weight polypeptide. The mode of action of the helper component is the subject of speculation.

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.

Helper components of two potyviruses are serologically distinct.

The specificity of antisera to helper component (HC) from tobacco vein mottling virus (TVMV)- or potato virus Y (PVY)-infected tobacco plants was tested in immunoprecipitation and immunoabsorption chromatography experiments. Treatment with the homologous antiserum abolished or drastically reduced the activity of either TVMV-HC or PVY-HC, as measured by their ability to effect aphid transmission of purified tobacco etch virus, while the heterologous antiserum had little or no effect on HC activity. Loss of TVMV-HC and PVY-HC activity in the immunoabsorption chromatography experiments was associated with the removal of a 53- and a 58-kDa polypeptide, respectively. The results indicate that serologically distinct HC proteins are produced in response to specific potyvirus infection and suggest that HC is virus coded.

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.

Molecular weights of maize mitochondrial and cytoplasmic ribosomal RNAs under denaturing conditions.

The molecular weights of maize cytoplasmic and mitochondrial rRNAs were determined by gel acrylamide electrophoresis under non-denaturing and denaturing conditions. The molecular weights of mitochondrial rRNAs (0.76-10-6 and 1.25-10-6) exceeded those of cytoplasmic rRNAs (0.67-10-6 and 1.19-10-6) when electrophoresed in 8 M urea at 60 degrees C. Electrophoresis in 1.1 M formaldehyde resulted in similar values except for heavy mitochondrial rRNA, which exhibited a higher molecular weight than observed in 8 M urea. The observed values for cytoplasmic rRNAs, especially the heavy component, represent a decrease from estimates obtained under non-denaturing conditions. This is the first report of the electrophoretic examination of higher plant rRNAs under denaturing conditions.