Thomas P. Pirone

A point mutation in the coat protein abolishes aphid transmissibility of a potyvirus

A nonaphid transmissible (NAT) variant of tobacco vein mottling virus (TVMV) was used to test the hypothesis that the viral coat protein (CP) plays a role in determining aphid transmissibility. Comparison of the nucleotide sequences in the coat protein cistron of an aphid transmissible isolate (TVMV-AT) with that of TVMV-NAT revealed a single nucleotide difference (G----A) at position 8445; this alters a single amino acid residue (G----E) at position 2747. A cDNA fragment representing the CP region of TVMV-NAT was substituted into the CP region of a full-length cDNA clone of TVMV-AT, and transcribed RNA was inoculated to tobacco plants. Aphids were unable to transmit the resultant hybrid virus which had the TVMV-NAT coat protein, although the concentration and infectivity of the hybrid virus in the source plants were similar to those of TVMV-AT. This is the first direct demonstration that a CP mutation affects aphid transmissibility of a potyvirus.

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.

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.

Context of the coat protein DAG motif affects potyvirus transmissibility by aphids

Previous work with tobacco vein mottling virus (TVMV) has established that a highly conserved three amino acid motif, asp-ala-gly (DAG), located near the N terminus of the coat protein (CP), is important for aphid transmission. However, several other potyviruses which have motifs other than DAG are aphid-transmissible. Creation of these motifs in TVMV through site-directed mutagenesis failed to render TVMV aphid-transmissible from infected plants, and the creation of a putative complementary motif in the helper component did not restore transmissibility. In an isolate of tobacco etch virus (TEV) that contains two consecutive DAG motifs separated by a single ala, transmissibility was abolished or reduced by mutations affecting the first motif, whereas mutations in the second motif had little or no effect. In a TEV mutant made non-transmissible due to an altered first motif, substitution of val for ala in the position immediately before the second DAG restored transmissibility, whereas changing val to ala in the location prior to the first DAG resulted in reduced TEV transmissibility. In contrast, a val to ala change in the position preceding the single DAG motif of TVMV did not affect transmission. Creation of another DAG motif at the beginning of the TVMV CP core, in a position where certain other potyviruses have a second DAG motif, did not restore transmissibility. Our results suggest that the mere presence of a DAG motif does not guarantee transmissibility and that the context in which the DAG or equivalent motif is found plays a role in the process.

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.

The effect of helper component on the uptake and localization of potyviruses in Myzus persicae.

125I-labeled virions were used to determine whether helper component (HC) affected the uptake or distribution of potyviruses in aphids. Aphids were allowed to acquire purified, 125I-labeled tobacco etch virus or potato virus Y mixed with HC or with inactivated HC. Helper component had no effect upon uptake of labeled virus, as measured by gamma counting. Autoradiography of freeze-sectioned aphids revealed, however, that in the presence of HC, label was associated with the maxillary stylets and with portions of the alimentary canal anterior to the gut, as well as with the gut region. Label accumulated only in the gut in control aphids. This selective localization of virus acquired in the presence of HC supports a binding mechanism for the mode of action of HC.

Acquisition factor required for aphid transmission of purified cauliflower mosaic virus.

Abstract Purified cauliflower mosaic virus (CIMV) could be transmitted only by aphids which had probed leaves infected with an aphid-transmissible isolate of CIMV before they probed into the purified virus. Aphids which had probed healthy leaves or leaves infected with a non-aphid-transmissible isolate of CIMV or aphids which first probed into purified virus and then into infected leaves did not transmit the purified virus. These results suggest that aphids obtain a factor (acquisition factor) from leaves infected with a transmissible isolate, which enables them to acquire, and to subsequently transmit, purified virus. The acquisition factor was shown not to be the virion of the transmissible isolate. Aphids which obtained the acquisition factor from CIMV infected leaves could transmit purified CIMV but not purified viruses of the potato Y (potyvirus) group; aphids which probed leaves infected with potyviruses could transmit other purified potyviruses but not purified CIMV.

Partial purification and characterization of the potato virus Y helper component.

Abstract Mg 2+ stabilized potato virus Y helper component during partial purification by precipitation using polyethyleneglycol. In solutions containing 0.02 M Mg 2+ the helper component retained most of its activity for 2 days at 4° and for at least 8 months at −15°. Activity was destroyed on incubation with pronase or trypsin, or by heating for 5 min at 55°, but not by incubation with ribonuclease. Incubation with its own antiserum strongly inhibited helper component activity, but antisera to potato virus Y coat protein or inclusion protein had no more effect than a control serum showing that the component was unrelated to these two proteins. Filtration through a Sephadex G-200 column resulted in a broad peak of activity which produced many protein-staining bands when electrophoresed on polyacrylamide gel. Gel filtration and ultrafiltration experiments both indicated a molecular weight of 100,000–200,000. Some helper component activity was retained by aphids allowed to probe into a sucrose solution for 20 min showing that the helper component is more firmly bound to the aphid than is the tobacco mosaic viruspoly- l -ornithine complex.

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.

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.