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Objectives: The aim of this study was to identify quantitative and qualitative differences between the reading and writing skills of children with developmental dyslexia and those of dyslexic children with a specific language impairment (SLI)., Background: It is suggested that although the etiology of developmental dyslexia and SLI may be diverse, dyslexic children with SLI and their language-intact peers are comparable on a behavioral level., Methods: Three groups of second-grade children were compared on reading and writing tests with single words and nonwords: 15 dyslexic children with a history of SLI (SLI group), 15 dyslexic children with a typical pattern of language development (non-SLI group), and a control group of 30 children with no clinical history of learning disabilities or communication disorders., Results: Analysis of the results revealed the performances of both SLI and non-SLI dyslexic groups to be comparable in terms of speed, accuracy, and error typology., Conclusions: This study confirms that there are parallels between dyslexic children with language disorders and their dyslexic peers with intact language skills, at least in terms of their performance on reading and writing tests.

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.

ABSTRACT The helper component (HC) protein of turnip mosaic virus (TuMV) was concentrated by differential centrifugation followed by ammonium sulfate precipitation. The partially purified HC was then loaded onto a Ni(2+)-resin column that bound the HC; a histidine tag was not required for binding. The HC eluted from the column migrated as a band of about 50 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In its native state, the HC did not pass through an ultrafiltration membrane with a molecular mass cutoff of 100 kDa, which suggested that the HC is in a multimeric form when it is biologically active. The molecular mass of the multimeric form was determined by gel filtration to be approximately 145 kDa. Purified HC retained its activity for several months at -20 degrees C. Using a protein blotting-overlay protocol, purified HC interacted in vitro with an aphid-transmissible TuMV isolate, but not with a non-aphid-transmissible isolate.

The coding sequence for a series of six histidines (his-tag) was inserted near the 5' terminus of the helper component (HC) coding region of tobacco etch potyvirus (TEV). Full length genomic clones containing the his-tag coding sequence were infectious and produced symptoms in tobacco (Nicotiana tabacuma) similar to those induced by wild-type TEV. The modified virus was genetically stable and the his-tag sequence was maintained through at least four cycles of aphid transmission. A protocol for purifying rapidly the HC protein, based on the affinity of its his-tag for Ni(2+)-charged resin, yielded large amounts of his-tagged HC protein that was fully functional as demonstrated by aphid transmission experiments.

Mutations of K --> E in the highly conserved 'KITC' motif of the potyvirus helper component (HC) protein result in loss of HC function in aphid transmission, presumably because of inability to interact with virions, stylets or both. In this study we show that HC of potato virus C (PVC), a naturally occurring variant of potato virus Y (PVY) that has the K --> E mutation, lacks the ability to be retained in stylets, whereas PVY HC is retained. The K --> E mutation in either PVC or a site-directed mutant of tobacco etch virus (TEV) did not hinder binding to capsid protein, nor did deletion of the N-terminal 107 aa of TEV HC. An additional mutation, F -->, L at aa 10 of TEV HC, which renders HC non-functional but does not affect binding to capsid protein, is reported. Collectively, the results suggest that the N-terminal domain is required for interaction of HC with stylets rather than for binding to virions.

Four aphid species were tested for their ability to transmit tobacco etch (TEV) and turnip mosaic (TuMV) potyviruses. Myzus persicae and Aphis gossypii transmitted both viruses efficiently from infected plants, whereas Lipaphis erysimi transmitted only TuMV and Myzus ascalonicus was a poor or non-transmitter of either virus. Similar electrically monitored probing patterns were produced by M. persicae, L. erysimi and M. ascalonicus, ruling out behavioural differences as the cause of differential transmission. Transmission results similar to those from infected plants were obtained when these aphids acquired homologous virus/helper component (HC) mixtures through membranes. With heterologous virus/HC mixtures, M. persicae remained a highly efficient vector and M. ascalonicus a non-vector, but L. erysimi became an efficient vector of TEV if acquired in the presence of TuMV HC and A. gossypii transmitted both viruses less efficiently when acquired with TuMV HC. Transmission was highly correlated with the retention of virus in the stylets, as determined by autoradiography of 125I-labelled virions. The results show that constituent(s) of or in the food canal of different aphid species differ in their ability to interact with specific HCs, leading to qualitative or quantitative differences in ability to retain and subsequently transmit specific potyviruses.

Mutants of tobacco vein mottling virus (TVMV) with substitutions of Lys or Arg for Asp in the DAG motif at position 5 in the coat protein (CP) failed to infect tobacco plants systemically, but replicated and produced virions in protoplasts. Occasional systemic infections occurred when Nicotiana benthamiana or transgenic tobacco plants expressing wild-type TVMV CP were inoculated with these mutants, but viral progeny contained reversions to negatively or non-charged amino acids at position 5 or substitutions of Glu for Lys at position 8. The compensatory nature of these mutations was demonstrated by recreating one of the most common alterations. Tobacco etch virus (TEV) mutants with substitutions of Lys for Asp in the two DAG motifs near the CP N terminus also failed to infect tobacco plants systemically, and in situ histochemical analysis showed limited movement. A certain net charge evidently must be maintained near the CP N terminus for systemic movement to occur.

The va gene is used in commercial Burley tobacco cultivars including cv TN 86 to confer resistance to tobacco vein mottling virus (TVMV) and, to some extent, other potyviruses. A naturally occurring strain of TVMV (TVMV-S), which overcomes this resistance, was isolated from TN 86 plants. To investigate the mechanism by which TVMV-S overcomes va gene resistance, a cDNA clone encompassing the complete genome of TVMV-S was produced. Using chimeric transcripts combining regions of TVMV-S and regions of the wild-type strain (TVMV-WT) to which TN 86 is resistant, it was demonstrated that a domain within the VPg protein is responsible for overcoming va resistance in TN 86. DNA sequence analysis revealed six amino acid differences between the two strains of TVMV within this domain. Inclusion of sequences for four of the TVMV-S VPg amino acids was sufficient to confer the resistance-overcoming phenotype to all corresponding transcripts. Coinoculation experiments suggested that the resistance of TN 86 to TVMV-WT was not due to the induction of a general host defense response. The results are compatible with the hypothesis that VPg must assume an appropriate configuration in order to interact with appropriate host components and facilitate systemic virus movement., (Copyright 1997 Academic Press.)

Infection of Tabasco pepper by the tobacco etch virus (TEV) typically causes wilting associated with root necrosis. However, a strain of TEV, designated TEV nonwilting (TEV NW), is able to infect Tabasco pepper plants but does not cause wilting. In order to locate the genetic determinants responsible for the wilting response, a full-length cDNA clone of TEV NW from which infectious transcripts can be derived was made. A number of chimeric constructs were prepared by substituting cDNA fragments between TEV HAT (which causes wilting) and TEV NW clones. This approach was used to identify two wilting determinants in TEV HAT: one encompasses the 3' one-third of the P3 encoding region; the other spans the 3' end of the CI, the 6-kDa protein, and the 5' end of the VPg-NIa coding regions. Substitutions of both these TEV NW fragments into TEV HAT resulted in infection but not wilting of Tabasco pepper, while the replacement of either of the fragments alone did not alter the wilting response. This indicates that both TEV NW regions contain determinants necessary but not sufficient to alter the wilting response and that both must be present in order to avoid the wilting response. There was no difference between the in vitro transcription-translation products derived from constructs containing these regions from TEV HAT and TEV NW.

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.

Aphids (Myzus persicae), fasted after removal from healthy rearing plants, transmitted tobacco etch potyvirus (TEV) more efficiently than unfasted aphids whether virus acquisition was from infected leaves or through membranes. There was no difference in uptake of 125I-labelled TEV by fasted or unfasted aphids as measured by liquid scintillation counting. When aphids acquired 125I-labelled TEV, label was retained in the stylets (as determined by autoradiographic light microscopy) by 51 % of 272 fasted aphids, as against 7.8% of 258 unfasted aphids. There was a close correlation between virus transmission by aphids and virion retention in stylets. The effect of pre-acquisition fasting disappeared when aphids reared on an artificial diet were used in virus transmission tests. The transmission rates obtained with such aphids were similar to the rates with fasted aphids reared on healthy plants. Our results support the hypothesis that fasting eliminates plant component(s) which interfere with the retention of virions in the food canal of 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.

A variety of noncirculatively transmitted viruses have evolved a vector transmission strategy that involves, in addition to virions, virus-encoded proteins that are not constituents of virions. These "helpers" and the genes encoding them have been characterized for viruses in the genera Potyvirus and Caulimovirus. Several lines of evidence support the hypothesis that these helpers act by mediating retention of virions in regions of the vector's alimentary tract from which they subsequently can be egested to initiate an infection. The possible advantage this convergently evolved strategy could confer to noncirculatively transmitted virus quasispecies is discussed.

The nature of the amino acids in the N-terminal 'DAGX' motif of the coat protein of tobacco vein mottling virus (TVMV) that have a direct effect on aphid transmissibility of the virion were further defined by site-directed mutagenesis. In the first position of the DAGX motif, Asp or Asn are required for aphid transmissibility. In the second position, the nonpolar residue Ala, but not the nonpolar Gly or Val or the polar Thr and Ser, is compatible with transmissibility. In the third position, the small, neutral, nonpolar Gly appears to be critical; even substitution of Ala, with a minimal side-chain, drastically reduces transmissibility. Although the amino acid following the DAG sequence is not highly conserved among potyviruses, the presence of an acidic Glu or Asp residue at this position in the TVMV coat protein drastically reduces or abolishes aphid transmissibility. An attempt was made to test the hypothesis that trypsin cleavage of the N terminus is involved in the aphid inoculation process by destroying a trypsin cleavage site downstream from the DAGX motif. While the predicted decrease in transmission occurred from infected plants, there was no effect on the transmission of purified virus. Of the 23 mutations in the DAGX region of TVMV reported here and previously, only two, substitutions of Lys and Arg for Asp, had a detectable adverse effect other than on aphid transmissibility. These, and perhaps other, residues near the N terminus function in some phase of the TVMV life cycle, in addition to aphid transmission.

We have previously provided evidence that amino acid substitutions within the N-terminal portion of the helper component-proteinase (HC-Pro) from tobacco vein mottling virus (TVMV), in particular at Lys-307, not only affect the aphid transmission activity of HC-Pro but also have a significant effect on TVMV virulence. In the present study amino acids which differ in their charge properties were substituted at position 307. A highly basic residue was required to retain helper component activity and virulence. Deletion and insertion mutagenesis in the 5' terminus of the HC-Pro gene suggested that this RNA domain may be an essential element for TVMV infectivity. Replacement of the TVMV HC-Pro gene with that from another potyvirus, zucchini yellow mosaic virus, maintained infectivity and aphid transmissibility of the chimeric virus, although symptoms were attenuated. Our results suggest that, in addition to its importance in aphid transmission, the HC-Pro gene may be of general importance in regulating virulence of potyviruses, possibly by interaction of these sequences with the host.

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.

The RNA genome of tobacco etch potyvirus (TEV) was engineered to express bacterial beta-glucuronidase (GUS) fused to the virus helper component proteinase (HC-Pro). It was shown previously that prolonged periods (approximately 1 month) of TEV-GUS propagation in plants resulted in the appearance of spontaneous deletion variants. Nine deletion mutants were identified by nucleotide sequence analysis of 40 cDNA clones obtained after polymerase chain reaction amplification. The mutants were missing between 1,741 and 2,074 nucleotides from TEV-GUS, including the sequences coding for most of GUS and the N-terminal region of HC-Pro. This region of HC-Pro contains determinants involved in helper component activity during aphid transmission, as well as a highly conserved series of cysteine residues. The deletion variants were shown to replicate and move systemically without the aid of a helper virus. Infectious viruses harboring the two largest HC-Pro deletions (termed TEV-2del and TEV-7del) were reconstructed by subcloning the corresponding mutated regions into full-length DNA copies of the TEV genome. Characterization of these and additional variants derived by site-directed mutagenesis demonstrated that deletion of sequences coding for the HC-Pro N-terminal domain had a negative effect on accumulation of viral RNA and coat protein. The TEV-2del variant possessed an aphid-nontransmissible phenotype that could be rescued partially by prefeeding of aphids on active HC-Pro from another potyvirus. These data suggest that the N-terminal domain of HC-Pro or its coding sequence enhances virus replication or genome expression but does not provide an activity essential for these processes. The function of this domain, as well as a proposed deletion mechanism involving nonhomologous recombination, is discussed.

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.

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.

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.

The cauliflower mosaic virus ORF II encoding the aphid transmission factor (ATF) was mutagenized to introduce a BamHI restriction site upstream from the initiation codon and then cloned into an eukaryotic viral expression vector (Autographa californica nuclear polyhedrosis virus). All recombinant viruses tested in Spodoptera frugiperda (SF21) cells expressed a protein of about 18 kD which comigrated in PAGE with ATF from infected plants. Western blotting using an oligopeptide antiserum to ATF confirmed the identity of the 18-kD protein from infected cells as the product of the ORF II sequences (P18). Subcellular fractionation of cells infected with the recombinant AcMNPV demonstrated that the expressed P18 accumulated intracellularly in an insoluble form. Antiserum was produced in rabbit against the partially purified P18 expressed in SF21 cells. When used to immunogold label ultrathin sections of cauliflower mosaic virus (CaMV)-infected turnip tissue, this antiserum was shown to be highly specific, labelling only the electronlucent inclusion bodies (containing P18) and not other plant cellular components.

Amino acids near the N terminus of the coat protein of tobacco vein mottling virus were deleted or altered by site-directed mutagenesis to determine the effect on aphid transmissibility of the virus. Deletion of a three amino acid sequence Asp-Ala-Gly, which is conserved in aphid-transmissible potyvirus isolates, abolished transmission. The mutation Ala----Thr in this triplet drastically reduced transmission, whereas the mutation Asp----Asn had no effect, and the mutation Asp----Lys consistently reverted to the wild-type residue. The mutation Lys----Glu, in the residue adjacent to the glycine of the triplet, drastically reduced transmission, whereas the mutation Gln----Pro, seven residues downstream from the glycine had no effect. Comparison of the sequences of other potyviruses suggests that the presence of a glycine residue at the third position of the Asp-Ala-Gly triplet is critical for aphid transmissibility and that certain changes in the residues adjacent to this position abolish or greatly reduce aphid transmissibility.

Polymerase chain reaction (PCR) based DNA amplification has replaced many time-consuming protocols in molecular biology. Here we describe a simple strategy to quickly select deletion mutants based on PCR methodology which then can be confirmed by nucleotide sequencing. A forward PCR primer is designed in such a way to recognize only the wild type sequences in the amplification reaction and thus a negative selection identifies the deletion in the samples.

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.

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.

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.

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.

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.

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.

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.

A deletion mutant of cauliflower mosaic virus (CaMV) isolate NY8153 deficient in aphid transmissibility was constructed by BAL-31 exonuclease treatment of XhoI linearized pCMS31 (a plasmid containing the entire CaMV genome cloned in the SalI site of pBR322), followed by ligation. The resulting mutant, pSA103, lacked about 100 by from the putative protein-coding region II of CaMV DNA. In turnips there was no difference in the number and appearance of starch lesions or hybridization lesions, or in the nature of symptoms in systemically infected leaves induced by pSA103 DNA, pCMS31 DNA, and NY8153 DNA. Systemic symptoms appeared later in plants infected with pSA103 (27 +/- 2 days) than in those infected with the parental pCMS31 DNA (19 +/- 2 days). Aphids fed on virus SA103-infected mustard plants were unable to transmit the virus to healthy plants while 30-70% transmission was observed from plants infected by the parent virus. Virions extracted from turnips infected with the mutant had DNA still containing the deletion. In addition, the single-stranded discontinuity in region II of NY8153 DNA was missing in this DNA. The results suggest that region II codes for a "helper component" required for aphid transmission of CaMV.

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.

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.

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.