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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.

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 use of polymerase chain reaction (PCR)-based mutagenesis to create chimeric genes is presently not cost-effective because of the size and number of primers as well as the number of PCRs required. We have developed two strategies based on inverse PCR that exploit limited homologies between two DNA molecules to create in-frame chimeric plant viral genes. This report also contains a compilation of information useful for determining possible restriction sites at a given common dipeptide coding motif between any genes of interest.

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.

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.

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.

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.

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.

Aphids transmitted poly-L-ornithine (PLO)-treated tobacco mosaic virus (TMV) when given acquistion and inoculation access periods as brief as 30 s and 2 min, respectively; the ability to transmit was lost within 90 min. Aphids without claws were able to transmit the virus. Transmission thus seems similar to that of nonpersistent viruses. The ratio of virus to polyamino acid, as well as the KCl concentration, markedly affected transmission. Transmission was best from mixtures which contained 250 mug/ml TMV, 2-5 MUG/ML PLO (mol. wt. 120000) and 0-6 M-KCl. A similar mixture favoured transmission when poly-L-lysine (mol. wt. 85000) was substituted for PLO, but with poly-L-lysine (mol. wt. 30 000) it was necessary to decrease the KCl to 0-3 M to obtain transmission. Less KCl (0-08 to 0-24 M) also favoured aphid transmission of PLO-treated potato virus X and tobacco rattle virus. PLO-treated TMV ultracentrifuged in the presence of, and resuspended in, 0-6 M-KCl remained aphid transmissible while PLO-treated virus in 2 M-DCl, which favours greater dissociation of the virus-PLO complex, was transmissible neither before nor after sedimentation by ultracentrifuging, and resuspension in 0-6 M-KCl. these results show that transmissibility is not due to a permanent alteration of the virus by PLO and indicate that the formation of a TMV-PLO complex is required for transmission. Sequential acquisition experiments suggest that PLO may act by binding TMV to receptor sites in aphids. However, the possibility that PLO affects the infection process was not ruled out.

Anesthetized aphids, whose stylets had been dipped into capillaries containing purified concentrated cucumber mosaic virus, acquired the virus and, after recovery from the anesthetic, were able to transmit it in a low percentage of cases. Although this study does not eliminate active probing as a means of virus acquisition, experimentally, it clearly establishes passive contamination of aphid mouthparts as a method of virus acquisition.