Purification and characterization of potyvirus helper component

Thornbury, David W.; Hellmann, Gary M.; Rhoads, Robert E.; Pirone, T. P.

doi:10.1016/0042-6822(85)90322-8

Cited by 152 publications

(102 citation statements)

References 13 publications

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“…However, it differs from the binding activity of the movement proteins (MP) of red clover necrotic mosaic virus (Osman et al, 1992) and tobacco mosaic virus (TMV; Citovsky et al, 1990) which bind to ssRNA and ssDNA equally well. The very low affinity for poly(A) is consistent with previous results obtained during purification of HC-Pro from infected tobacco plants (Thornbury et al, 1985). The significance of this finding remains unexplained.…”

Section: Discussionsupporting

confidence: 82%

“…RNA binding could be competitively inhibited by a 1000-fold (w/w) excess of bovine tRNA (lane 8), indicating that binding activity of MBP:HC-Pro lacks nucleic acid sequence specificity. However, protein-RNA complex formation was apparently unaffected when a 1000-fold (w/w) excess of poly(A) was added to the binding reaction (lane 7), in agreement with a previous report (Thornbury et al, 1985) suggesting that HC-Pro has little, if any, affinity for poly(A).…”

Section: Determination Of the Rna-binding Properties Of Mbp :Hc-prosupporting

confidence: 75%

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Nucleic acid-binding properties of a bacterially expressed potato virus Y helper component-proteinase

Maia

Bernardi

1996

Journal of General Virology

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The potyvirus helper component-proteinase (HC-Pro) is a multifunctional protein previously reported to have affinity for polyribonucleotides. To investigate further the ability of HC-Pro to bind nucleic acids, the potato virus Y (PVY) LYE84 isolate HC-Pro gene was amplified, cloned in an Escherichia coli expression vector and sequenced. HC-Pro was expressed as a fusion with the maltose-binding protein and purified by affinity chromatography. Electrophoretic mobility-shift assays demonstrated that HC-Pro acts as a sequence nonspecific RNA-binding protein and suggest that more than one molecule of protein was bound per molecule of RNA. The HC-Pro RNA-binding activity was stable in 400 mu-NaC1 and temperature sensitive. The recombinant protein preferentially bound ssRNA over DNA or dsRNA and showed little, if any, affinity for poly(A). The possible implications of the RNA-binding activity of HC-Pro in potyvirus replication and movement are discussed.

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Section: Discussionsupporting

confidence: 82%

Section: Determination Of the Rna-binding Properties Of Mbp :Hc-prosupporting

confidence: 75%

Nucleic acid-binding properties of a bacterially expressed potato virus Y helper component-proteinase

Maia

Bernardi

1996

Journal of General Virology

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“…The HC protein is involved in the aphid transmission of potyviruses and must be acquired by the insect in conjunction with the virus Hiebert et al, 1984b;Thornbury et al, 1985;Berger & Pirone, 1986). The HC protein has been associated with the amorphous inclusions that are induced by some potyviruses by several criteria (Hiebert et al, 1984b;De Mejia et al, 1985a, b;Baunoch et al, 1990), although functional studies have suggested that either the inclusion-bound form of this protein has been inactivated or, alternatively, the helper component activity is associated with a modified form of the inclusion protein Thornbury et al, 1985;. The biologically active form of the HC protein is believed to be a dimer (Hellmann et al, 1983;Thornbury et al, 1985).…”

Section: Virus Spreadmentioning

confidence: 99%

Highlights and prospects of potyvirus molecular biology

Riechmann

Laı́n

Garcı́a

1992

Journal of General Virology

576

355

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“…The properties of PVY HC and TVMV HC, both of which are capable of effecting the transmission of TVMV-AT and TEV-HAT, have been described (Thornbury et al, 1985(Thornbury et al, , 1990, as have the properties of the transmission-defective PVC HC (Thornbury et al, 1990).…”

Section: Methodsmentioning

confidence: 99%

“…Virus purification was by the method of Murphy et al (1990), and HC purification, through the sucrose gradient step, followed our previously described procedures (Thornbury et al, 1985(Thornbury et al, , 1990. The intrinsic infectivity of virus preparations was tested by mechanical inoculation; TVMV preparations were assayed on tobacco (Nieotiana tabacum cv.…”

Section: Methodsmentioning

confidence: 99%

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

Wang

Ammar

Thornbury

et al. 1996

Journal of General Virology

100

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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- (HC). TVMV virions were detected, by electron microscopic examination of immunogoldlabelled 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 nontransmissible 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.

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Purification and characterization of potyvirus helper component

Cited by 152 publications

References 13 publications

Nucleic acid-binding properties of a bacterially expressed potato virus Y helper component-proteinase

Nucleic acid-binding properties of a bacterially expressed potato virus Y helper component-proteinase

Highlights and prospects of potyvirus molecular biology

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

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