title: Cylindrical inclusion protein of potato virus A is associated with a subpopulation of particles isolated from infected plants.
abstract: Potato virus A (PVA) particles were purified by centrifugation through a 30 % sucrose cushion and the pellet (P1) was resuspended and sedimented through a 5-40 % sucrose gradient. The gradient separation resulted in two different virus particle populations: a virus fraction (F) that formed a band in the gradient and one that formed a pellet (P2) at the bottom of the gradient. All three preparations contained infectious particles that retained their integrity when visualized by electron microscopy (EM). Western blotting of the P1 particles revealed that the viral RNA helicase, cylindrical inclusion protein (CI), co-purified with virus particles. This result was confirmed with co-immunoprecipitation experiments. CI was detected in P2 particle preparations, whereas F particles were devoid of detectable amounts of CI. ATPase activity was detected in all three preparations with the greatest amount in P2. Results from immunogold-labelling EM experiments suggested that a fraction of the CI present in the preparations was localized to one end of the virion. Atomic force microscopy (AFM) studies showed that P1 and P2 contained intact particles, some of which had a protruding tip structure at one end, whilst F virions were less stable and mostly appeared as beaded structures under the conditions of AFM. The RNA of the particles in F was translated five to ten times more efficiently than RNA from P2 particles when these preparations were subjected to translation in wheat-germ extracts. The results are discussed in the context of a model for CI-mediated functions.
authors.affiliation: Department of Applied Chemistry and Microbiology, FIN-00014 University of Helsinki, Finland.
authors.forename: Rasa
authors.initials: R
authors.lastname: Gabrenaite-Verkhovskaya
authors.affiliation: Plant Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK.
authors.forename: Igor A
authors.initials: IA
authors.lastname: Andreev
authors.affiliation: A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119992, Russia.
authors.forename: Natalia O
authors.initials: NO
authors.lastname: Kalinina
authors.affiliation: Plant Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK.
authors.forename: Lesley
authors.initials: L
authors.lastname: Torrance
authors.affiliation: Plant Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Taliansky
authors.affiliation: Department of Applied Chemistry and Microbiology, FIN-00014 University of Helsinki, Finland.
authors.forename: Kristiina
authors.initials: K
authors.lastname: Mäkinen
mesh_terms: D000595:Amino Acid Sequence; D002499:Centrifugation, Density Gradient; D047468:Immunoprecipitation; D018625:Microscopy, Atomic Force; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010935:Plant Diseases / Q000821:virology*; D017800:Potyvirus / Q000378:metabolism*; D011198:Solanum tuberosum / Q000821:virology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*; D014771:Virion / Q000302:isolation & purification* / Q000378:metabolism* / Q000472:pathogenicity
chemical_list: D014764:Viral Proteins; C406275:cylindrical inclusion protein, potyvirus