Umbravirus-encoded movement protein induces tubule formation on the surface of protoplasts and binds RNA incompletely and non-cooperatively.
Various functions of the cell-to-cell movement protein (MP) of Groundnut rosette virus (GRV) were analysed. The GRV ORF4-encoded protein was shown by immunofluorescence microscopy to generate tubular structures that protrude from the surface of the protoplast. The protein encoded by ORF4 was assessed also for RNA-binding properties. This protein was tagged at its C terminus with six histidine residues, produced in Escherichia coli using an expression vector and purified by affinity chromatography. Gel retardation analysis demonstrated that, in contrast to many other viral MPs, including the 3a MP of Cucumber mosaic virus (CMV), the ORF4-encoded protein bound non-cooperatively to viral ssRNA and formed complexes of low protein:RNA ratios. Competition binding experiments showed that the ORF4-encoded protein bound to both ssRNA and ssDNA without sequence specificity, but did not bind to dsDNA. UV cross-linking and nitrocellulose membrane-retention assays confirmed that both the GRV and the CMV MPs formed complexes with ssRNA and that these complexes showed similar stability in NaCl. Probing the MP-RNA complexes by atomic force microscopy demonstrated that the ORF4-encoded protein bound RNA incompletely, leaving protein-free RNA segments of varying length, while the CMV 3a protein formed highly packed complexes. The significance of the two properties of limited RNA binding and tubule formation of the umbraviral MP is discussed.
Nurkiyanova
Kulpash M
KM
Unit of Virology, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK1.
Ryabov
Eugene V
EV
Unit of Virology, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK1.
Kalinina
Natalia O
NO
Unit of Virology, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK1.
Fan
Yongchang
Y
Department of Electronic Engineering and Physics, University of Dundee, Dundee DD1 4NH, UK2.
Andreev
Igor
I
Department of Electronic Engineering and Physics, University of Dundee, Dundee DD1 4NH, UK2.
Fitzgerald
Alexander G
AG
Department of Electronic Engineering and Physics, University of Dundee, Dundee DD1 4NH, UK2.
Palukaitis
Peter
P
Unit of Virology, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK1.
Taliansky
Michael
M
Unit of Virology, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK1.
D010367:Arachis / Q000821:virology*; D017799:Cucumovirus / Q000502:physiology; D018625:Microscopy, Atomic Force; D016366:Open Reading Frames; D053830:Plant Viral Movement Proteins; D010942:Plant Viruses / Q000502:physiology*; D011523:Protoplasts / Q000502:physiology*; D012313:RNA / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*
D053830:Plant Viral Movement Proteins; D014764:Viral Proteins; D012313:RNA