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 ro sette 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 assesse d also for RNA-binding properties. This protein was tagged at its C terminu s with six histidine residues, produced in Escherichia coli using an expres sion vector and purified by affinity chromatography. Gel retardation analys is demonstrated that, in contrast to many other viral MPs, including the 3a MP of Cucumber mosaic virus (CMV), the ORF4-encoded protein bound non-coop eratively to viral ssRNA and formed complexes of low protein: RNA ratios. C ompetition binding experiments showed that the ORF4-encoded protein bound t o both ssRNA and ssDNA without sequence specificity, but did not bind to ds DNA. UV cross-linking and nitrocellulose membrane-retention assays confirme d that both the GRV and the CMV MPs formed complexes with ssRNA and that th ese complexes showed similar stability in NaCl. Probing the MP-RNA complexe s by atomic force microscopy demonstrated that the ORF4-encoded protein bou nd RNA incompletely, leaving protein-free RNA segments of varying length, w hile the CMV 3a protein formed highly packed complexes. The significance of the two properties of limited RNA binding and tubule formation of the umbr aviral MP is discussed.