Imaging the early events of TMV infection (original) (raw)
2008, Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology
Almost nothing is known of the early stages of TMV infection. To address this, we directly labelled the viral RNA of TMV by incorporation of UTP-Cy3 and injected it onto the cytoplasm of living tobacco trichome cells. The Cy3-labelled virions were infectious and the viral genome trafficked from cell-to-cell. However, neither labelled vRNA nor coinjected GFP were able to pass out of the initial injected trichome, indicating that virus movement out of trichomes is not accompanied by passive plasmodesmatal gating. Both Cy3-virions and uncoated Cy3-vRNA formed granules that became anchored to the motile cortical ER/ actin network of the trichome cell within minutes of injection. Movement of vRNA granules on the actin/ER was arrested by inhibitors of the actin cytoskeleton (cytochalasin). TMV capping was shown to be required for vRNA anchoring to the ER. Virions, or vRNA, lacking the 5′ cap failed to form RNA transport granules and were degraded in the host-cell cytoplasm. Deleting the 3′ prime UTR region from TMV virions did not affect the initial formation or anchoring of vRNA granules. We subsequently generated dual-labelled infectious TMV virions in which the vRNA was labelled with Cy3 (red) and the capsid protein was labelled with Cy2 (green). Following injection, both red and green signals were located on the same ER-bound granules, with a subsequent loss of the green signal only, indicating that in natural infections TMV virions are anchored to the ER prior to uncoating of the viral genome.
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