The 5’cap of Tobacco Mosaic Virus (TMV) is required for virion attachment to the actin/ER network during early infection

Almost nothing is known of the earliest stages of plant virus infections. To address this, we microinjected Cy3 (UTP)-labelled tobacco mosaic virus (TMV) into living tobacco trichome cells. The Cy3-virions were infectious, and the viral genome trafficked cell-to-cell. However, neither the fluorescent vRNA pool nor co-injected GFP left the injected trichome, indicating that the synthesis of unlabelled progeny viral (v)RNA is required to initiate cell-cell movement, and that virus movement is not accompanied by passive plasmodesmatal gating. Cy3-vRNA formed granules that became anchored to the motile cortical actin/ER network within minutes of injection. Granule movement on actin/ER was arrested by actin inhibitors indicating actindependent RNA movement. The 5’ methylguanosine TMV cap was shown to be required for vRNA anchoring to the ER. TMV vRNA lacking the 5’cap failed to form granules and was degraded in the cytoplasm. Removal of the 3’UTR and replicase both inhibited replication but did not prevent granule formation and movement. Dual-labelled TMV virions in which the vRNA and the coat protein were highlighted with different fluorophores showed both fluorescent signals to be initially located on the same ER-bound granules, indicating that TMV virions may become attached to the ER prior to uncoating of the viral genome.