Replication of tobacco mosaic virus on endoplasmic reticulum and role of the cytoskeleton and virus movement protein in intracellular distribution ofviral RNA

Little is known about the mechanisms of intracellular targeting of viral nu cleic acids within infected cells. We used in situ hybridization to visuali ze the distribution of tobacco mosaic virus (TMV) viral RNA (vRNA) in infec ted tobacco protoplasts. Immunostaining of the ER lumenal binding protein ( BiP) concurrent with in situ hybridization revealed that VRNA colocalized w ith the ER, including perinuclear ER. At midstages of infection, vRNA accum ulated in large irregular bodies associated with cytoplasmic filaments whil e at late stages, vRNA was dispersed throughout the cytoplasm and was assoc iated with hair-like protrusions from the plasma membrane containing ER. TM V movement protein (MP) and replicase colocalized with vRNA, suggesting tha t viral replication and translation occur in the same subcellular sites. Im munostaining with tubulin provided evidence of colocalization of vRNA with microtubules, while disruption of the cytoskeleton with pharmacological age nts produced severe changes in vRNA localization. Mutants of TMV lacking fu nctional MP accumulated vRNA, but the distribution of vRNA was different fr om that observed in wild-type infection. MP was not required for associatio n of vRNA with perinuclear ER, but was required for the formation of the la rge irregular bodies and association of vRNA with the hair-like protrusions .