Visualization and Characterization of the Intracellular Movement of Vaccinia Virus Intracellular Mature Virions

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Abstract
Previous work indicated that vaccinia intracellular mature virus (IMV) utilizes microtubules to move from the viral factory to the site of intracellular envelopment and that expression of the viral A27 protein is required for this transport. To investigate further the role of A27 in IMV intracellular transport, a recombinant vaccinia virus was constructed that had the A27L gene deleted and expressed a yellow fluorescent protein (YFP)-A4 chimera in place of the normal A4 protein. The resulting recombinant, vYFP-A4/ΔA27, produced relatively normal quantities of virus in a one-step growth curve but had a small plaque phenotype. Subsequent experiments demonstrated that vYFP-A4/ΔA27 was severely defective in envelope virus production. Despite the absence of A27, live digital video fluorescent microscopy visualized YFP-labeled IMV movement in cells infected with the recombinant. Virion movement approached 3 μm/s and was sensitive to the microtubule depolymerizing drug nocodazole. In addition, IMV could be discerned transiting away from and back towards viral factories. Immunofluorescent staining determined that the distance traveled by A27-deficient virions was sufficient for transport to the site of envelopment. These results indicate that IMVs are capable of bidirectional movement on microtubules, suggesting that they are able to interact with both kinesin and dynein microtubule motors in the absence of A27 and that the distance traveled is sufficient to deliver IMV to the site of wrapping.