The budded form of Autographa californica M nucleopolyhedrovirus enters permissive cells via adsorptive endocytosis. Shortly after nucleocapsid penetration into the cytoplasm, thick actin cables form, which frequently project toward the nucleus. These actin cables are transient structures, formed in association with viral nucleocapsids prior to viral gene expression and concomitant with nucleocapsid transport to the nucleus. In this paper we report that nucleocapsids are capable of nucleating actin polymerization in vitro in a concentration-dependent manner. Two viral-encoded capsid proteins, p39 and p78/83, were found to bind actin directly and therefore could be involved in the observed acceleration of actin polymerization. When nucleocapsids were added to actin in the presence of cytochalasin D, actin polymerization was reduced to levels below those obtained with actin and cytochalasin D alone, suggesting that the nucleocapsids bound to the pointed ends of actin filaments. Finally, treatment of infected cells with the myosin inhibitor 2,3-butanedione monoxime delayed nucleocapsid transport to the nucleus. We postulate that upon entering the cytoplasm, AcMNPV nucleocapsids induce the polymerization of actin cables, which, in conjunction with a myosin-like motor, facilitate their transport to and/or into the nucleus.
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The authors thank David Drubin for allowing us to use his Hitachi F-4010 fluorometer and for his guidance, Christopher Richardson and Jorge Vialard for the antibody to p78/83 and for the bacterial expression plasmid pT7-7 containing AcMNPV ORF8 encoding p78, Paul Janmey for gelsolin, and Velia Fowler for the antibody to tropomodulin. We also thank Beth Luna for consultation and for reviewing this manuscript, Jan Washburn for his editorial comments, and Sharyl Wong for her assistance with the Latrunculin B and CD time course experiments. Equipment used for fluorescence microscopy was made available by Center for Biological Imaging, University of California, Berkeley. Financial support for these studies was provided by USDA NRICG 97-35302-4340, by federal Regional Research and HATCH funds, and by NIH Training Grant GM07232.