Actin Rearrangement-Inducing Factor of Baculoviruses Is Tyrosine Phosphorylated and Colocalizes to F-Actin at the Plasma Membrane

In previous studies we have identified actin rearrangement-inducing factor 1 as an early gene product of Autographa californica multicapsid nuclear polyhedrosis virus that is involved in the remodeling of the actin cytoskeleton. We have constructed viral recombinants with a mutated Arif-1 open reading frame that confirm the causal link of Arif-1 expression and the actin rearrangement observed as accumulation of F-actin at the plasma membrane at 3 to 7 h postinfection. Infection with Arif mutant viruses leads to the loss of actin accumulation at the plasma membrane in TN-368 cells, although in the course of infection, early actin cables and nuclear F-actin are observed as in wild-type-infected cells. By immunofluorescence studies, we have demonstrated the localization of Arif-1 at the plasma membrane, and confocal imaging reveals the colocalization to F-actin. Accordingly, the ;47-kDa Arif-1 protein is observed exclusively in membrane fractions prepared at 4 to 48 h postinfection, with a decrease at 24 h postinfection. Phosphatase treatment suggests that Arif-1 is modified by phosphorylation. Antibodies against phosphotyrosine precipitate Arif-1 from membrane fractions, indicating that Arif-1 becomes tyrosine phosphorylated during the early and late phases of infection. In summary, our results indicate that functional Arif-1 is tyrosine phosphorylated and is located at the plasma membrane as a component of the actin rearrangement-inducing complex. During their life cycle, viruses can interact specifically with the actin cytoskeleton of their host cells, resulting in a variety of alterations. Those alterations that are distinct from the effects that follow the virus-induced breakdown of the cells have been postulated to play a role in viral genome transcription and replication, virion assembly, and viral budding (for a review, see reference 5). Extensive changes of the actin cytoskeleton have been described in cells infected with the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). The different stages of actin rearrangement include the induction of actin cables, followed by a second step of reorganizing the microfilaments, and the appearance of nuclear filamentous (F)-actin (3). The functional role of the virus-induced changes is still speculative. AcMNPV belongs to the large DNA viruses and infects