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ABSTRACTThe intracellular enveloped form of vaccinia virus (IEV) induces the formation of actin tails that are strikingly similar to those seen in Listeriaand Shigellainfections. In contrast to the case for Listeriaand Shigella, the vaccinia virus protein(s) responsible for directly initiating actin tail formation remains obscure. However, previous studies with recombinant vaccinia virus strains have suggested that the IEV-specific proteins A33R, A34R, A36R, B5R, and F13L play an undefined role in actin tail formation. In this study we have sought to understand how these proteins, all of which are predicted to have small cytoplasmic domains, are involved in IEV assembly and actin tail formation. Our data reveal that while deletion of A34R, B5R, or F13L resulted in a severe reduction in IEV particle assembly, IEVs formed by the ?B5R and ?F13L deletion strains, but not ?A34R, were still able to induce actin tails. The ?A36R deletion strain produced normal amounts of IEV particles, although these were unable to induce actin tails. Using several different approaches, we demonstrated that A36R is a type Ib membrane protein with a large, 195-amino-acid cytoplasmic domain exposed on the surface of IEV particles. Finally, coimmunoprecipitation experiments demonstrated that A36R interacts with A33R and A34R but not with B5R and that B5R forms a complex with A34R but not with A33R or A36R. Using extracts from ?A34R- and ?A36R-infected cells, we found that the interaction of A36R with A33R and that of A34R with B5R are independent of A34R and A36R, respectively. We conclude from our observations that multiple interactions between IEV membrane proteins exist which have important implications for IEV assembly and actin tail formation. Furthermore, these data suggest that while A34R is involved in IEV assembly and organization, A36R is critical for actin tail formation.

Our understanding of the interactions between the actin cytoskeleton and cellular membranes at the molecular level is rudimentary. One system that offers an opportunity to examine these interactions in greater detail is provided by vaccinia virus, which induces the nucleation of actin tails from the outer membrane surrounding the virion. To further understand the mechanism of their formation and how they generate motility, we have examined the structure of these actin tails in detail. Actin filaments in vaccinia tails are organized so they splay out at up to 45° from the centre of the tail and are up to 0.74 μm in length, which is considerably longer than those reported in the Listeria system. Actin filaments show unidirectional polarity with their barbed filament ends pointing towards the surface of the virus particle. Rhodamine-actin incorporation experiments show that the first stage of tail assembly involves a polarized recruitment of G-actin, and not pre-formed actin filaments, to the membrane surrounding the virion. Incorporation of actin into the tail only occurs by nucleation from the viral surface, suggesting filament ends in the tail are blocked against further actin addition. As virus particles fuse with the plasma membrane during the extention of projections, actin nucleation sites previously in the viral membrane become localized to the plasma membrane, where they are able to nucleate actin polymerization in a manner analogous to the leading edge of motile cells.