INTRODUCTION A key mechanism in the virulence of any cellular pathogen is its ability to move from one cell to another to facilitate the spread of infection. A group of unrelated bacterial pathogens, Shigella, Listeria and Rickettsia, that are the causative agents of a number of important diseases including meningitis, septi- caemia, bacillary dysentery and spotted fever, have indepen- dently developed a similar actin-based mechanism that is essential for their intercellular spread (for reviews see Tilney and Tilney, 1993; Cossart and Kocks, 1994; Cossart, 1995). These bacteria are not the only infectious agents that have profound effects on the actin cytoskeleton of their host. Several viruses have been shown to disrupt or stabilize the actin cytoskeleton (Tyrell and Norrby, 1978; Giuffre et al., 1982; Murti et al., 1985; Bohn et al., 1986). The best studied example is that of vaccinia virus, the prototype member of the orthopox genus and a close relative of smallpox (Stokes, 1976; Hiller et al., 1979, 1981; Krempien et al., 1981; Cudmore et al., 1995). Vaccinia, one of the largest and most complex viruses known, has a programmed series of interactions with the host cell that involve wrapping by two membrane cisternae of different subcellular origin during viral assembly and matura- tion. An intriguing characteristic of the vaccinia virus assembly process is that it results in two different infectious forms. The first of these, the intracellular mature virus (IMV), is sur- rounded by a membrane cisterna derived from the intermedi- ate compartment (Sodeik et al., 1993). Infectious IMV are released when the cell lyses due to the cytotoxic effects of infection. Alternatively, a small proportion of IMV, which varies according to virus strain and cell type, can undergo a second wrapping step by a cisternal domain derived from the trans-Golgi network (Payne, 1980; Payne and Kristensson, 1985; Schmelz et al., 1994). This form is called the intracellu- lar enveloped virus (IEV) and escapes from the cell by fusion of its outer membrane with the plasma membrane of the host, thereby releasing the second infectious form called the extra- cellular enveloped virus (EEV) (Dales, 1971; Morgan, 1976; Payne, 1980; Blasco and Moss, 1991). During the fusion of IEV with the plasma membrane, a small proportion of EEV particles are not released into the medium but remain associ- ated with the outside of the cell. These particles are refered to as cell-associated enveloped virus (CEV) (Blasco and Moss, 1992). The first indication that vaccinia virus was able to interact with the cytoskeleton during its complex assembly process came from high voltage electron microscopy studies which showed virus particles at the tips of large microvilli-like pro- jections in infected cells (Stokes, 1976). Subsequent studies confirmed that these vaccinia-tipped projections contained actin, as well as the actin cross-linking proteins α-actinin, filamin and fimbrin but not tropomyosin or myosin (Hiller et al., 1979, 1981). Furthermore, inhibition of virus assembly prevented the formation of the large microvilli suggesting that these structures were virally induced (Krempien et al., 1981). In light of the effects of bacterial pathogens on the actin 1739 Journal of Cell Science 109, 1739-1747 (1996) Printed in Great Britain © The Company of Biologists Limited 1996 JCS4226 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 con- siderably 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. Incor- poration 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 polymer- ization in a manner analogous to the leading edge of motile cells. Key words: Vaccinia virus, Actin tail, Membrane SUMMARY Vaccinia virus: a model system for actin-membrane interactions Sally Cudmore, Inge Reckmann, Gareth Griffiths and Michael Way* Cell Biology Programme, EMBL, Meyerhofstrasse 1, Heidelberg 69117, Germany *Author for correspondence (e-mail: way@embl-heidelberg.de)