Budding of Marburgvirus is associated with filopodia Larissa Kolesnikova, 1,2 Aparna B. Bohil, 3 Richard E. Cheney 3 and Stephan Becker 1,2 * 1 Institute of Virology, Marburg Philipps University, Marburg, Germany. 2 Robert Koch-Institut, Berlin, Germany. 3 Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, NC 27599, USA. Summary Viruses exploit the cytoskeleton of host cells to trans- port their components and spread to neighbouring cells. Here we show that the actin cytoskeleton is involved in the release of Marburgvirus (MARV) particles. We found that peripherally located nucleo- capsids and envelope precursors of MARV are located either at the tip or at the side of filopodial actin bundles. Importantly, viral budding was almost exclusively detected at filopodia. Inhibiting actin poly- merization in MARV-infected cells significantly dimin- ished the amount of viral particles released into the medium. This suggested that dynamic polymerization of actin in filopodia is essential for efficient release of MARV. The viral matrix protein VP40 plays a key role in the release of MARV particles and we found that the intracellular localization of recombinant VP40 and its release in form of virus-like particles were strongly influenced by overexpression or inhibition of myosin 10 and Cdc42, proteins important in filopodia forma- tion and function. We suggest that VP40, which is capable of interacting with viral nucleocapsids, pro- vides an interface of MARV subviral particles and filopodia. As filopodia are in close contact with neigh- bouring cells, usurpation of these structures may facilitate spread of MARV to adjacent cells. Introduction Eukaryotic cells are surrounded by a layer of F-actin underlying the plasma membrane, the actin cortex. This structure is important for the maintenance of cell shape and for cell movement (Mitchison and Cramer, 1996). The actin cortex can also either facilitates or hinders the move- ment and release of viruses that bud at the plasma membrane. For example, the power of actin polymeriza- tion is used to propel vaccinia virus away from the cell surface, thus facilitating spread to uninfected cells (Cudmore et al., 1995; Rottger et al., 1999; Hollinshead et al., 2001). On the other hand, short-term actin depoly- merization stimulated the release of equine infectious anaemia virus suggesting that the intact actin cortex can hinder the release of some viral particles (Chen et al., 2004). Additionally, several studies suggest that viruses use an actin-myosin-based transport step during their egress (Sasaki et al., 1995; van Leeuwen et al., 2002). The presence of actin in purified virus particles is usually considered as indicator that actin is involved in the budding process (Smith and Enquist, 2002). Actin was detected in particles of several members of the order Mononegavirales including Marburgvirus (MARV; Kolesni- kova et al., 2002), respiratory syncytial virus (Fernie and Gerin, 1982; Burke et al., 1998; Ulloa et al., 1998), human parainfluenza viruses 2/3 (Cowley and Barry, 1983; Pani- grahi et al., 1987), and rabies virus (Naito and Matsu- moto, 1978; Sagara et al., 1995). In all these cases, the identity of actin-based structures that accompany release of viral particles remains unknown. Marburgvirus (family Filoviridae, order Mononegavi- rales) causes a fulminant haemorrhagic disease in humans and non-human primates, resulting in high mor- tality rates (Peters, 2005). Infection with MARV is accom- panied by haemostatic impairment, hepatic dysfunction, severe lymphoid depletion and generalized shock. Out- breaks of MARV disease in sub-Saharan Africa underline the emerging potential of MARV (Borchert et al., 2002; Bausch et al., 2003; Colebunders et al., 2004; CDC, 2005; Ligon, 2005). Like the other members of the order Mononegavirales, MARV contains a single-stranded negative-sense RNA genome, which is encapsidated by the nucleoprotein (NP). Seven structural proteins are encoded by the genome (Feldmann et al., 1992; Bukreyev et al., 1995). The polymerase (L), VP35 and VP30 associate with NP to generate the helical ribonucleocapsid structure (Sanchez et al., 1992; Becker et al., 1998; Mavrakis et al., 2002). The glycoprotein (GP), which is inserted in the viral enve- lope, mediates cell entry (Becker et al., 1995; Marzi et al., 2004). VP40 and VP24 represent matrix proteins and play a key role in virion assembly (Kolesnikova et al., 2002; 2004a,b; Bamberg et al., 2005). Morphogenesis of MARV is associated with formation of viral inclusions located in the perinuclear region (Peters Received 30 May, 2006; revised 25 September, 2006; accepted 26 September, 2006. *For correspondence. E-mail beckerst@rki.de; Tel. (+49) 030 4547 2373; Fax (+49) 030 4547 2181. Cellular Microbiology (2007) 9(4), 939–951 doi:10.1111/j.1462-5822.2006.00842.x First published online 28 November 2006 © 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd