Bars, 5 m. SEPT7 at 18 h after infection with WR or the YdF virus. Error bars represent SEM from three independent experiments. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Bar, 1 cm. Although we have a good molecular understanding of how CEVs induce actin polymerization, we still lack crucial insights into the events taking place when IEVs fuse with the plasma membrane during viral spread. Previous observations from genome-wide high-throughput RNAi-based screens demonstrate that knockdown of septins enhances vaccinia replication and/or spread (Sivan et al., 2013; Beard et al., 2014). Septins are a family of cytoskeletal proteins found in animals and fungi (Mostowy and Cossart, 2012). In humans, there are 13 septins, which are subdivided into four different homology groups (SEPT2, SEPT3, SEPT6, and SEPT7; Saarikangas and Barral, 2011; Mostowy and Cossart, 2012; Neubauer and Zieger, 2017). Septins form heterooligomers that assemble into nonpolar filaments and ring-like structures in the cytoplasm and on the plasma membrane (Kinoshita et al., 2002; Sirajuddin et al., 2007; Bertin et al., 2008; Bridges et al., 2014). All higher-order septin structures contain SEPT2 and SEPT6 family members but are critically dependent on SEPT7 (Sirajuddin et al., 2007). Septins play a variety of roles in many cellular processes including cell division and migration as well as membrane trafficking by virtue of their ability to associate with lipids, microtubules, and actin filaments (Saarikangas SBI-553 and Barral, 2011; Mostowy and Cossart, 2012). Septins can also inhibit bacterial infection by forming cage-like structures around intracellular pathogens such as (Mostowy et al., 2010; Sirianni et al., 2016). We now report that septins are recruited to vaccinia virus after its fusion with the plasma membrane and act to suppress the release of the virus from infected cells. Moreover, the Nck-mediated recruitment of dynamin to the virus as well as formin-driven actin polymerization displaces septins, thereby overcoming their antiviral effect. Results Septins suppress the release and SBI-553 cell-to-cell spread of vaccinia To understand the role of septins during vaccinia infection, we examined the impact of the loss of SEPT7 on the release and spread of the Western Reserve (WR) strain of vaccinia virus. The knockdown efficiency of SEPT7, which is essential for septin filament formation SBI-553 and function (Sirajuddin et al., 2007), was confirmed by immunoblot analysis (Fig. 1 B). We found that loss of SEPT7 leads to a significantly larger WR plaque diameter in confluent A549 cell monolayers with liquid (Fig. 1 C) or semisolid overlay (Fig. S1, A and B); the latter measures only direct cell-to-cell spread. It is also striking that loss of SEPT7 leads to the formation of extensive plaque comets in liquid overlay, which are seen as a diffuse spray emanating from a central round plaque. This phenomenon is indicative of enhanced virus release in liquid overlay conditions (Yakimovich et al., 2015). In SBI-553 agreement with their assembly into functional heteromeric complexes, we BRIP1 found that RNAi-mediated loss of SEPT2 or SEPT11 also increases the size of plaques induced by WR (Fig. S1 C). The increase in plaque size in the absence of SEPT7 is not restricted to WR, as it is also apparent in cells infected with WR expressing A36-YdF (designated as YdF), a vaccinia strain deficient in actin tail formation and cell-to-cell spread since A36 cannot be phosphorylated on tyrosine 112 or 132 (Rietdorf et al., 2001; Ward and Moss, 2001; Fig. 1 C and Fig. S1 A). In both cases, there was also a concomitant increase in virus release when SEPT7 was depleted (Fig. 1 D). This increase in release is not related to virus production, as septin loss actually reduces the number of intracellular virions (Fig. 1 D). RNAi-mediated depletion of SEPT7 in HeLa cells has no appreciable impact on the actin cytoskeleton (Fig. 2, A and B), and vaccinia infection does not affect the level of septin expression (Fig. 2 C). However, loss of SEPT7 increases the number of CEV inducing actin tails (35.5 1.7% compared with 23.9 0.5%), which are also significantly longer (3.9 0.1 m compared with 3.0 0.1 m; Fig. 2 D and Fig. S2 A). Loss of SEPT2, SEPT9, or SEPT11 also results in more CEV-inducing actin tails that are again longer than normal (Fig. S2 B). The velocity and directionality of actin tails remained the same in the absence of SEPT7 and functional septins (Fig. 2 E). However, the time required for actin tail formation after the virus reached the cell periphery decreased (62.1 5.4 s compared with 80.2 7.5 s). It was also noticeable that actin tails had a significantly longer lifetime, lasting on average 3.8 0.1 min compared with 2.9 .