Clathrin Potentiates Vaccinia-Induced Actin Polymerization to Facilitate Viral Spread

Cell Host & Microbe

Article Clathrin Potentiates Vaccinia-Induced Actin Polymerization to Facilitate Viral Spread Ashley C. Humphries,1 Mark P. Dodding,1,3 David J. Barry,1 Lucy M. Collinson,2 Charlotte H. Durkin,1 and Michael Way1,* 1Cell

Motility Laboratory Microscopy Department Cancer Research UK, London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3LY, UK 3Present address: Randall Division of Cell and Molecular Biophysics, King’s College, London SE1 1UL, UK *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chom.2012.08.002 2Electron

SUMMARY

During their egress, newly assembled vaccinia virus particles fuse with the plasma membrane and enhance their spread by inducing Arp2/3-dependent actin polymerization. Investigating the events surrounding vaccinia virus fusion, we discovered that vaccinia transiently recruits clathrin in a manner dependent on the clathrin adaptor AP-2. The recruitment of clathrin to vaccinia dramatically enhances the ability of the virus to induce actin-based motility. We demonstrate that clathrin promotes clustering of the virus actin tail nucleator A36 and host N-WASP, which activates actin nucleation through the Arp2/3 complex. Increased clustering enhances N-WASP stability, leading to more efficient actin tail initiation and sustained actin polymerization. Our observations uncover an unexpected role for clathrin during virus spread and have important implications for the regulation of actin polymerization. INTRODUCTION During vaccinia virus infection, newly assembled intracellular enveloped virus (IEV) particles are transported from their perinuclear site of assembly to the plasma membrane in a microtubuledependent fashion by kinesin-1 (Dodding et al., 2011; Geada et al., 2001; Hollinshead et al., 2001; Rietdorf et al., 2001; Ward and Moss, 2001a, 2001b). When IEV reach the cell periphery, they undergo actin-dependent movements in the cell cortex prior to fusing with the plasma membrane (Arakawa et al., 2007a). This fusion event is associated with loss of kinesin-1 as well as the IEV-associated proteins E2 and F12 (Dodding et al., 2009; Newsome et al., 2004) and results in the liberation of virus particles from the infected cell. However, a proportion of particles are not immediately released into the culture medium but remain attached to the outside of the cell. These cell-associated enveloped viruses (CEV) are able to induce Arp2/3 complex-dependent actin polymerization to enhance their spread to neighboring cells (Cudmore et al., 1995, 1996; Doceul et al., 2010; Frischknecht et al., 1999; Hollinshead et al., 2001; Ward and Moss, 2001a). CEV stimulate actin polymerization by inducing an outside-in signaling cascade

that locally activates Src and Abl family kinases (Frischknecht et al., 1999; Newsome et al., 2004, 2006; Reeves et al., 2005). Activation of these kinases results in the phosphorylation of tyrosine 112 and 132 of A36, which is localized beneath CEV after IEV fuse with the plasma membrane (Frischknecht et al., 1999; Newsome et al., 2004, 2006; Reeves et al., 2005). Phosphorylation of A36 mediates release of kinesin-1 and recruitment of Nck and Grb2 to tyrosine 112 and 132, respectively (Frischknecht et al., 1999; Newsome et al., 2004; Scaplehorn et al., 2002; Weisswange et al., 2009). Nck and Grb2 in turn recruit a complex of WIP and N-WASP, the latter of which is able to activate the Arp2/3 complex to induce actin polymerization beneath the CEV and enhance the spread of infection (Cudmore et al., 1995, 1996; Doceul et al., 2010; Frischknecht et al., 1999; Moreau et al., 2000; Scaplehorn et al., 2002; Snapper et al., 2001; Weisswange et al., 2009). In the current study, we examined the events at the plasma membrane prior to formation of vaccinia-induced actin tails, which still remain largely uncharacterized. We found that AP-2 and clathrin are recruited to the virus after IEV fuse with the plasma membrane, but are left behind when CEV initiate actinbased motility. The transient recruitment of AP-2 and clathrin, however, impacts on the ability of the virus to induce and sustain actin-based motility. Our data demonstrate that clathrin helps cluster A36 and its associated signaling network to promote robust actin polymerization. RESULTS AP-2 and Clathrin Are Recruited by Extracellular Virus Prior to Actin Nucleation Ultrastructural analysis of vaccinia-infected cells revealed the presence of a spiky structure, highly reminiscent of a clathrin coat surrounding viral particles in the cell periphery (Figure 1A). Immunofluorescence analysis confirmed that clathrin and its plasma membrane adaptor AP-2 are associated with a subset of DAPI positive virus particles (Figure 1B). Labeling prior to permeabilization with an antibody against the viral protein B5 demonstrated that AP-2 and clathrin are in fact associated with a similar proportion of CEV, suggesting that both proteins are recruited concurrently (Figure 1B). Consistent with this, AP-2 and clathrin were colocalized on DAPI positive viral particles (Figure 1B). Our observations suggest that AP-2 and clathrin are recruited to viral particles after they fuse with the plasma membrane.

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Cell Host & Microbe Clathrin Clusters N-WASP to Enhance Vaccinia Spread

Figure 1. Clathrin and AP-2 Localize to Vaccinia Virus during Egress

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(A) Electron micrographs showing virus at the periphery of cells with an apparent clathrin coat (yellow arrows). Scale bar = 250 nm. (B) Immunofluorescence images showing that extracellular virus particles (Ex-virus) detected with anti-B5 prior to permeabilization and positive for DNA (DAPI) recruit clathrin heavy chain (CHC) and the plasma membrane adaptor protein-2 (AP-2) (white arrows). CHC and AP-2 colocalize on DNApositive viral particles (yellow arrows). Quantification of the colocalization of CHC and AP-2 on DAPIpositive extracellular virus particles is shown. Error bars represent SEM from 30 cells in three independent experiments. Scale bars = 1 and 10 mm.

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Consistent with this, we did not detect AP-2 or clathrin on kinesin1 positive IEV, indicative of particles undergoing microtubule transport (Figure 2A). Additionally, neither AP-2 nor clathrin were associated with actin tails (Figure 2B). This suggests that either both proteins are recruited to the virus prior to actin nucleation or, alternatively, those particles that recruit clathrin do not go on to induce actin tails. To resolve this issue, we performed live imaging of cells expressing GFP-tagged clathrin light chain (GFP-LCa) infected with a virus expressing RFP-A3 (a core viral protein). We found that virus particles acquire clathrin following the cessation of rapid, microtubule-dependent transport (Figure 2C, Movie S1). The clathrin signal, however, is left behind when the virus initiates actin-based motility. Our live cell observations demonstrate that clathrin is transiently recruited to virus particles between their microtubule and actin-dependent motilities. To pinpoint the precise stage of clathrin recruitment, relative to IEV fusion with the plasma membrane and actin tail formation, we performed live imaging on infected cells in media containing an Alexa 488-conjugated B5 antibody (B5-488). This antibody detects extracellular viral particles after they fuse with the plasma membrane in live unfixed cells (Figure S1). We found that clathrin (Cherry-LCa) only accumulated on viral particles that had previously recruited the Alexa 488-conjugated B5 antibody (Figure 3A, Movie S2). Clathrin is therefore recruited to viral particles only after they fuse with the plasma membrane.

To investigate the relationship between clathrin and actin-based movement, we imaged cells stably expressing LifeActCherry infected with a virus expressing RFP-A3. The difference in shape and signal intensities between LifeAct-Cherry n.s. and RFP-A3 allows visualization of virus particles and actin tails in the same fluorescent channel in live cells (Figure 3B, Movie S3). Clathrin was readily detected on stationary viral particles. However, it was left behind and its signal subsequently dissipated when virus particles underwent actin-based motility (Figure 3B, Movie S3). To examine whether the association of clathrin with CEV changes in the absence of virus-induced actin polymerization, we inhibited actin tail formation using two different methods. In the first, we compared mouse embryonic fibroblast cells lacking either Nck or N-WASP (Bladt et al., 2003; Snapper et al., 2001), both essential components of the actin-signaling complex, to their wild-type counterparts. In the second, we treated HeLa cells with cytochalasin D. We found that all three approaches resulted in a significant increase in the colocalization of AP-2 with extracellular virus particles (Figure 3C). This increased colocalization, together with our observations in live cells, suggests that actin polymerization is responsible for disrupting the association of AP-2 and clathrin with the virus. AAPP --22

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AP-2 and Clathrin Promote Actin Tail Formation Given the transient recruitment of clathrin prior to actin nucleation, we wanted to determine whether it plays a functional role in virus-induced actin polymerization. We depleted clathrin using siRNA oligos against the clathrin heavy chain and AP-2 by targeting either its a or m2 subunit, as depletion of one subunit markedly reduces the level of the other (Motley et al., 2003) (Figure 4A). Loss of the AP-2 subunits resulted in a dramatic reduction in the colocalization of AP-2 and clathrin with extracellular virus particles (Figures 4B and S2). As expected, depletion of the clathrin heavy chain had no impact on AP-2 recruitment (Figures 4B and S2). In both cases, the loss of AP-2 or clathrin resulted in a 40% reduction in the number of actin tails formed

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Figure 2. Clathrin Recruitment Follows Kinesin-1 Loss but Precedes Actin Tail Formation (A) CHC and AP-2 are recruited to DAPI-positive virus particles that lack kinesin-1 (RFP-KLC2) (yellow arrows). White arrows highlight kinesin-1-positive virus. (B) CHC and AP-2 are absent on virus particles inducing actin tails (white arrows). (C) Images from Movie S1 showing that GFP-tagged clathrin light chain (GFP-LCa) is recruited to RFP-A3 virus particles when they stop moving on microtubules (yellow arrow), but is left behind as the virus initiates actin-based motility (white arrow). The trajectories of moving virus before and after clathrin recruitment are indicated by the white dotted lines. The time and the average velocity of virus motility between frames are shown. Scale bar = 1 mm.

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Figure 3. Actin Polymerization Disrupts Clathrin Association (A) Images from Movie S2 showing clathrin (Cherry-LCa) recruitment to an extracellular virus detected with the Alexa 488-conjugated B5 antibody (B5-488) (white arrows) in live cells. See also Figure S1. (B) Images from Movie S3 showing that GFP-LCa is left behind and subsequently disappears (yellow arrows) when the virus (RFP-A3) induces an actin tail (LifeAct-Cherry). In (A) and (B) the time is indicated and scale bar = 1 mm. (C) Images showing the recruitment of AP-2 to extracellular virus particles (Ex-virus) in Nck+/+ or Nck / MEFs, N-WASP+/+ or N-WASP / MEFs, and HeLa cells treated with DMSO or cytochalasin D. The graphs show the quantification of the number of DAPI-positive extracellular virus recruiting AP-2 in the presence (Nck+/+, N-WASP+/+, or DMSO) and absence (Nck / , N-WASP / , or cytochalasin D) of actin tail formation. Error bars represent SEM from 30 cells in three independent experiments. A p value of < 0.001 is indicated by ***. Scale bars = 1 and 10 mm.

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AP-2 Modulates Actin Tail Assembly and Disassembly 20 Kinetics 10 To investigate the role of clathrin in actin tail formation, we performed live cell 0 imaging of RFP-A3 virus particles in LifeAct-Cherry-expressing cells treated with siRNA against the a subunit of AP-2. In N-WASP +/+ MEF N-WASP -/- MEF *** 30 the absence of AP-2, virus particles took 1.653 longer to initiate an actin tail once 20 they reached the cell periphery (Figure 5A). 10 Concomitant with longer length, there was a 21% increase in the speed of actin0 based viral movement in the absence of AP-2 (Figure 5A). However, there was +DMSO +Cytochalasin D a 33% decrease in the average duration 30 *** of their movement relative to controls 20 (Figure 5A). Our observations indicate that in the absence of AP-2 (clathrin) 10 recruitment, it is more difficult for a virus 0 to initiate and sustain an actin tail. Given their longer length, we investigated whether actin filament disassembly is also altered in the absence of clathrin recruitment. A focused 405 nm laser by viruses that had fused with the plasma membrane (Figure 4C). was used to photoactivate GFPPACherry-b-actin directly Consistent with this reduction, we found that AP-2 knockdown beneath YFP-A3 virus particles inducing actin tails (visualized also impaired viral spread in confluent monolayers of A549 cells using the Cherry-actin signal). The intensity of the photoacti(Figure 4D). Intriguingly, however, those actin tails that did form vated GFP-actin patch was monitored over time and the decay were significantly longer than those formed in controls (Fig- kinetics calculated (Figures 5B and 5C, Movie S4). Actin tails ure 4C). The formation of longer tails suggests that AP-2 and formed in the absence of AP-2 had a significantly slower rate clathrin limit the extent and/or rate of actin polymerization on of disassembly (Figure 5C). Our data indicate that changes in individual viruses, despite their recruitment clearly increasing the both the assembly and disassembly rate of actin tails the number of viruses that are capable of forming an actin tail. contribute to their longer length in the absence of AP-2. 3030

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Figure 4. Clathrin Promotes Actin Tail Formation (A) Immunoblot analysis shows the level of clathrin heavy chain (CHC) and AP-2 subunits after siRNA treatment with the indicated RNAi oligos (top). (B) Quantification of the colocalization of AP-2 and clathrin with DAPI-positive extracellular virus in cells treated with CHC or AP-2 RNAi oligos. See also Figure S2. (C) Immunofluorescence images and magnified inserts showing actin tails (red) induced by extracellular viruses (green) in cells treated with the indicated RNAi oligos. Scale bars = 1 and 10 mm. The graphs show the quantification of the number of extracellular virus particles inducing actin tails and their length in the different conditions. In (B) and (C), the error bars represent SEM from 30 cells in three independent experiments. A p value of < 0.001 is indicated by ***. (D) Images of plaques formed on A549 cell monolayers 2 days postinfection after transfection with control or AP-2 a RNAi oligos. The graph shows the quantification of plaque size in the two conditions. Error bars represent SEM from three independent experiments in which a total of 100 plaques were measured. A p value of < 0.01 is indicated by **.

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AP-2 Stabilizes N-WASP at the Tip of Actin Tails Given the changes in actin tail dynamics in the absence of AP-2, we examined whether there were any differences in the vaccinia-signaling network inducing actin polymerization. Immunofluorescence analysis revealed that Nck, Grb2, WIP, and N-WASP are all present at the tip of actin tails in control and AP-2 siRNA-treated cells (Figure S3). As N-WASP turnover is a major determinant in defining the rate of vaccinia actinbased motility (Weisswange et al., 2009), we investigated whether N-WASP turnover was altered in AP-2 siRNA-treated cells. We found that its rate of exchange was significantly faster in the absence of AP-2 (Figure 5D). Analysis of laser-scanning confocal images also revealed that the distribution of N-WASP was more dispersed and had lower peak intensity in the absence of AP-2 (Figure 5E). Taken together, these observations suggest that the recruitment of clathrin may contribute to the organization of the actin-signaling complex beneath viral particles. Clathrin Clusters A36 and N-WASP to Promote Actin Polymerization In contrast to the other integral IEV membrane proteins, A36 has a large cytoplasmic domain (Ro¨ttger et al., 1999). Given this, we investigated whether A36 can interact with AP-2. We found that GST-tagged cytoplasmic domain of A36 (residues 24–221) was capable of associating with AP-2 (Figure 6A). As A36 initiates actin nucleation, any clathrin-mediated changes in its spatial distribution may account for the observed changes in N-WASP turnover and actin tail dynamics in the absence of AP-2. To examine the spatial distribution of A36, we performed highresolution imaging on a DeltaVision OMX structured illumination microscope (Gustafsson et al., 2008). We found that in the majority of cases A36 is distributed around the virus particle as defined by a distinct ring of the inner viral protein A27 (Figures 6B and 6C). When the virus induces an actin tail, there is a considerable polarization of A36 toward the site of actin polymerization (Figures 6B and 6C). In the absence of AP-2, A36 still polarizes, but it is significantly less focused (Figure 6C). The polarization of N-WASP toward the site of actin tail formation is also reduced in AP-2 siRNA-treated cells (Figures 6B and 6C). We took advantage of cells lacking N-WASP to confirm that clathrin acts independently of actin to promote A36 clustering. Consistent with our previous observations (Figure 6C), we found that polarization of A36 still occurs in the absence of N-WASP (actin polymerization), and this is significantly reduced in AP-2 siRNA-treated cells (Figure 6D). Our data suggest that clathrin-mediated clustering of A36 and N-WASP helps promote robust vaccinia-induced actin polymerization. To test this hypothesis, we examined whether mimicking the loss of AP-2 by reducing the density of functional A36 molecules results in a similar phenotype. Cells infected with the DA36R virus were transfected with plasmids encoding wildtype A36 and increasing amounts of one of three different A36 derivatives that cannot induce actin polymerization: the transmembrane domain of A36 fused to Cherry (A36TM) or the kinesin-1 binding region of calsyntenin-1 (A36TM-CSTN 879-971) as well as the A36-YdF mutant. Immunofluorescence analysis revealed that increasing amounts of fluorescent signal from the defective A36 derivative is incorporated into virus particles as

the ratio of defective to wild-type A36 is increased (Figures 7A and 7B). The increase in fluorescent signal from the defective A36 derivative means that the density of functional A36 must be progressively reduced. Consistent with this, there was a corresponding decrease in wild-type A36 (detected with an antibody directed against the cytoplasmic domain of the protein) (Figure 7A). In each case, we found that increasing the amount of defective variant to wild-type A36 led to an increase in actin tail length (Figures 7B and 7C). Furthermore, live imaging showed that actin tails in cells expressing a 1:5 ratio of wildtype A36 to A36-TM-Cherry moved significantly faster than the control (Figure 7D). Reducing the density of functional A36 molecules clearly results in an actin tail phenotype similar to that observed with the loss of clathrin recruitment. DISCUSSION Clathrin Recruitment Promotes Vaccinia Actin Tail Formation In our efforts to obtain more insights into the events at the plasma membrane, we have now discovered that the transient recruitment of AP-2 and clathrin to extracellular virus particles enhances the ability of the virus to induce robust actin polymerization by promoting clustering of A36. The recruitment of clathrin to extracellular virus at this stage of infection might be considered somewhat unexpected, as its canonical role is in endocytosis, a process of internalization that recycles, traffics, or removes transmembrane proteins and lipids from the surface of cells (McMahon and Boucrot, 2011; Reider and Wendland, 2011; Traub, 2009). During endocytosis, clathrin is recruited to the plasma membrane by AP-2, which interacts with sorting signals in the cytoplasmic domains of transmembrane proteins, leading to their selective incorporation into the developing/invaginating endosome. AP-2 provides the selectivity while the self-assembly of the clathrin lattice acts as a structural scaffold to drive endocytosis (McMahon and Boucrot, 2011; Reider and Wendland, 2011; Traub, 2009). We envisage that the recruitment of AP-2 and clathrin to extracellular virus particles reflects the internalization mechanisms involved in recycling IEV proteins back to the Golgi when the virus is released from the cell (Husain and Moss, 2003, 2005; Ward and Moss, 2000). We found that the cytoplasmic domain of A36 can associate with AP-2, consistent with the presence of several potential endocytic sorting signals. The recruitment of clathrin to the virus is, however, likely to involve additional interactions, as F13 can also interact with AP-2 (Husain and Moss, 2003). The internalization of B5 from the plasma membrane also involves tyrosine 130 and an adjacent dileucine motif (Ward and Moss, 2000). Understanding the molecular basis of clathrin recruitment will not be easy, as it is likely to involve multiple weak interactions with a variety of sorting signals in several different IEV-associated proteins. These multiple interactions presumably help ensure that the IEV proteins can be recycled from the plasma membrane to the Golgi independently of their interactions with one another. It was originally thought that clathrin-based endocytosis was only capable of driving internalization of relatively small membrane-bound structures. However, in recent years clathrin has also been shown to play an important role in the

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Cell Host & Microbe Clathrin Clusters N-WASP to Enhance Vaccinia Spread

internalization of a number of different pathogens that are considerably larger than an endosome (Bonazzi et al., 2011; Chan et al., 2009; Cureton et al., 2009; Eto et al., 2008; Law et al., 2011; Mercer et al., 2010; Mishima and Sharma, 2011; Moreno-Ruiz et al., 2009; Veiga and Cossart, 2005; Veiga et al., 2007). We now find that clathrin can also promote actin polymerization and contribute to pathogen spread. The faster rate of vaccinia-induced actin tail initiation, together with their increased stability, accounts for the increased numbers of actin tails per cell in the presence of AP-2 and clathrin. Loss of AP-2 or clathrin recruitment results in significantly longer and faster actin tails. The slower rate of actin tail disassembly in the absence of AP-2 will contribute to their increased length. Changes in actin filament organization such as increased branching and/or crosslinking would also lead to a slower disassembly of the tail. However, it is more likely that the filaments within the tail are longer and require more time to disassemble given the increased speed of the virus. The faster rate of virus movement also suggests that the net actin polymerization beneath the extracellular virus must be increased. This could be achieved by increasing the number of filament nucleation events and/or the extent of filament elongation prior to capping. The question is how AP-2 and clathrin recruitment brings about these changes in actin tail behavior. The Density of A36 beneath the Virus Affects the Output of the Signaling Network Vaccinia virus promotes Arp2/3-dependent actin polymerization by recruiting a signaling complex of Nck, Grb2, WIP, and N-WASP (Frischknecht et al., 1999; Moreau et al., 2000; Scaplehorn et al., 2002; Snapper et al., 2001; Weisswange et al., 2009). One of the outstanding questions is how the organization and cooperativity within this signaling network affect the output, namely actin polymerization. Using structured illumination microscopy, we have found that AP-2 recruitment induces increased clustering of A36 and N-WASP at the site of actin tail formation. The increased clustering of N-WASP in turn enhances its stability, leading to more efficient actin tail initiation and robust actin polymerization. Conversely, reduced clustering of A36 and N-WASP clearly makes it harder for the virus to initiate an actin tail, and there is increased fragility within the system. We envisage that these differences arise because there is a threshold and optimal density of phosphorylated A36 required to initiate and sustain actin tails. In the wild-type situation, clathrin helps facilitate a level of A36 clustering that assembles an organized signaling network sufficient to induce sustainable actin polymerization. Consistent with this hypothesis, previous

observations have shown that the density of artificially clustered Nck or its SH3 domains impacts on its ability to induce actin polymerization (Blasutig et al., 2008; Campellone et al., 2004; Rivera et al., 2004, 2009). A similar synergistic activation of actin polymerization is also induced by clustering increasing numbers of EspFu repeats, which bind N-WASP to stimulate its ability to activate the Arp2/3 complex (Campellone et al., 2008; Cheng et al., 2008; Sallee et al., 2008). In the absence of clathrin recruitment, fewer viruses reach the threshold of A36 clustering required to efficiently nucleate actin polymerization. Moreover, even when the threshold is achieved, the organization of the signaling network is suboptimal, resulting in an altered output and changes in actin tail morphology and stability, as we have previously reported with Yaba-like disease virus (Dodding and Way, 2009). The loss of clathrin recruitment leads to significantly longer actin tails and a faster rate of virus movement. We suggest this is a direct consequence of reduced A36 clustering as artificially decreasing the density of wild-type phosphorylation competent A36 also increases the length and velocity of actin tails. Differences in the degree of A36 clustering must ultimately translate into changes in the density of N-WASP and its ability to stimulate Arp2/3-dependent actin polymerization beneath the virus. Consistent with this, in vitro motility assays using functionalized beads or giant unilamellar vesicles demonstrate that the density of N-WASP or its VCA domain determines their velocity and type of movement (continuous or saltatory) (Bernheim-Groswasser et al., 2002; Delatour et al., 2008; Wiesner et al., 2003). This dependence must in part be due to the stoichiometric relationship between WASP/N-WASP and the Arp2/3 complex, as WASP/N-WASP dimers are more effective at activating the Arp2/3 complex than monomers (Higgs and Pollard, 2000; Padrick et al., 2008, 2011; Ti et al., 2011). Increased clustering of N-WASP would therefore favor increased activation of the Arp2/3 complex, as it increases the likelihood that dimers will form (Padrick and Rosen, 2010). It will also impact on the ability of the WH2 domains of N-WASP to interact with the ends of growing actin filaments, which will help maintain a connection between the virus and the actin tail (Co et al., 2007; Delatour et al., 2008). However, this interaction will reduce the turnover of N-WASP beneath the virus and the rate of virus movement (Weisswange et al., 2009). It is also possible that overclustering of N-WASP could become limiting, if its turnover is required to ensure a continuous supply of activated Arp2/3 beneath the virus. We envisage that the transient recruitment of clathrin modulates the rate of virus movement, as there is an optimal balance between N-WASP-mediated Arp2/3 activation and

Figure 5. Loss of Clathrin Recruitment Alters N-WASP and Actin Tail Dynamics (A) Quantification of the time taken for virus to induce actin tails as well as the speed and duration of actin-based movement in cells treated with control or AP-2 a RNAi oligos. One hundred and fifty actin tails were tracked in three independent experiments. (B) Images from Movie S4 showing the actin-based movement of the YFP-A3 virus (yellow arrow) in a cell expressing GFPPACherry-b-actin (red when not photoactivated). The white arrow indicates the position of photoactivated GFPPACherry-b-actin (green when activated). Scale bar = 1 mm. (C) Comparison of the decay kinetics of photoactivated GFPPACherry-b-actin in actin tails in control and a-AP-2 siRNA-treated cells. n = 41 from three independent experiments. (D) Comparison of the recovery kinetics of GFP-N-WASP after photobleaching in control and a-AP-2 siRNA-treated cells. n = 52 from three independent experiments. See also Figure S3. (E) Quantification of the area covered by GFP-N-WASP on viral particles in N-WASP / MEFs treated with control and a-AP-2 siRNA. n = 150 in three independent experiments. Heat maps show the mean values of N-WASP intensity on virus particles in the two different conditions and the parameters used to fit a normal distribution to the data set (n = 300). In all quantifications error bars represent SEM; *** and ** represent p values of < 0.001 and < 0.01, respectively.

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Bound

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354 Cell Host & Microbe 12, 346–359, September 13, 2012 ª2012 Elsevier Inc.

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Cell Host & Microbe Clathrin Clusters N-WASP to Enhance Vaccinia Spread

N-WASP turnover that is dependent on its local density and organization. To obtain a complete molecular understanding of the system, it will be necessary to determine the stoichiometry of the components in the signaling network (Nck, WIP, NWASP, and Arp2/3) and how their higher organization impacts on activation of the Arp2/3 complex and ultimately actin filament growth. The Role of Clathrin in Vaccinia Actin Formation Is Distinct from That in EPEC It is now clear that a variety of different pathogens can recruit clathrin to promote their efficient internalization, often in an actin-dependent fashion (Bonazzi et al., 2011; Chan et al., 2009; Cureton et al., 2009; Eto et al., 2008; Law et al., 2011; Mercer et al., 2010; Mishima and Sharma, 2011; Moreno-Ruiz et al., 2009; Veiga and Cossart, 2005; Veiga et al., 2007). Clathrin is also recruited to enteropathogenic Escherichia coli (EPEC)induced actin pedestals (Veiga et al., 2007), which, like vaccinia actin tails, are dependent on Nck and N-WASP-mediated activation of the Arp2/3 complex (Campellone et al., 2004; Gruenheid et al., 2001; Kalman et al., 1999; Lommel et al., 2001, 2004). EPEC recruits a number of clathrin-associated proteins, including CD2AP, Dab2, Eps15, Epsin1, and Hip1R (Bonazzi et al., 2011; Guttman et al., 2010; Lin et al., 2011). Dab2, Eps15, and Epsin1 function as adaptors to recruit clathrin, which promotes the formation of an actin pedestal by recruiting Hip1R and CD2AP (Bonazzi et al., 2011; Guttman et al., 2010; Lin et al., 2011; Veiga et al., 2007). Interestingly, in contrast to vaccinia, AP-2 is neither recruited to EPEC nor involved in actin pedestal formation (Lin et al., 2011). The absence of AP-2 highlights an essential difference in how clathrin is used by the two pathogens. In the case of EPEC, clathrin appears to act as part of a signaling scaffold to recruit proteins required for actin nucleation. In contrast, vaccinia uses clathrin to cluster A36, to promote viral spread in a process that appears to mimic the early stages of clathrin-pit formation. Additional studies are required to determine if the exploitation of clathrin contributes to the spread of other pathogens that use actin-based motility. However, it is curious that Hip1R, which links clathrin to actin, was identified in a screen for regulators of Rickettsia actin-based motility (Serio et al., 2010). Depletion of Hip1R resulted in fewer but significantly longer actin tails, but the role of clathrin in actin-based motility of Rickettsia remains to be explored. In conclusion, we have demonstrated that AP-2 and clathrin are transiently recruited to extracellular viral particles and help prime the virus for actin nucleation by clustering A36 and its signaling network (N-WASP). Further studies aimed at analyzing

how the density of A36 directly correlates with signaling output offer an excellent opportunity to further our understanding of the initiation and regulation of phosphotyrosine-based signaling networks, as well as the parameters that define continuous actin propulsion in a freely exchanging system. EXPERIMENTAL PROCEDURES Infection, Drug Treatments, and Immunofluorescence Analysis HeLa cells were infected with the indicated viruses for 9 hr before being processed for immunofluorescence or treated with cytochalasin D as described previously (Arakawa et al., 2007a, 2007b; Weisswange et al., 2009). Antibodies against A27, A33, A36, B5, WIP, and N-WASP have been described (Hiller and Weber, 1985; Moreau et al., 2000; Rodriguez et al., 1985; Ro¨ttger et al., 1999). The following antibodies were also used for immunofluorescence: CHC (21679) and AP-2 (2730) (AbCam), Nck (Millipore), and Grb2 (C-23) (Santa Cruz Biotechnology). Actin tails were stained with Alexa 488 or Texas red phalloidin (Invitrogen). Immunofluorescence images were acquired and figures prepared as previously described (Dodding et al., 2011). The quantification of percentage colocalization with extracellular virus and the percentage of extracellular virus-inducing actin tails was performed by manual counting; all other analyses were performed using Metamorph (Molecular Devices). RNAi Transfections, Plaque Assays, Pull-Downs, and Immunoblot Analysis The following RNAi were used: All-Star control (QIAGEN) and a-AP-2 (Motley et al., 2003), m-AP-2 (J-008170-06-0005), and CHC (D-004001-01 and D-004001-03) (Dharmacon). HeLa cells were transfected with 20 nM of each oligo using the HiPerFect fast-forward protocol (QIAGEN). After 48 hr the cells were infected and either fixed 9 hr later or processed for immunoblot analysis. A549 cells were infected with virus 48 hr after transfection with a-AP-2 RNAi, during which time they grew to confluence. Two days later plaques were revealed using anti-B5 and HRP and quantified as described previously (Dodding et al., 2009; Earl et al., 2001). Pull-downs using the GST-tagged cytoplasmic domain of A36 (residues 24–221) were performed as previously described (Dodding et al., 2011), except cells were lysed in 50 mM Tris (pH 7.5), 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40, 0.5% Triton X-100 with protease inhibitors. Immunoblot analysis was performed using CHC (21679), a-Adaptin (MA1-064; Cambridge Biosciences), b-actin (AC-74) and GST (G7781) (Sigma-Aldrich), and m-Adaptin (AP50) and Grb2 (610112) (BD Biosciences). Vector Construction and Generation of Stable Cell Lines The GFP-LCa and Cherry-LCa expression constructs were generated by cloning the clathrin light-chain ORF into the NotI-EcoRI sites of pELGFP and pELCherry vectors, which drive expression of the insert under the control of a synthetic early-late hybrid promoter (EL) (Frischknecht et al., 1999). The pEL-GFPPACherry-b-actin expression vector was created by PCR using pAREK1 GPAC (variant1)-b-actin as a template (Welman et al., 2010). The PCR product was cloned into NotI-BamHI sites of the pELGFP vector in place of GFP. A LifeAct-Cherry construct was generated by linker cloning LifeAct (Riedl et al., 2008) into a pLVX-Cherry-puro vector (Dodding et al., 2011). Lentiviruses were generated with the pLVX-LifeAct-Cherry-puro vector and subsequently used to establish stable HeLa cell lines under 1 mg/ml puromycin

Figure 6. Clathrin Recruitment Promotes A36 and N-WASP Clustering (A) Immunoblot analysis with the indicated antibodies of glutathione Sepharose pull-downs reveals that AP-2 copurifies with GST-tagged cytoplasmic domain of A36 but not GST alone. F13, but not B5 and A33, also copurifies with A36 and AP-2. (B) Representative structured illumination images showing the distribution of A36 and N-WASP (green) on virus particles (A27, red) in control and a-AP-2 siRNA-treated cells. Yellow arrowheads highlight actin tails. Scale bar = 500 nm. (C) Graphs showing the distribution of A36 and N-WASP around virus particles in the two different conditions. Error bars represent SEM from 50 particles. (D) Representative structured illumination images showing the distribution of A36 on virus particles (A27) in N-WASP / MEFs treated with control or a-AP-2 siRNA. Magnified inserts highlight examples of polar and nonpolar A36 distribution; the percentage of viral particles at the periphery of cells that have A36 polarization is quantified. Scale bar = 1 mm; error bars represent SEM from three peripheral areas in ten different cells. In (C) and (D) a p value of < 0.001 is indicated by ***.

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A36

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Figure 7. Reducing the Density of Functional A36 Induces Longer, Faster Actin Tails (A) Images demonstrating the increased incorporation of Cherry fluorescent protein into virus particles in cells infected with the DA36R virus and expressing the indicated ratios of wild-type A36 (A36) and Cherry fused to the transmembrane domain of A36 (TM-Cherry). The transition from green to red (left to right) indicates there is also corresponding decrease in wild-type A36. (B) Immunofluorescence images showing that increasing the proportion of TM-Cherry to A36 increases actin tail length. Scale bar = 10 mm. (C) Quantification of actin tail length in DA36R-infected cells expressing different ratios of wild-type and one of three fluorescently tagged A36 constructs deficient in actin tail formation (A36-transmembrane, A36-transmembrane fused to the kinesin-1 binding region of calsyntenin, and the A36-YdF mutant). Error bars represent SEM from 150 tails in three independent experiments. A p value of < 0.05, < 0.01, and < 0.001 are represented by *, **, and ***, respectively. (D) Quantification of the speed of actin tails in DA36R-infected cells expressing either A36 or a ratio of 1:5 A36 to TM-Cherry. Error bars represent SEM of 75 tracked tails from three independent experiments. A p value of < 0.01 is represented by **.

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Cell Host & Microbe Clathrin Clusters N-WASP to Enhance Vaccinia Spread

selection as described previously (Weisswange et al., 2009). HeLa cells stably expressing RFP-KLC2 have been described (Dodding et al., 2011). Live Cell Imaging, FRAP, and Photoactivation At 4 hr postinfection, HeLa cells were transfected with pEL expression vectors using Effectene (QIAGEN). Cells were imaged from 5 hr later and, where required, the Alexa 488 B5 antibody, which was generated using the Molecular Probes Alexa Fluor 488 Monoclonal Antibody Labeling kit (Invitrogen), was added to the media before imaging. Images were collected using a PlanAchromat 633/1.40 lens (Carl Zeiss) and an Evolve 512 camera (Photometrics, Tucson, AZ) on an Axio Observer Microscope controlled by Slidebook (Intelligent Imaging Innovations, Denver, CO). FRAP experiments were performed using a vector high-speed point scanner on the same microscope. The GFP-N-WASP signal was bleached using five iterations at 100% 488 nm laser power, and its recovery was measured using Slidebook software and analyzed as previously described (Weisswange et al., 2009). The time taken for a RFP-A3 virus to induce an actin tail was determined from the point the virus stopped moving in the cell periphery until it initiated an actin tail in cells stably expressing LifeAct-Cherry. Photoactivation experiments were performed on HeLa cells expressing GFPPACherry-b-actin and infected with the YFP-A3 virus (Arakawa et al., 2007a). GFPPACherry-b-actin was activated using five iterations at 100% 405 nm laser power. The fluorescence intensity was determined and normalized against background fluorescence intensity using Metamorph. Analysis and plotting were carried out using Prism software (GraphPad). Analysis and preparation of stacks were performed using Metamorph (Molecular Devices). Analysis and Preparation of N-WASP Intensity Profiles N-WASP / mouse fibroblasts expressing GFP-N-WASP (Weisswange et al., 2009) treated with control or AP-2 RNAi for 2 days were infected and processed for immunofluorescence as above. Confocal stacks were acquired on a LSM 780 microscope using a 633/1.46 Plan-Achromat lens controlled by the Zen software (Zeiss, Germany). Virus particles were detected in the 3D stacks by fitting a Gaussian curve around local maxima in the A27 signal. The maximum intensity of the N-WASP signal in the neighborhood of each particle was then located, and a 32 3 32 pixel area about this point was cropped. The cropped areas were then averaged for each condition to produce characteristic N-WASP distributions. Quantitative parameters for these distributions were estimated by assuming approximately Gaussian distributions and fitting curves accordingly. Fifteen fields of view containing 300 virus particles were analyzed for each RNAi treatment. Super-Resolution Microscopy and Electron Microscopy Control and RNAi-treated HeLa and N-WASP null MEFs were plated on fibronectin-coated 0.17 mm certified coverslips and infected with WR. Cells were processed for immunofluorescence, and the coverslips were mounted using Vectashield (Vector Laboratories). Images were acquired using an OMX V3 microscope (Applied Precision, Inc.) with UPLANSAPO C-achromatic 1003/ 1.40 oil lens (Olympus), under softWoRx software (Applied Precision, Inc.). Images were reconstructed and a projection comprising five stacks centered over the virus particle was created using softWoRx. To quantify the degree of dispersion around the virus particle, the spread of N-WASP/A36 signal around the virus was compared to the circumference of A27 signal, which was set to 100%. Preparation of samples for electron microscopy was as previously described (Dodding et al., 2009). Artificially Altering Functional A36 Density One hour after infection with the DA36R virus, HeLa cells were transfected with varying ratios of the pEL expression vectors: A36, Cherry-A36TM, CherryA36TM-CSTN 879-917, and A36-YdF-YFP (Dodding et al., 2011; Frischknecht et al., 1999). Eight hours later cells were processed for immunofluorescence or used in live imaging, and actin tail lengths or rate of movement were quantified.

ACKNOWLEDGMENTS We wish to thank Steve Royle (Liverpool University) for providing Cherry-LCa and GFP-LCa, Arkalusz Welman and Margaret Frame (University of Edinburgh) for providing photoactivatable GFP Cherry actin, and Anne Vaahtokari (LRI, Cancer Research UK) for help with super resolution imaging. We would like to thank Tony Pawson (Samuel Lunenfeld Research Institute) and Scott Snapper (Department of Medicine and Immunology, Massachusetts General Hospital) for Nck and N-WASP null cell lines. We also wish to thank all members of the Way lab as well as Christien Merrifield (LEBS) for constructive comments on the manuscript. Received: April 19, 2012 Revised: May 30, 2012 Accepted: August 13, 2012 Published: September 12, 2012 REFERENCES Arakawa, Y., Cordeiro, J.V., Schleich, S., Newsome, T.P., and Way, M. (2007a). The release of vaccinia virus from infected cells requires RhoAmDia modulation of cortical actin. Cell Host Microbe 1, 227–240. Arakawa, Y., Cordeiro, J.V., and Way, M. (2007b). F11L-mediated inhibition of RhoA-mDia signaling stimulates microtubule dynamics during vaccinia virus infection. Cell Host Microbe 1, 213–226. Bernheim-Groswasser, A., Wiesner, S., Golsteyn, R.M., Carlier, M.-F., and Sykes, C. (2002). The dynamics of actin-based motility depend on surface parameters. Nature 417, 308–311. Bladt, F., Aippersbach, E., Gelkop, S., Strasser, G.A., Nash, P., Tafuri, A., Gertler, F.B., and Pawson, T. (2003). The murine Nck SH2/SH3 adaptors are important for the development of mesoderm-derived embryonic structures and for regulating the cellular actin network. Mol. Cell. Biol. 23, 4586–4597. Blasutig, I.M., New, L.A., Thanabalasuriar, A., Dayarathna, T.K., Goudreault, M., Quaggin, S.E., Li, S.S., Gruenheid, S., Jones, N., and Pawson, T. (2008). Phosphorylated YDXV motifs and Nck SH2/SH3 adaptors act cooperatively to induce actin reorganization. Mol. Cell. Biol. 28, 2035–2046. Bonazzi, M., Vasudevan, L., Mallet, A., Sachse, M., Sartori, A., Prevost, M.C., Roberts, A., Taner, S.B., Wilbur, J.D., Brodsky, F.M., and Cossart, P. (2011). Clathrin phosphorylation is required for actin recruitment at sites of bacterial adhesion and internalization. J. Cell Biol. 195, 525–536. Campellone, K.G., Rankin, S., Pawson, T., Kirschner, M.W., Tipper, D.J., and Leong, J.M. (2004). Clustering of Nck by a 12-residue Tir phosphopeptide is sufficient to trigger localized actin assembly. J. Cell Biol. 164, 407–416. Campellone, K.G., Cheng, H.C., Robbins, D., Siripala, A.D., McGhie, E.J., Hayward, R.D., Welch, M.D., Rosen, M.K., Koronakis, V., and Leong, J.M. (2008). Repetitive N-WASP-binding elements of the enterohemorrhagic Escherichia coli effector EspF(U) synergistically activate actin assembly. PLoS Pathog. 4, e1000191. Chan, Y.G., Cardwell, M.M., Hermanas, T.M., Uchiyama, T., and Martinez, J.J. (2009). Rickettsial outer-membrane protein B (rOmpB) mediates bacterial invasion through Ku70 in an actin, c-Cbl, clathrin and caveolin 2-dependent manner. Cell. Microbiol. 11, 629–644. Cheng, H.C., Skehan, B.M., Campellone, K.G., Leong, J.M., and Rosen, M.K. (2008). Structural mechanism of WASP activation by the enterohaemorrhagic E. coli effector EspF(U). Nature 454, 1009–1013. Co, C., Wong, D.T., Gierke, S., Chang, V., and Taunton, J. (2007). Mechanism of actin network attachment to moving membranes: barbed end capture by NWASP WH2 domains. Cell 128, 901–913. Cudmore, S., Cossart, P., Griffiths, G., and Way, M. (1995). Actin-based motility of vaccinia virus. Nature 378, 636–638.

SUPPLEMENTAL INFORMATION

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Supplemental Information includes three figures and can be found with this article online at http://dx.doi.org/10.1016/j.chom.2012.08.002.

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