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• W2025328992 abstract "Prior to being released from the infected cell, intracellular enveloped vaccinia virus particles are transported from their perinuclear assembly site to the plasma membrane along microtubules by the motor kinesin-1. After fusion with the plasma membrane, stimulation of actin tails beneath extracellular virus particles acts to enhance cell-to-cell virus spread. However, we lack molecular understanding of events that occur at the cell periphery just before and during the liberation of virus particles. Using live cell imaging, we show that virus particles move in the cell cortex, independently of actin tail formation. These cortical movements and the subsequent release of virus particles, which are both actin dependent, require F11L-mediated inhibition of RhoA-mDia signaling. We suggest that the exit of vaccinia virus from infected cells has strong parallels to exocytosis, as it is dependent on the assembly and organization of actin in the cell cortex. Prior to being released from the infected cell, intracellular enveloped vaccinia virus particles are transported from their perinuclear assembly site to the plasma membrane along microtubules by the motor kinesin-1. After fusion with the plasma membrane, stimulation of actin tails beneath extracellular virus particles acts to enhance cell-to-cell virus spread. However, we lack molecular understanding of events that occur at the cell periphery just before and during the liberation of virus particles. Using live cell imaging, we show that virus particles move in the cell cortex, independently of actin tail formation. These cortical movements and the subsequent release of virus particles, which are both actin dependent, require F11L-mediated inhibition of RhoA-mDia signaling. We suggest that the exit of vaccinia virus from infected cells has strong parallels to exocytosis, as it is dependent on the assembly and organization of actin in the cell cortex. Vaccinia virus, a close relative of Variola virus, the causative agent of smallpox, is a large double-stranded DNA virus that undergoes a complex replication cycle in the cytoplasm of the infected cell (Harrison et al., 2004Harrison S.C. Alberts B. Ehrenfeld E. Enquist L. Fineberg H. McKnight S.L. Moss B. O'Donnell M. Ploegh H. Schmid S.L. et al.Discovery of antivirals against smallpox.Proc. Natl. Acad. Sci. USA. 2004; 101: 11178-11192Crossref PubMed Scopus (70) Google Scholar, Smith et al., 2003Smith G.L. Murphy B.J. Law M. Vaccinia virus motility.Annu. Rev. Microbiol. 2003; 57: 323-342Crossref Scopus (80) Google Scholar). Replication, which occurs in viral factories anchored at or near the microtubule-organizing center, results in the formation of two types of cytoplasmic virus particles, the intracellular mature virus (IMV) and the intracellular enveloped virus (IEV) (Harrison et al., 2004Harrison S.C. Alberts B. Ehrenfeld E. Enquist L. Fineberg H. McKnight S.L. Moss B. O'Donnell M. Ploegh H. Schmid S.L. et al.Discovery of antivirals against smallpox.Proc. Natl. Acad. Sci. USA. 2004; 101: 11178-11192Crossref PubMed Scopus (70) Google Scholar, Smith et al., 2003Smith G.L. Murphy B.J. Law M. Vaccinia virus motility.Annu. Rev. Microbiol. 2003; 57: 323-342Crossref Scopus (80) Google Scholar). IMV move throughout the cytoplasm in a bidirectional manner at speeds up to 3 μm·s−1, consistent with a microtubule-based transport mechanism (Ward, 2005Ward B.M. Visualization and characterization of the intracellular movement of vaccinia virus intracellular mature virions.J. Virol. 2005; 79: 4755-4763Crossref Scopus (70) Google Scholar). IEV are formed when IMV are wrapped by membrane cisternae derived from the trans-Golgi network or endosomal compartments containing a subset of integral viral membrane proteins (Harrison et al., 2004Harrison S.C. Alberts B. Ehrenfeld E. Enquist L. Fineberg H. McKnight S.L. Moss B. O'Donnell M. Ploegh H. Schmid S.L. et al.Discovery of antivirals against smallpox.Proc. Natl. Acad. Sci. USA. 2004; 101: 11178-11192Crossref PubMed Scopus (70) Google Scholar, Smith et al., 2003Smith G.L. Murphy B.J. Law M. Vaccinia virus motility.Annu. Rev. Microbiol. 2003; 57: 323-342Crossref Scopus (80) Google Scholar). IEV recruit the plus end-directed microtubule motor kinesin-1 (conventional kinesin) and are subsequently transported to the plasma membrane on microtubules in a saltatory fashion at speeds of up to 3 μm·s−1 (Geada et al., 2001Geada M.M. Galindo I. Lorenzo M.M. Perdiguero B. Blasco R. Movements of vaccinia virus intracellular enveloped virions with GFP tagged to the F13L envelope protein.J. Gen. Virol. 2001; 82: 2747-2760PubMed Google Scholar, Hollinshead et al., 2001Hollinshead M. Rodger G. Van Eijl H. Law M. Hollinshead R. Vaux D.J. Smith G.L. Vaccinia virus utilizes microtubules for movement to the cell surface.J. Cell Biol. 2001; 154: 389-402Crossref PubMed Scopus (178) Google Scholar, Rietdorf et al., 2001Rietdorf J. Ploubidou A. Reckmann I. Holmström A. Frischknecht F. Zettl M. Zimmermann T. Way M. Kinesin dependent movement on microtubules precedes actin based motility of vaccinia virus.Nat. Cell Biol. 2001; 3: 992-1000Crossref PubMed Scopus (231) Google Scholar, Ward and Moss, 2001aWard B.M. Moss B. Vaccinia virus intracellular movement is associated with microtubules and independent of actin tails.J. Virol. 2001; 75: 11651-11663Crossref PubMed Scopus (142) Google Scholar, Ward and Moss, 2001bWard B.M. Moss B. Visualization of intracellular movement of vaccinia virus virions containing a green fluorescent protein-B5R membrane protein chimera.J. Virol. 2001; 75: 4802-4813Crossref PubMed Scopus (132) Google Scholar). Fusion of the IEV with the plasma membrane results in the formation of the extracellular cell-associated enveloped virus (CEV) (Harrison et al., 2004Harrison S.C. Alberts B. Ehrenfeld E. Enquist L. Fineberg H. McKnight S.L. Moss B. O'Donnell M. Ploegh H. Schmid S.L. et al.Discovery of antivirals against smallpox.Proc. Natl. Acad. Sci. USA. 2004; 101: 11178-11192Crossref PubMed Scopus (70) Google Scholar, Smith et al., 2003Smith G.L. Murphy B.J. Law M. Vaccinia virus motility.Annu. Rev. Microbiol. 2003; 57: 323-342Crossref Scopus (80) Google Scholar). CEV that remain attached to the cell are able to induce an outside-in signaling cascade to locally activate Src and Abl family kinases (Frischknecht et al., 1999Frischknecht F. Moreau V. Röttger S. Gonfloni S. Reckmann I. Superti-Furga G. Way M. Actin based motility of vaccinia virus mimics receptor tyrosine kinase signalling.Nature. 1999; 401: 926-929Crossref PubMed Scopus (339) Google Scholar, Newsome et al., 2004Newsome T.P. Scaplehorn N. Way M. SRC mediates a switch from microtubule- to actin-based motility of vaccinia virus.Science. 2004; 306: 124-129Crossref PubMed Scopus (133) Google Scholar, Newsome et al., 2006Newsome T.P. Weisswange I. Frischknecht F. Way M. Abl collaborates with Src family kinases to stimulate actin-based motility of vaccinia virus.Cell. Microbiol. 2006; 8: 233-241Crossref PubMed Scopus (75) Google Scholar, Reeves et al., 2005Reeves P.M. Bommarius B. Lebeis S. McNulty S. Christensen J. Swimm A. Chahroudi A. Chavan R. Feinberg M.B. Veach D. et al.Disabling poxvirus pathogenesis by inhibition of Abl-family tyrosine kinases.Nat. Med. 2005; 11: 731-739Crossref PubMed Scopus (184) Google Scholar). This activation results in the tyrosine phosphorylation of A36R (Frischknecht et al., 1999Frischknecht F. Moreau V. Röttger S. Gonfloni S. Reckmann I. Superti-Furga G. Way M. Actin based motility of vaccinia virus mimics receptor tyrosine kinase signalling.Nature. 1999; 401: 926-929Crossref PubMed Scopus (339) Google Scholar, Newsome et al., 2004Newsome T.P. Scaplehorn N. Way M. SRC mediates a switch from microtubule- to actin-based motility of vaccinia virus.Science. 2004; 306: 124-129Crossref PubMed Scopus (133) Google Scholar, Newsome et al., 2006Newsome T.P. Weisswange I. Frischknecht F. Way M. Abl collaborates with Src family kinases to stimulate actin-based motility of vaccinia virus.Cell. Microbiol. 2006; 8: 233-241Crossref PubMed Scopus (75) Google Scholar), an integral vaccinia membrane protein that becomes localized in the plasma membrane beneath CEV (Röttger et al., 1999Röttger S. Frischknecht F. Reckmann I. Smith G.L. Way M. Interactions between vaccinia virus IEV membrane proteins and their roles in IEV assembly and actin tail formation.J. Virol. 1999; 73: 2863-2875PubMed Google Scholar, Smith et al., 2002Smith G.L. Vanderplasschen A. Law M. The formation and function of extracellular enveloped vaccinia virus.J. Gen. Virol. 2002; 83: 2915-2931PubMed Google Scholar, van Eijl et al., 2000van Eijl H. Hollinshead M. Smith G.L. The vaccinia virus A36R protein is a type Ib membrane protein present on intracellular but not extracellular enveloped virus particles.Virology. 2000; 271: 26-36Crossref PubMed Scopus (90) Google Scholar). Phosphorylation of A36R facilitates the release of kinesin-1 (Newsome et al., 2004Newsome T.P. Scaplehorn N. Way M. SRC mediates a switch from microtubule- to actin-based motility of vaccinia virus.Science. 2004; 306: 124-129Crossref PubMed Scopus (133) Google Scholar), and the subsequent recruitment of a signaling complex consisting of Grb2, Nck, WIP (WASP-interacting protein), and N-WASP (Frischknecht et al., 1999Frischknecht F. Moreau V. Röttger S. Gonfloni S. Reckmann I. Superti-Furga G. Way M. Actin based motility of vaccinia virus mimics receptor tyrosine kinase signalling.Nature. 1999; 401: 926-929Crossref PubMed Scopus (339) Google Scholar, Moreau et al., 2000Moreau V. Frischknecht F. Reckmann I. Vincentelli R. Rabut G. Stewart D. Way M. A complex of N-WASP and WIP integrates signalling cascades that lead to actin polymerization.Nat. Cell Biol. 2000; 2: 441-448Crossref PubMed Scopus (266) Google Scholar, Scaplehorn et al., 2002Scaplehorn N. Holmstrom A. Moreau V. Frischknecht F. Reckmann I. Way M. Grb2 and nck act cooperatively to promote actin-based motility of vaccinia virus.Curr. Biol. 2002; 12: 740-745Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, Snapper et al., 2001Snapper S.B. Takeshima F. Anton I. Liu C.H. Thomas S.M. Nguyen D. Dudley D. Fraser H. Purich D. Lopez-Ilasaca M. et al.N-WASP deficiency reveals distinct pathways for cell surface projections and microbial actin-based motility.Nat. Cell Biol. 2001; 3: 897-904Crossref PubMed Scopus (257) Google Scholar, Zettl and Way, 2002Zettl M. Way M. The WH1 and EVH1 domains of WASP and Ena/VASP family members bind distinct sequence motifs.Curr. Biol. 2002; 12: 1617-1622Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The recruitment of N-WASP stimulates the actin-nucleating activity of the Arp2/3 complex, resulting in actin tail formation beneath the CEV (Frischknecht and Way, 2001Frischknecht F. Way M. Surfing pathogens and the lessons learned for actin polymerization.Trends Cell Biol. 2001; 11: 30-38Abstract Full Text Full Text PDF Scopus (179) Google Scholar). The stimulation of actin tails then acts to enhance cell-to-cell spread of the virus (Cudmore et al., 1995Cudmore S. Cossart P. Griffiths G. Way M. Actin-based motility of vaccinia virus.Nature. 1995; 378: 636-638Crossref PubMed Scopus (368) Google Scholar, Cudmore et al., 1996Cudmore S. Reckmann I. Griffiths G. Way M. Vaccinia virus: A model system for actin-membrane interactions.J. Cell Sci. 1996; 109: 1739-1747PubMed Google Scholar, Hollinshead et al., 2001Hollinshead M. Rodger G. Van Eijl H. Law M. Hollinshead R. Vaux D.J. Smith G.L. Vaccinia virus utilizes microtubules for movement to the cell surface.J. Cell Biol. 2001; 154: 389-402Crossref PubMed Scopus (178) Google Scholar, Ward and Moss, 2001aWard B.M. Moss B. Vaccinia virus intracellular movement is associated with microtubules and independent of actin tails.J. Virol. 2001; 75: 11651-11663Crossref PubMed Scopus (142) Google Scholar). It has been calculated that an IEV particle would take 5.7 hr to move 10 μm in the cytoplasm of an infected cell, in the absence of an active transport mechanism (Sodeik, 2000Sodeik B. Mechanisms of viral transport in the cytoplasm.Trends Microbiol. 2000; 8: 465-472Abstract Full Text Full Text PDF Scopus (244) Google Scholar). Clearly, cell-to-cell spread of vaccinia would be extremely inefficient if the virus was only dependent on diffusion. Fortuitously for the virus, it has evolved the ability to use both microtubule- and actin-based motility during its exit from the infected cell. Although actin-based motility provides an extra advantage to the virus, it is microtubule-based transport that plays the most important role during the spread of infection. In the absence of microtubule transport, IEV would only be released when the cell undergoes lysis or when a virus particle encounters the plasma membrane due to a diffusion-driven process. Microtubule-based transport thus ensures that IEV particles are efficiently delivered to the plasma membrane, where they are ultimately released from the infected cell. We have shown that vaccinia not only uses the microtubule cytoskeleton for its transport but also stimulates increased microtubule dynamics during infection (Arakawa et al., 2007Arakawa Y. Cordeiro J.V. Way M. F11L-mediated inhibition of RhoA-mDia signaling stimulates microtubule dynamics during vaccinia virus infection.Cell Host & Microbe. 2007; 1 (this issue): 213-226Abstract Full Text Full Text PDF Scopus (59) Google Scholar [this issue of Cell Host & Microbe]). Vaccinia achieves these changes through the action of F11L, which binds to RhoA and blocks its ability to signal to mDia (Arakawa et al., 2007Arakawa Y. Cordeiro J.V. Way M. F11L-mediated inhibition of RhoA-mDia signaling stimulates microtubule dynamics during vaccinia virus infection.Cell Host & Microbe. 2007; 1 (this issue): 213-226Abstract Full Text Full Text PDF Scopus (59) Google Scholar, Valderrama et al., 2006Valderrama F. Cordeiro J.V. Schleich S. Frischknecht F. Way M. Vaccinia virus-induced cell motility requires F11L-mediated inhibition of RhoA signaling.Science. 2006; 311: 377-381Crossref PubMed Scopus (92) Google Scholar). Infection also increases the targeting of microtubules to the cell periphery, suggestive of possible changes in the cell cortex. In this paper we sought to examine if changes in peripheral microtubule dynamics and their cortical targeting contribute to the exit of vaccinia from infected cells. Our observations have uncovered a role for RhoA-mDia signaling in the regulation of cortical actin and an actin-dependent movement of virus particles, which is likely to be a myosin-driven event, in the cell cortex prior to their release. To test whether changes in microtubule dynamics might contribute to the spread of infection, we examined the effect of nocodazole and paclitaxel on vaccinia actin tail formation. We found that depolymerization of microtubules by 2 hr treatment with nocodazole, at 8 hr postinfection with Western Reserve (WR), results in a dramatic reduction in the number of actin tails and extracellular virus particles (CEV), indicative of a lack of IEV transport to the plasma membrane (Figures 1A–1C), as previously reported (Geada et al., 2001Geada M.M. Galindo I. Lorenzo M.M. Perdiguero B. Blasco R. Movements of vaccinia virus intracellular enveloped virions with GFP tagged to the F13L envelope protein.J. Gen. Virol. 2001; 82: 2747-2760PubMed Google Scholar, Hollinshead et al., 2001Hollinshead M. Rodger G. Van Eijl H. Law M. Hollinshead R. Vaux D.J. Smith G.L. Vaccinia virus utilizes microtubules for movement to the cell surface.J. Cell Biol. 2001; 154: 389-402Crossref PubMed Scopus (178) Google Scholar, Ward and Moss, 2001aWard B.M. Moss B. Vaccinia virus intracellular movement is associated with microtubules and independent of actin tails.J. Virol. 2001; 75: 11651-11663Crossref PubMed Scopus (142) Google Scholar, Ward and Moss, 2001bWard B.M. Moss B. Visualization of intracellular movement of vaccinia virus virions containing a green fluorescent protein-B5R membrane protein chimera.J. Virol. 2001; 75: 4802-4813Crossref PubMed Scopus (132) Google Scholar). Consistent with these observations, reinfection plaque assays demonstrated that there was a corresponding decrease (>95%) in the number of infectious virus particles released into the media of nocodazole-treated cells (Figure 1D). Stabilization of microtubules with paclitaxel also resulted in a significant reduction in the number of WR-infected cells with actin tails and loss of extracellular CEV (Figures 1A–1C). There was also a corresponding ∼60% reduction in the number of infectious virus particles released from paclitaxel-treated cells (Figure 1D). The effects of nocodazole and paclitaxel on virus release are not dependent on actin tail formation during WR infection. Treatment with either drug also reduced the number of extracellular CEV and infectious virus particles released into the media from cells infected with the recombinant virus strain A36R-YdF, which is unable to induce actin tails (Figures 1C and 1D). The effects of nocodazole on CEV formation and virus release are readily explained by a lack of microtubule-dependent transport during both the formation of IEV and their subsequent transport to the cell periphery. A similar defect in microtubule-dependent transport of IEV to the plasma membrane could also account for the effects of paclitaxel treatment if kinesin-1 is unable to move on stabilized microtubules. However, this does not appear to be the case, as video analysis reveals that YFP-tagged IEV are still transported along microtubules in the presence of paclitaxel (Figure 2A; Movie S1 in the Supplemental Data available with this article online). It was noticeable, however, that the characteristic accumulation of virus particles at the edge of infected cells was absent in paclitaxel-treated cells (Figure 2A; Movie S1). Focusing on the bottom of paclitaxel-treated cells reveals that IEV particles are dispersed throughout the cell and also had great difficulty in approaching to within ∼4 μm of the plasma membrane at the edge of the cell (Figure 2A). The region immediately beneath the plasma membrane, which is known as the cell cortex, consists of a dense meshwork of actin filaments. One possible explanation for the effects of paclitaxel on intracellular virus distribution is that stabilization of microtubule cytoskeleton results in the reorganization of the actin in the cell cortex, which then acts as a barrier to prevent the virus from reaching the plasma membrane. To explore this hypothesis, we examined whether modulation of actin dynamics by the addition of Latrunculin B would rescue the effects of paclitaxel on virus release. We found that inhibition of actin polymerization with low concentrations of Latrunculin B (0.1 μM) could partially rescue the release of infectious virus particles from paclitaxel-treated cells (Figure 2B). Our observations are consistent with the hypothesis that the organization of cortical actin regulates the ability of the virus to reach the plasma membrane during its release. It is well established that the actin cytoskeleton plays an important role at multiple stages during exocytosis. We therefore wondered whether the release of virus particles at the plasma membrane is also dependent on actin, given they are a similar dimension to secretory granules. We found that a 2 hr treatment with a low (0.1 μM) concentration of Latrunculin B at 8 hr postinfection did not affect the number of WR-infected cells with actin tails or inhibit release of infectious virus particles into the media (Figure 2C). Indeed, video analysis and quantitative analysis of peripheral virus distribution reveals that this low concentration of Latrunculin B actually stimulates movement of virus particles in the cell periphery toward the plasma membrane (Figures 2D and 2E; Movie S2). In contrast, a similar treatment with high (1 μM) concentration of Latrunculin B was found to reduce the number of actin tails and the release of infectious virus particles (Figure 2C). Treatment of WR-infected cells with Cytochalasin D, which, in contrast to Latrunculin B, can cap the fast-growing end of actin filaments, in addition to sequestering actin monomers, was found to have more potent effects on the inhibition of actin tails and virus release (Figure 2C). Our observations indicate that virus release is dependent on the organization and dynamics of the actin cytoskeleton in the cell cortex. To obtain more insights into the nature of virus particle movements occurring in the cell periphery, we performed video analysis at a higher sampling frequency on cells infected with the A36R-YdF virus, which cannot make actin tails. In the absence of drug treatments, we found that there were two distinct types of virus movement in addition to static virus particles (Figure 3A; Movies S3–S5). The first type of movement, which tended to occur over longer distances and in linear fashion, had an average speed of 1.06 ± 0.24 μm/s (n = 87), consistent with microtubule-based transport. The second form of motility, which was more random and tended to occur closer to the edge of the cell, had an average speed of 0.35 ± 0.1 μm/s (n = 301). When cells were treated with paclitaxel, there was a noticeable reduction in the second form of motility (Figure 3A; Movie S3). This paclitaxel-mediated inhibition of movement could be reversed by addition of low concentrations of Latrunculin B (Figure 3A; Movie S3). Consistent with these live observations, we found that low concentrations of Latrunculin B could partially rescue the effects of paclitaxel on the distribution of virus particles in the cell periphery (Figure 3B). The speed and behavior of this second form of virus motility is highly suggestive of an actin-based transport. To confirm this hypothesis, we examined the effect of modulating actin dynamics on virus particle movements in the cell periphery. We found that depolymerization or stabilization of actin filaments with Cytochalashin D or Jaspaklinolide, respectively, results in an increase in the number of static particles and reduction in the number of the slow random virus movements (Figure 3C; Movies S4 and S5). The observation that addition of paclitaxel inhibits actin-dependent virus movements in the cell periphery suggests that microtubule dynamics is linked to regulation of cortical actin. This is not unexpected given the intimate and reciprocal relationship between microtubule and actin cytoskeletons. Live imaging and quantitative analysis on infected cells expressing YFP-actin reveal that addition of paclitaxel stimulates actin polymerization in cell cortex (Figures 3D and 3E). The ability of paclitaxel to stimulate actin polymerization in the cortex of infected cells is highly suggestive of RhoA activation. Quantitative western blot analysis of Rhotekin-pull-down assays on extracts from WR-infected cells treated with paclitaxel confirmed that stabilization of microtubules increases the level of GTP-bound Rho at 8 hr postinfection (Figure 3F). Our observations suggest that the organization of cortical actin regulates an actin-dependent transport step in the cell cortex, which is required for virus particles to reach the plasma membrane. Given the ability of paclitaxel to activate RhoA and stimulate peripheral actin polymerization, we investigated whether RhoA signaling also regulates virus release. We found that expression of constitutively active RhoA-V14, but not dominant-negative RhoA-N19, at 8 hr postinfection dramatically reduces the number of CEV and their associated actin tails in WR-infected cells (Figures 4A and 4B). Reinfection assays confirmed that expression of RhoA-V14 also reduces the release of infectious virus particles (Figure 4C). Consistent with our earlier observations, we found that the effect of RhoA-V14 expression on virus release could be reversed by treating infected cells with low concentrations of Latrunculin B (Figure 4C). Likewise, expressing dominant-negative RhoA-N19 neutralized the effects of paclitaxel treatment on virus release (Figure 4C). Our observations suggest that RhoA signaling plays an important role in regulating the organization of the actin cytoskeleton during virus release. Our observations suggest that RhoA signaling acts to inhibit virus release by stimulating actin polymerization in the cell cortex. However, during the course of a normal infection, the viral protein F11L binds to RhoA to inhibit its downstream signaling (Arakawa et al., 2007Arakawa Y. Cordeiro J.V. Way M. F11L-mediated inhibition of RhoA-mDia signaling stimulates microtubule dynamics during vaccinia virus infection.Cell Host & Microbe. 2007; 1 (this issue): 213-226Abstract Full Text Full Text PDF Scopus (59) Google Scholar, Valderrama et al., 2006Valderrama F. Cordeiro J.V. Schleich S. Frischknecht F. Way M. Vaccinia virus-induced cell motility requires F11L-mediated inhibition of RhoA signaling.Science. 2006; 311: 377-381Crossref PubMed Scopus (92) Google Scholar). Our previous observations have demonstrated that F11L is required for efficient virus particle assembly. Consequently, to examine if F11L-mediated inhibition of RhoA signaling is required for virus release, we examined the consequences of overexpressing an F11L mutant (F11L-VK) that is unable to bind RhoA. We found that overexpression of F11L-VK, at 8 hr postinfection, results in a dramatic reduction in the number of extracellular virus particles (CEV) and actin tails in WR-infected cells (Figures 5A–5C). There was also a corresponding reduction in the number of infectious virus particles released into the media (Figure 5D). The effects of F11L-VK on virus release could be rescued by inhibiting RhoA signaling with TAT-C3. F11L-VK mutant appears to be acting as a dominant negative to prevent the F11L-mediated inhibition of RhoA signaling during vaccinia infection. Our previous observations have shown that F11L binds RhoA in a similar fashion to ROCK (Valderrama et al., 2006Valderrama F. Cordeiro J.V. Schleich S. Frischknecht F. Way M. Vaccinia virus-induced cell motility requires F11L-mediated inhibition of RhoA signaling.Science. 2006; 311: 377-381Crossref PubMed Scopus (92) Google Scholar), suggesting that F11L may also function as a dimer. We therefore performed pull-down assays to examine if F11L can interact with itself. We found that GST-tagged F11L and F11L-VK were equally effective pulling down endogenous F11L from WR-infected cells (Figure 5E). We believe that the formation of F11L and F11L-VK complexes that are unable to inhibit RhoA signaling is likely to account for the dominant-negative effect of F11L-VK overexpression on virus release. Previous studies have established a role for Myosin II during exocytosis. As Myosin II is downstream of RhoA-ROCK signaling, we wondered whether ROCK participates in regulating cortical actin during virus release. We found that the formation of actin tails and release of infectious virus particles from paclitaxel-treated cells is rescued by inhibiting RhoA signaling with TAT-C3 exoenzyme (Figures 6A–6C). In contrast, inhibition of ROCK with Y-27632 did not rescue the effects of paclitaxel on WR-infected cells (Figures 6A–6C). Given the absence of an effect of Y-27632 on paclitaxel-treated cells, we examined whether expression of active mDia1 (mDia1 ΔN3) might inhibit release of infectious virus particles. We found that expression of the active form of mDia1 but not ROCK1 (ROCK1 Δ3) reduced the number of extracellular virus particles and actin tails in WR-infected cells (Figures 7A and 7B). Active mDia1 also reduced the number of infectious particles released into the media (Figure 7C). The effect of active mDia1 on virus release could be rescued by treating infected cells with low concentrations of Latrunculin B (Figure 7C). To obtain further insights into the role of RhoA-mDia signaling during virus release, we performed video analysis on infected cells expressing active RhoA or mDia1 at 8 hr postinfection (Figure 7D; Movie S6). In contrast to controls, we found that virus particles did not accumulate at the edge of cells expressing active RhoA or mDia1. It was also apparent that virus particles in the cell periphery were also largely static and did not undergo slow actin-dependent movements (Figure 7D). Taken together, our observations suggest that F11L-mediated inhibition of RhoA-mDia signaling leads to changes in organization of actin in the cell cortex, which acts to facilitate virus release (Figure 7E).Figure 7mDia-Mediated Actin Polymerization Inhibits Virus Particle ReleaseShow full caption(A) Immunofluorescence images of the actin cytoskeleton, CEV (B5R), or IEV and CEV (F13L) particles in WR-infected cells expressing GFP-tagged active forms of mDia1 (mDia1 ΔN3) and ROCK1 (ROCK1 Δ3), in the presence or absence of 0.1 μM Latrunculin B.(B) Quantitative analyses of the actin tail positive cell rate and the number of actin tails in WR-infected cells expressing mDia1 ΔN3 and ROCK1 Δ3.(C) Quantitative analyses of the number of infectious particles released from WR-infected cells expressing the indicated GFP-tagged protein in the presence or absence of 0.1 μM Latrunculin B.(D) Movement of IEV particles in cells infected with A36R-YdF-YFP and expressing mDia1 ΔN3 and RhoA V14. Smaller panels represent movie stills from the highlighted area in main cell. Color-coded tracks of the movements of virus particles over 26 s for each drug regime are indicated above each movie still (Movie S6).(E) Schematic representation of F11L-mediated inhibition of RhoA-mDia signaling, which results in increased microtubules dynamics and changes in cortical actin that promote virus particle release.Error bars represent SEM; ∗∗∗p < 0.0001.View Large Image Figure ViewerDownload Hi-res" @default.
• W2025328992 created "2016-06-24" @default.
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• W2025328992 creator A5012396048 @default.
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• W2025328992 creator A5057211161 @default.
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• W2025328992 date "2007-05-01" @default.
• W2025328992 modified "2023-10-16" @default.
• W2025328992 title "The Release of Vaccinia Virus from Infected Cells Requires RhoA-mDia Modulation of Cortical Actin" @default.
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