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• W2072730267 abstract "•Vaccinia virus F11 protein contains a central PDZ-like domain that regulates RhoA binding•F11 PDZ-like domain is required to inhibit RhoA signaling and promote viral spread•F11 PDZ-like domain interacts with the PDZ binding motif of the RhoGAP Myosin-9A•Myosin-9A GAP activity downregulates RhoA to promote viral spread The vaccinia F11 protein promotes viral spread by modulating the cortical actin cytoskeleton by inhibiting RhoA signaling via an unknown mechanism. PDZ domains are widely conserved protein interaction modules whose occurrence in viral proteins is unprecedented. We found that F11 contains a central PDZ-like domain that is required to downregulate RhoA signaling and enhance viral spread. The PDZ-like domain interacts with the PDZ binding motif of the Rho GTPase-activating protein (GAP) Myosin-9A. In the absence of Myosin-9A, RhoA signaling is not inhibited, resulting in fewer actin tails and reduced virus release concomitant with less viral spread. The loss of Myosin-9A GAP activity or its ability to bind F11 also reduces actin tail formation. Furthermore, the ability of Myosin-9A to promote viral spread depends on F11 binding RhoA. Thus, F11 acts as a functional PDZ-containing scaffolding protein to inhibit RhoA signaling by binding Myosin-9A. The vaccinia F11 protein promotes viral spread by modulating the cortical actin cytoskeleton by inhibiting RhoA signaling via an unknown mechanism. PDZ domains are widely conserved protein interaction modules whose occurrence in viral proteins is unprecedented. We found that F11 contains a central PDZ-like domain that is required to downregulate RhoA signaling and enhance viral spread. The PDZ-like domain interacts with the PDZ binding motif of the Rho GTPase-activating protein (GAP) Myosin-9A. In the absence of Myosin-9A, RhoA signaling is not inhibited, resulting in fewer actin tails and reduced virus release concomitant with less viral spread. The loss of Myosin-9A GAP activity or its ability to bind F11 also reduces actin tail formation. Furthermore, the ability of Myosin-9A to promote viral spread depends on F11 binding RhoA. Thus, F11 acts as a functional PDZ-containing scaffolding protein to inhibit RhoA signaling by binding Myosin-9A. Given that RhoGTPases play an essential role in regulating a wide variety of cellular processes, it is not surprising that their signaling is modulated by many different viruses, especially during actin-dependent entry (Chandran, 2010Chandran B. Early events in Kaposi’s sarcoma-associated herpesvirus infection of target cells.J. Virol. 2010; 84: 2188-2199Crossref PubMed Scopus (123) Google Scholar, Favoreel et al., 2007Favoreel H.W. Enquist L.W. Feierbach B. Actin and Rho GTPases in herpesvirus biology.Trends Microbiol. 2007; 15: 426-433Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, Mercer and Helenius, 2012Mercer J. Helenius A. Gulping rather than sipping: macropinocytosis as a way of virus entry.Curr. Opin. Microbiol. 2012; 15: 490-499Crossref PubMed Scopus (152) Google Scholar, Mercer et al., 2010bMercer J. Schelhaas M. Helenius A. Virus entry by endocytosis.Annu. Rev. Biochem. 2010; 79: 803-833Crossref PubMed Scopus (718) Google Scholar, Quetglas et al., 2012Quetglas J.I. Hernáez B. Galindo I. Muñoz-Moreno R. Cuesta-Geijo M.A. Alonso C. Small rho GTPases and cholesterol biosynthetic pathway intermediates in African swine fever virus infection.J. Virol. 2012; 86: 1758-1767Crossref PubMed Scopus (32) Google Scholar, Sánchez et al., 2012Sánchez E.G. Quintas A. Pérez-Núñez D. Nogal M. Barroso S. Carrascosa A.L. Revilla Y. African swine fever virus uses macropinocytosis to enter host cells.PLoS Pathog. 2012; 8: e1002754Crossref PubMed Scopus (118) Google Scholar, Stolp and Fackler, 2011Stolp B. Fackler O.T. How HIV takes advantage of the cytoskeleton in entry and replication.Viruses. 2011; 3: 293-311Crossref PubMed Scopus (50) Google Scholar, Taylor et al., 2011Taylor M.P. Koyuncu O.O. Enquist L.W. Subversion of the actin cytoskeleton during viral infection.Nat. Rev. Microbiol. 2011; 9: 427-439Crossref PubMed Scopus (273) Google Scholar, Van den Broeke and Favoreel, 2011Van den Broeke C. Favoreel H.W. Actin’ up: herpesvirus interactions with Rho GTPase signaling.Viruses. 2011; 3: 278-292Crossref PubMed Scopus (11) Google Scholar, Van den Broeke et al., 2009Van den Broeke C. Radu M. Deruelle M. Nauwynck H. Hofmann C. Jaffer Z.M. Chernoff J. Favoreel H.W. Alphaherpesvirus US3-mediated reorganization of the actin cytoskeleton is mediated by group A p21-activated kinases.Proc. Natl. Acad. Sci. USA. 2009; 106: 8707-8712Crossref PubMed Scopus (68) Google Scholar). In the case of Vaccinia virus, entry transiently activates Cdc42, Rac, and RhoA signaling (Locker et al., 2000Locker J.K. Kuehn A. Schleich S. Rutter G. Hohenberg H. Wepf R. Griffiths G. Entry of the two infectious forms of vaccinia virus at the plasma membane is signaling-dependent for the IMV but not the EEV.Mol. Biol. Cell. 2000; 11: 2497-2511Crossref PubMed Scopus (143) Google Scholar, Mercer and Helenius, 2008Mercer J. Helenius A. Vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells.Science. 2008; 320: 531-535Crossref PubMed Scopus (586) Google Scholar, Mercer et al., 2010aMercer J. Knébel S. Schmidt F.I. Crouse J. Burkard C. Helenius A. Vaccinia virus strains use distinct forms of macropinocytosis for host-cell entry.Proc. Natl. Acad. Sci. USA. 2010; 107: 9346-9351Crossref PubMed Scopus (123) Google Scholar). During the later stages of infection, however, Vaccinia downregulates the steady-state level of GTP-bound Cdc42, Rac, and RhoA (Arakawa et al., 2007bArakawa 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: 213-226Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The mechanism by which Vaccinia reduces Cdc42 and Rac signaling during infection remains to be determined. In contrast, we have previously demonstrated that Vaccinia inhibits RhoA signaling using F11, a viral protein that is expressed from as early as 2 hr postinfection (Arakawa et al., 2007bArakawa 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: 213-226Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, Cordeiro et al., 2009Cordeiro J.V. Guerra S. Arakawa Y. Dodding M.P. Esteban M. Way M. F11-mediated inhibition of RhoA signalling enhances the spread of vaccinia virus in vitro and in vivo in an intranasal mouse model of infection.PLoS ONE. 2009; 4: e8506https://doi.org/10.1371/journal.pone.0008506Crossref PubMed Scopus (46) 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 (95) Google Scholar). F11 interacts directly with RhoA using a motif that is also found in the RhoA effector ROCK (Cordeiro et al., 2009Cordeiro J.V. Guerra S. Arakawa Y. Dodding M.P. Esteban M. Way M. F11-mediated inhibition of RhoA signalling enhances the spread of vaccinia virus in vitro and in vivo in an intranasal mouse model of infection.PLoS ONE. 2009; 4: e8506https://doi.org/10.1371/journal.pone.0008506Crossref PubMed Scopus (46) 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 (95) Google Scholar). F11-mediated inhibition of RhoA signaling is responsible for stimulating Vaccinia-induced cell migration (Morales et al., 2008Morales I. Carbajal M.A. Bohn S. Holzer D. Kato S.E. Greco F.A. Moussatché N. Krijnse Locker J. The vaccinia virus F11L gene product facilitates cell detachment and promotes migration.Traffic. 2008; 9: 1283-1298Crossref PubMed Scopus (27) 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 (95) Google Scholar). Moreover, the introduction of the Vaccinia F11L gene into the genomes of Modified Vaccinia Ankara (MVA) and Myxoma viruses, which do not possess an F11 ortholog promotes virus-induced cell migration (Irwin and Evans, 2012Irwin C.R. Evans D.H. Modulation of the myxoma virus plaque phenotype by vaccinia virus protein F11.J. Virol. 2012; 86: 7167-7179Crossref PubMed Scopus (21) Google Scholar, Zwilling et al., 2010Zwilling J. Sliva K. Schwantes A. Schnierle B. Sutter G. Functional F11L and K1L genes in modified vaccinia virus Ankara restore virus-induced cell motility but not growth in human and murine cells.Virology. 2010; 404: 231-239Crossref PubMed Scopus (14) Google Scholar). F11-dependent inhibition of RhoA signaling also stimulates increased microtubule dynamics and targeting to the plasma membrane (Arakawa et al., 2007bArakawa 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: 213-226Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). These changes aid newly assembled viruses, undergoing kinesin-1-dependent microtubule transport, to reach the plasma membrane from their perinuclear site of assembly (Dodding et al., 2011Dodding M.P. Mitter R. Humphries A.C. Way M. A kinesin-1 binding motif in vaccinia virus that is widespread throughout the human genome.EMBO J. 2011; 30: 4523-4538Crossref PubMed Scopus (69) Google Scholar, 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 (180) 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 (234) 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 (144) 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 (133) Google Scholar). Inhibition of RhoA signaling to mDia, a key regulator of actin polymerization, also enhances Vaccinia release from infected HeLa cells by modulating the cortical actin beneath the plasma membrane (Arakawa et al., 2007aArakawa Y. Cordeiro J.V. Schleich S. Newsome T.P. Way M. The release of vaccinia virus from infected cells requires RhoA-mDia modulation of cortical actin.Cell Host Microbe. 2007; 1: 227-240Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, Cordeiro et al., 2009Cordeiro J.V. Guerra S. Arakawa Y. Dodding M.P. Esteban M. Way M. F11-mediated inhibition of RhoA signalling enhances the spread of vaccinia virus in vitro and in vivo in an intranasal mouse model of infection.PLoS ONE. 2009; 4: e8506https://doi.org/10.1371/journal.pone.0008506Crossref PubMed Scopus (46) Google Scholar). The cortical actin, which is regulated by RhoA signaling and provides the cell with mechanical resilience (Bergert et al., 2012Bergert M. Chandradoss S.D. Desai R.A. Paluch E. Cell mechanics control rapid transitions between blebs and lamellipodia during migration.Proc. Natl. Acad. Sci. USA. 2012; 109: 14434-14439Crossref PubMed Scopus (228) Google Scholar, de Curtis and Meldolesi, 2012de Curtis I. Meldolesi J. Cell surface dynamics—how Rho GTPases orchestrate the interplay between the plasma membrane and the cortical cytoskeleton.J. Cell Sci. 2012; 125: 4435-4444Crossref PubMed Scopus (74) Google Scholar, Fritzsche et al., 2013Fritzsche M. Lewalle A. Duke T. Kruse K. Charras G. Analysis of turnover dynamics of the submembranous actin cortex.Mol. Biol. Cell. 2013; 24: 757-767Crossref PubMed Scopus (141) Google Scholar, Salbreux et al., 2012Salbreux G. Charras G. Paluch E. Actin cortex mechanics and cellular morphogenesis.Trends Cell Biol. 2012; 22: 536-545Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar), represents a physical barrier that has to be actively remodeled during exocytosis (Gutiérrez, 2012Gutiérrez L.M. New insights into the role of the cortical cytoskeleton in exocytosis from neuroendocrine cells.Int. Rev. Cell. Mol. Biol. 2012; 295: 109-137Crossref PubMed Scopus (45) Google Scholar, Nightingale et al., 2012Nightingale T.D. Cutler D.F. Cramer L.P. Actin coats and rings promote regulated exocytosis.Trends Cell Biol. 2012; 22: 329-337Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, Wollman and Meyer, 2012Wollman R. Meyer T. Coordinated oscillations in cortical actin and Ca2+ correlate with cycles of vesicle secretion.Nat. Cell Biol. 2012; 14: 1261-1269Crossref PubMed Scopus (90) Google Scholar). It is also a significant obstacle to the spread of viral infection, as it has to be traversed or manipulated by newly assembled virus particles before they can fuse with the plasma membrane (Radtke et al., 2006Radtke K. Döhner K. Sodeik B. Viral interactions with the cytoskeleton: a hitchhiker’s guide to the cell.Cell. Microbiol. 2006; 8: 387-400Crossref PubMed Scopus (295) Google Scholar, Taylor et al., 2011Taylor M.P. Koyuncu O.O. Enquist L.W. Subversion of the actin cytoskeleton during viral infection.Nat. Rev. Microbiol. 2011; 9: 427-439Crossref PubMed Scopus (273) Google Scholar, Ward, 2011Ward B.M. The taking of the cytoskeleton one two three: how viruses utilize the cytoskeleton during egress.Virology. 2011; 411: 244-250Crossref PubMed Scopus (32) Google Scholar). Consistent with this, the loss of F11 expression and the absence of RhoA inhibition impair Vaccinia spread both in cell monolayers and in vivo, in an intranasal mouse model of infection (Cordeiro et al., 2009Cordeiro J.V. Guerra S. Arakawa Y. Dodding M.P. Esteban M. Way M. F11-mediated inhibition of RhoA signalling enhances the spread of vaccinia virus in vitro and in vivo in an intranasal mouse model of infection.PLoS ONE. 2009; 4: e8506https://doi.org/10.1371/journal.pone.0008506Crossref PubMed Scopus (46) Google Scholar). Conversely, a recombinant Myxoma virus expressing F11 is significantly more effective in cell-to-cell spread, as plaques expand ∼6-fold faster and are four times larger than the control (Irwin and Evans, 2012Irwin C.R. Evans D.H. Modulation of the myxoma virus plaque phenotype by vaccinia virus protein F11.J. Virol. 2012; 86: 7167-7179Crossref PubMed Scopus (21) Google Scholar). The presence of F11 homologs in divergent chordopoxviruses (http://www.poxvirus.org/) suggests that F11-mediated inhibition of RhoA signaling plays a conserved role in promoting viral spread. F11 interacts directly with RhoA (Arakawa et al., 2007bArakawa 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: 213-226Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, Cordeiro et al., 2009Cordeiro J.V. Guerra S. Arakawa Y. Dodding M.P. Esteban M. Way M. F11-mediated inhibition of RhoA signalling enhances the spread of vaccinia virus in vitro and in vivo in an intranasal mouse model of infection.PLoS ONE. 2009; 4: e8506https://doi.org/10.1371/journal.pone.0008506Crossref PubMed Scopus (46) 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 (95) Google Scholar). Nevertheless, we still do not know the mechanistic basis of F11-mediated inhibition of RhoA signaling during vaccinia infection. In the current study, we have demonstrated that F11 interacts with Myosin-9A using a central PDZ-like domain. This interaction and the GAP activity of Myosin-9A are required to downregulate RhoA and enhance the spread of infection. Our data demonstrate that F11 represents an unprecedented example of a viral protein with a functional PDZ domain that acts as a scaffolding protein to inhibit RhoA signaling. Comparison of the amino acid sequence of Vaccinia F11 with orthologs in other chordopoxviruses shows that the central portion is more conserved than the rest of the molecule (Figure 1A). Structural predictions of residues 118–205 of Vaccinia F11 using the Phyre server (http://www.sbg.bio.ic.ac.uk/∼phyre/) suggest that this region would adopt a PDZ-like fold (Figure 1B). Consistent with this, Vaccinia F11 and its orthopoxvirus homologs contain a K/RxxxxxGF motif that is often a characteristic feature of PDZ domains, as it is involved in direct interactions with PDZ binding motifs (PBMs) (Figure 1B) (Luck et al., 2012Luck K. Charbonnier S. Travé G. The emerging contribution of sequence context to the specificity of protein interactions mediated by PDZ domains.FEBS Lett. 2012; 586: 2648-2661Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, Nourry et al., 2003Nourry C. Grant S.G. Borg J.P. PDZ domain proteins: plug and play!.Sci. STKE. 2003; 2003 (RE7)Crossref PubMed Scopus (425) Google Scholar). In addition, there is a highly conserved GD motif that is found in nearly all PDZ domains, as it appears to be required for the structural integrity of the PDZ fold (Figure 1B) (Kalyoncu et al., 2010Kalyoncu S. Keskin O. Gursoy A. Interaction prediction and classification of PDZ domains.BMC Bioinformatics. 2010; 11: 357Crossref PubMed Scopus (37) Google Scholar, Ponting, 1997Ponting C.P. Evidence for PDZ domains in bacteria, yeast, and plants.Protein Sci. 1997; 6: 464-468Crossref PubMed Scopus (193) Google Scholar). Interestingly, the amino acid sequence of the vaccinia F11 C terminus also corresponds to a class II PBM (Figure 1C). To investigate whether the central PDZ-like domain interacts with the C-terminal PBM, we performed in vitro PBM peptide pull-down assays on the F11-PDZ-like domain produced in E. coli. We found that a peptide containing the C-terminal PBM of F11 is capable of interacting with the F11 PDZ-like domain (Figure 2A). Mutation of the conserved GF motif within PDZ domains will weaken and/or abolish PBM interactions, while loss of the GD motif is predicted to disrupt the PDZ fold. To investigate whether mutation of the GF and GD motifs impacts on F11-PBM binding, we generated recombinant Western Reserve (WR) viruses expressing F11-GF/AA and F11-GD/AA (Figure 2B). Using these viruses together with WR (wild-type F11), we performed peptide pull-down assays on infected cell lysates (Figure 2C). We found that alanine substitution of the GF motif substantially reduces the interaction of full-length F11 with its C-terminal PBM, while mutation of the GD motif largely abrogated all binding (Figure 2C). Given these data, we wondered whether an intramolecular interaction between the PDZ-like domain and the PBM regulates the ability of F11 to interact with RhoA. To explore this possibility, we performed in vitro pull-down assays using GTP-loaded GST-RhoA on E. coli extracts containing full-length, untagged F11 and a truncated version of the protein lacking its C-terminal PBM (F11-ΔPBM) (Figure 2D). The truncated F11 showed markedly increased RhoA binding compared to the full-length wild-type protein. However, this effect was abrogated by the introduction of the GF/AA and GD/AA mutations into the PDZ-like domain (Figure 2D). This latter result was unexpected, as the RhoA binding site in F11 is located toward the C terminus of the molecule (residues 299–312), away from the central PDZ-like domain (residues 118–205) (Figure 1A). The RhoA binding site in F11 is strikingly similar to that of ROCK, which is found within a coiled-coil dimer interface that involves two molecules (Dvorsky et al., 2004Dvorsky R. Blumenstein L. Vetter I.R. Ahmadian M.R. Structural insights into the interaction of ROCKI with the switch regions of RhoA.J. Biol. Chem. 2004; 279: 7098-7104Crossref PubMed Scopus (108) Google Scholar). Given that F11 also interacts with itself (Arakawa et al., 2007aArakawa Y. Cordeiro J.V. Schleich S. Newsome T.P. Way M. The release of vaccinia virus from infected cells requires RhoA-mDia modulation of cortical actin.Cell Host Microbe. 2007; 1: 227-240Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), we wondered whether the lack of RhoA binding by the F11 GF/AA and GD/AA mutants is because the PDZ-like domain is required for this self-association. Consistent with this notion, pull-down assays from infected cell lysates reveal that F11-GF/AA and F11-GD/AA have reduced self-association when compared to wild-type full-length F11 (Figure 2E). Pull-down assays on extracts from cells coexpressing GFP and FLAG-tagged PDZ-like domain or the C-terminal region of F11 reveal that they are also capable of interacting with themselves (Figure 2F). Our data suggest that an interaction between PDZ-like domains and the C-terminal PBM regulates the ability of F11 to interact with RhoA. Consistent with our in vitro pull-down assays using GST-RhoA, we found that in contrast to WR, but similar to ΔF11L, the F11-GF/AA or F11-GD/AA viruses did not reduce the level of GTP-bound RhoA at 8 hr postinfection (Figure 3A). Our previous observations have shown that F11-mediated inhibition of RhoA signaling enhances the ability of intracellular enveloped virions (IEVs) to traverse the cortical actin and fuse with the plasma membrane, prior to inducing actin tails or being released from the infected cell (Arakawa et al., 2007bArakawa 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: 213-226Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, Cordeiro et al., 2009Cordeiro J.V. Guerra S. Arakawa Y. Dodding M.P. Esteban M. Way M. F11-mediated inhibition of RhoA signalling enhances the spread of vaccinia virus in vitro and in vivo in an intranasal mouse model of infection.PLoS ONE. 2009; 4: e8506https://doi.org/10.1371/journal.pone.0008506Crossref PubMed Scopus (46) Google Scholar). The average number of actin tails per cell can therefore be used to assess the ability of F11 to downregulate RhoA signaling. We found that the ΔF11L, F11-GF/AA, and F11-GD/AA viruses induce significantly fewer actin tails than the WR virus from which they are derived (Figure 3B). These mutant viruses also had reduced cell-to-cell spread as they formed smaller plaques than WR on confluent cell monolayers (Figure 3C). In addition, the release of infectious virus particles into the media is equally reduced for all three viruses compared to WR (Figure 3D). Collectively, our observations demonstrate that the PDZ-like domain is required for F11 to downregulate RhoA signaling and enhance the spread of infection. Our earlier observations demonstrate that an interaction between the PDZ-like domain and the C-terminal PBM regulates the ability of F11 to bind RhoA (Figure 2D). Given this, we wondered whether removal of the C-terminal PBM would render F11 constitutively active for RhoA binding and enhance viral spread. To investigate if this is the case, we generated a recombinant WR virus expressing F11-ΔPBM (Figure 4A). Unexpectedly, cells infected with the ΔF11L and F11-ΔPBM viruses had significantly fewer actin tails than those infected with WR (Figure 4B). They also released less infectious virus into the media than WR (Figure 4C). PBMs play an important role in targeting proteins to specific cellular compartments (Ivarsson, 2012Ivarsson Y. Plasticity of PDZ domains in ligand recognition and signaling.FEBS Lett. 2012; 586: 2638-2647Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, Luck et al., 2012Luck K. Charbonnier S. Travé G. The emerging contribution of sequence context to the specificity of protein interactions mediated by PDZ domains.FEBS Lett. 2012; 586: 2648-2661Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, Nourry et al., 2003Nourry C. Grant S.G. Borg J.P. PDZ domain proteins: plug and play!.Sci. STKE. 2003; 2003 (RE7)Crossref PubMed Scopus (425) Google Scholar, Subbaiah et al., 2011Subbaiah V.K. Kranjec C. Thomas M. Banks L. PDZ domains: the building blocks regulating tumorigenesis.Biochem. J. 2011; 439: 195-205Crossref PubMed Scopus (72) Google Scholar). We therefore investigated whether loss of its PBM impacts on the cytosolic and membrane distribution of F11. We found that F11-ΔPBM had reduced membrane association as compared to F11 (Figure 4D). We assume that the decrease in the number of actin tails and release of infectious virus is because the reduced membrane association of F11-ΔPBM impairs its ability to downregulate RhoA signaling at the plasma membrane. It is possible that the function of the PDZ-like domain is to promote dimerization of F11 to allow it to sequester RhoA to inhibit its downstream signaling. However, WR infection leads to an F11-dependent reduction in the steady-state levels of GTP-bound RhoA (Arakawa et al., 2007bArakawa 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: 213-226Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, Cordeiro et al., 2009Cordeiro J.V. Guerra S. Arakawa Y. Dodding M.P. Esteban M. Way M. F11-mediated inhibition of RhoA signalling enhances the spread of vaccinia virus in vitro and in vivo in an intranasal mouse model of infection.PLoS ONE. 2009; 4: e8506https://doi.org/10.1371/journal.pone.0008506Crossref PubMed Scopus (46) 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 (95) Google Scholar). This suggests that the F11 PDZ-like domain has a more direct role in downregulating RhoA signaling, presumably by interacting with an unknown binding partner. In recent years, it has become clear that the cellular targeting of multiple RhoGTPase-activating Rho-GEFs is dependent on their interaction with PDZ-containing scaffolding proteins (García-Mata and Burridge, 2007García-Mata R. Burridge K. Catching a GEF by its tail.Trends Cell Biol. 2007; 17: 36-43Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). It is also striking that PBMs are also enriched in Rho-GAPs, which promote inactivation of RhoGTPases, when compared to other protein families (Giallourakis et al., 2006Giallourakis C. Cao Z. Green T. Wachtel H. Xie X. Lopez-Illasaca M. Daly M. Rioux J. Xavier R. A molecular-properties-based approach to understanding PDZ domain proteins and PDZ ligands.Genome Res. 2006; 16: 1056-1072Crossref PubMed Scopus (41) Google Scholar). Eighteen of the 80 Rho-GAPs in the human genome have a predicted PBM at their C terminus (see Figure S1A online). Given our earlier observations, we hypothesized that the F11 PDZ-like domain mediates downregulation of RhoA signaling by interacting with a class II PBM containing Rho-GAP. To explore this possibility, we performed in vitro PBM peptide pull-down assays with the seven Rho-GAPs that have a class II PBM (Figure 5A). We found that only β-Chimaerin (CHN2) and Myosin-9A interacted with the PDZ-like domain of F11. We focused our attention on Myosin-9A, as it has been reported to regulate RhoA activity and cortical actin during cell-cell adhesion (Chieregatti et al., 1998Chieregatti E. Gärtner A. Stöffler H.E. Bähler M. Myr 7 is a novel myosin IX-RhoGAP expressed in rat brain.J. Cell Sci. 1998; 111: 3597-3608PubMed Google Scholar, Omelchenko and Hall, 2012Omelchenko T. Hall A. Myosin-IXA regulates collective epithelial cell migration by targeting RhoGAP activity to cell-cell junctions.Curr. Biol. 2012; 22: 278-288Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), while β-Chimaerin is a Rac-specific GAP (Yang and Kazanietz, 2007Yang C. Kazanietz M.G. Chimaerins: GAPs that bridge diacylglycerol signalling and the small G-protein Rac.Biochem. J. 2007; 403: 1-12Crossref PubMed Scopus (12) Google Scholar). In vitro pull-down assays reveal that the deletion of the PBM in a peptide corresponding to the C terminus of Myosin-9A abolishes its interaction with the isolated F11 PDZ-like domain produced in E. coli (Figure 5B). The native Myosin-9A PBM peptide was also unable to interact with full-length F11-GF/AA and F11-GD/AA from infected cell lysates (Figure 5B). This interaction is not unique to the Myosin-9A PBM peptide, as endogenous Myosin-9A coimmunoprecipitates with F11 from infected cell lysates (Figure 5C, Figure S1B). Moreover, GFP-Trap pull-downs reveal that deletion of the C-terminal PBM of Myosin-9A abolishes its interaction with endogenous F11 (Figure 5C). To determine the functional significance of the interaction between F11 and Myosin-9A, we examined the consequence of RNAi-mediated depletion of Myosin-9A on F11-dependent inhibition of RhoA signaling (Figures 6A and 6B ). Loss of Myosin-" @default.
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• W2072730267 title "Vaccinia Virus F11 Promotes Viral Spread by Acting as a PDZ-Containing Scaffolding Protein to Bind Myosin-9A and Inhibit RhoA Signaling" @default.
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