Src Mediates a Switch from Microtubule- to Actin-Based Motility of Vaccinia Virus

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Science  01 Oct 2004:
Vol. 306, Issue 5693, pp. 124-129
DOI: 10.1126/science.1101509


The cascade of events that leads to vaccinia-induced actin polymerization requires Src-dependent tyrosine phosphorylation of the viral membrane protein A36R. We found that a localized outside-in signaling cascade induced by the viral membrane protein B5R is required to potently activate Src and induce A36R phosphorylation at the plasma membrane. In addition, Src-mediated phosphorylation of A36R regulated the ability of virus particles to recruit and release conventional kinesin. Thus, Src activity regulates the transition between cytoplasmic microtubule transport and actin-based motility at the plasma membrane.

The microtubule cytoskeleton provides many viruses with an efficient way to reach their site of replication, as well as a means for newly assembled viruses to leave their unwilling host (1, 2). In the case of vaccinia virus, recruitment of conventional kinesin by intracellular enveloped virus (IEV) particles results in their microtubule-dependent transport from perinuclear assembly sites to the cell periphery (37). Upon reaching the plasma membrane, vaccinia induces localized actin polymerization that acts to enhance cell-to-cell spread of the virus (4, 5, 79). Vaccinia actin tail formation, which occurs beneath the extracellular cellassociated enveloped virus (CEV) at the plasma membrane (4, 5, 10), appears to mimic receptor kinase signaling at the leading edge of motile cells (1114). Src-dependent phosphorylation of Tyr112 and Tyr132 of A36R results in the generation of binding sites for the SH2 (Src homology 2) domains of the adapter proteins Nck and Grb2, respectively (11, 14). Nck is recruited to the virus as a complex with WASP interacting protein (WIP) and NWASP; this complex is stabilized by additional interactions with Grb2 (11, 12, 1416). Ultimately, recruitment of N-WASP leads to stimulation of the actin-nucleating activity of the Arp2/3 complex and actin tail formation (13).

To address how the microtubule and actin phases of virus transport are coordinated at the plasma membrane, we examined the regulation of A36R phosphorylation, which is the most upstream event of actin tail formation. Src activity is required for actin tail formation (11), but it is not clear whether A36R is a Src substrate. In in vitro kinase assays using purified components, Src was able to phosphorylate Tyr112 of A36R (residues 24 to 118) directly, and this led to the binding of Nck (Fig. 1A) (17). Neither Src-mediated phosphorylation of A36R (residues 24 to 118) nor the subsequent binding of Nck disrupted its interaction with conventional kinesin in vitro (18) (Fig. 1A). Endogenous Src kinase was localized beneath CEV particles that were promoting actin polymerization. Src kinase recruited to CEV was also phosphorylated at Tyr418, indicating the active conformation of the kinase (Fig. 1B). Activated Src was not detected on kinesin-positive IEV or in cells that lack Src (Figs. 1C and 2A) (17). To determine whether A36R phosphorylation was restricted to the plasma membrane in response to Src recruitment, we raised antibodies against a phosphopeptide corresponding to Tyr132 of A36R (anti–A36R-Y132PO4) (17). Anti–A36R-Y132PO4 detected a signal beneath actin tail–inducing CEV at the plasma membrane, but not on A36R-Y132F virus (Fig. 1, D and E). No signal was observed with anti–A36R-Y132PO4 on kinesin-positive IEV in the cytoplasm (Fig. 2C). The pattern of A36R phosphorylation reflected a subset of the total distribution of A36R, which was also observed on the perinuclear trans-Golgi network as well as on IEV being transported to the plasma membrane on microtubules (Figs. 1D and 2C). The absence of a signal on kinesin-positive IEV suggests that Src-mediated phosphorylation of A36R occurred only when the virus reached the plasma membrane, and not before.

Fig. 1.

Src phosphorylates A36R at the plasma membrane. (A) Immunoblot of in vitro phosphorylated GST-A36R. Recombinant Src directly phosphorylated A36R (residues 24 to 118) at Tyr112, which was sufficient to mediate binding to Nck (left and center panels). A36R phosphorylation was detected by mAb 4G10 to phosphotyrosine. The interaction between A36R (residues 24 to 118) and GFP-TPR was independent of A36R phosphorylation and Nck binding (right panel). The degree of A36R phosphorylation and Nck binding was proportional to the amount of Src added (0.0, 0.4, 1.2, and 3.6 units in right panel). (B) Src kinase (detected by mAb 3-27) was locally recruited and activated (detected by anti–Src-Y418PO4) at the plasma membrane beneath actin tail–inducing CEV in WR-infected cells. (C) Activated Src was observed on B5R-positive (detected by mAb 19C2) WR and A36R-YdF virus particles in infected Src parental cells (solid arrowheads) but not in SYF cells, which lack Src, Yes, and Fyn kinases (open arrowheads). (D) Tyrosine-phosphorylated A36R was restricted to the growing tips of actin tails (arrowheads). (E) Preincubation of anti–A36R-Y132PO4 with the phosphorylated but not the unphosphorylated Tyr132 peptide abrogated the signal. Anti–A36R-Y132PO4 did not label CEV in cells infected with A36R-Y132F virus. Scale bars, 1 μm [(B) and (E)], 10 μm [(C) and (D)].

Fig. 2.

Vaccinia activates Src upstream of actin tail formation. (A) Conventional kinesin and phospho-Src recruitment to virus particles was mutually exclusive in WR-infected but not A36R-YdF–infected cells. (B) WR-infected whole-cell extracts displayed a marked increase in phosphorylation of Src and cortactin. In contrast, A36R-YdF stimulated activation of Src without corresponding cortactin phosphorylation. (C) The presence of phospho-A36R (solid arrowheads) on B5R-positive virus particles was mutually exclusive to conventional kinesin (open arrowhead). In A36R-YdF–infected cells, kinesin-positive virus particles accumulated at the cell periphery. Scale bars, 1 μm (A), 10 μm (B).

To confirm that Src recruitment and activation occur upstream of A36R phosphorylation, we looked for evidence of the presence of the kinase on A36R-YdF virus particles that cannot induce actin tails because Tyr112 and Tyr132 of A36R have been mutated to Phe (5). A36R-YdF virus particles recruited active Src, indicating that its activation was independent of interactions with cellular components in the vaccinia actin tail–promoting complex (Figs. 1C and 2A). In contrast to WR (the wild-type Western Reserve strain), kinesin was still present beneath CEV particles that had recruited Src in A36R-YdF–infected cells (Fig. 2A). However, both WR and A36R-YdF viruses were potent stimulators of Src activation, as judged by the increase in phosphorylation of Tyr418 by immunoblot analysis (Fig. 2B) (17). Consistent with elevated Src activity, we found a substantial increase in tyrosine phosphorylation of cortactin in WR-infected cells (Fig. 2B). Cortactin is a Src substrate that is recruited to sites of dynamic actin polymerization, including pathogen-induced actin tails (13). In contrast, cortactin phosphorylation in A36R-YdF–infected cells was not increased, which suggests that Src-mediated phosphorylation of cortactin requires the prior recruitment of the vaccinia actin tail–nucleating complex (Fig. 2B). Thus, the localized recruitment and activation of Src at the plasma membrane, and subsequent phosphorylation of A36R, determines the site of vaccinia actin tail formation.

Our observations in vitro suggested that phosphorylation of A36R and binding of Nck did not affect its ability to interact with the kinesin light chain (Fig. 1A). In WR-infected cells, however, the presence of activated Src and phosphorylated A36R beneath CEV particles was always mutually exclusive with that of conventional kinesin (Fig. 2, A and C). The exception to this was the A36R-YdF virus, which accumulated active Src in addition to conventional kinesin (Fig. 2A). One possible explanation would be that phosphorylation of A36R by Src defines a switch required to regulate conventional kinesin release at the plasma membrane, before the initiation of actin polymerization. Consistent with this idea, kinesin was not released when the virus reached the plasma membrane in HeLa cells when Src activity was inhibited by a Src kinase inhibitor (PP2) or in the absence of Src in SYF cells (19).

We examined the effects of overexpression of Src-GFP (chicken Src fused to green fluorescent protein) on the ability of IEV to recruit kinesin. In cells expressing Src-GFP, A36R was ectopically phosphorylated at the site of IEV assembly, and virus particles were concentrated in the perinuclear region, having failed to recruit kinesin and be transported to the cell surface (Fig. 3, A and B) (17). Premature phosphorylation of A36R severely inhibited microtubule-dependent transport of virus particles to the cell periphery by blocking their ability to recruit conventional kinesin (Fig. 3D). In contrast, overexpression of Src-GFP did not affect the ability of the A36R-YdF virus to recruit kinesin and disperse to the cell periphery. Furthermore, the motor was not released when the A36R-YdF virus reached the plasma membrane (Figs. 2C and 3A). Thus, the phosphorylation of A36R, and not conventional kinesin or the recruitment of Src to IEV, inhibits microtubule-dependent movement of virus particles to the cell periphery. Consistent with this idea, dominant-negative Src, which inhibits actin tail formation (11), was recruited to the site of IEV assembly in WR-infected cells but did not block their transport to the cell periphery (Fig. 3, C and D). Thus, in the absence of Src-mediated phosphorylation of A36R, conventional kinesin remains bound to virus particles, which then accumulate in the cell periphery. Conversely, premature phosphorylation of A36R blocks kinesin recruitment and IEV egress.

Fig. 3.

Src regulates vaccinia recruitment and release of conventional kinesin. (A) Overexpression of Src-GFP inhibited kinesin recruitment and IEV dispersal to the cell periphery in WR-infected but not A36R-YdF–infected cells. (B) Src-GFP was recruited to the perinuclear trans-Golgi network in WR-infected cells and induced premature tyrosine phosphorylation of A36R. (C) Dominant-negative Src 527Kin was recruited to IEV particles and their site of assembly but did not inhibit their dispersal to the cell periphery. (D) Quantification of microtubule-dependent viral egress in cells expressing GFP, GFP-Src, or Src 527Kin (Src-DN). Bars represent the percentage of cells with at least a single kinesin-positive virus particle for each condition at 8 hours after infection. Error bars represent SD of the mean from three experiments. Scale bars, 10 μm.

Src-mediated phosphorylation of A36R at the plasma membrane is essential in vivo to achieve release of conventional kinesin and the subsequent switch to actin-based motility. However, in vitro phosphorylation of A36R did not affect its direct binding to the kinesin light chain (Fig. 1A). It is thus possible that phosphorylation of A36R also modulates its interaction with additional proteins. The binding site for kinesin on A36R (residues 81 to 111) overlaps with that of the integral viral membrane protein A33R (residues 91 to 111 of A36R) (18, 20). Moreover, the interaction of A33R with A36R is mutually exclusive with that of kinesin (18). It is possible that Src-mediated phosphorylation of A36R promotes or modulates its interaction with A33R, resulting in release of kinesin.

One possible explanation for vaccinia-induced activation of Src at the plasma membrane is that extracellular CEV induces a localized outside-in signaling cascade. If this were the case, we would predict that the viral protein responsible is present on the surface of CEV. The lumenal tails of only four integral viral membrane proteins—A33R, A34R, A56R and B5R—are exposed on the surface of CEV (21). Previous studies suggest that B5R is the best candidate to induce outside-in signaling, because deletion of its lumenal domain, which consists of four short consensus repeats (SCRs), does not inhibit the ability of the virus to reach the plasma membrane but does result in a loss of actin tail formation (22, 23). Furthermore, a recombinant virus expressing SCR domains 1 to 3 of B5R is not able to promote actin tail formation, even though the virus is able to reach the plasma membrane (23). We constructed two recombinant viruses, B5R-SCR4 and B5R-P189S, to test the role of the SCR4 domain in vaccinia actin tail formation (Fig. 4A) (17). In cells infected with the B5R-SCR4 virus, which lacks the first three SCR domains, CEV induced actin tails with associated phospho-A36R and Src (Fig. 4, B and C). Immunoblot analysis revealed that the B5R-SCR4 virus also activated Src and induced tyrosine phosphorylation of A36R and cortactin (Fig. 4D). In contrast, the B5R-P189S virus (24), which contains a Pro189 → Ser amino acid substitution in the structural fold of the SCR4 domain, did not induce increased activation of Src, A36R phosphorylation, or actin tail formation (Fig. 4, B to D). Thus, the SCR4 domain of B5R exposed on the surface of extracellular CEV was required to activate Src at the plasma membrane.

Fig. 4.

The SCR4 domain of B5R is required to activate Src. (A) Schematic representation of wild-type and recombinant B5R reading frames, indicating the position of the N-terminal signal peptide (red), the four short consensus repeat domains (SCR, yellow), and the single transmembrane domain (blue). (B) WR and B5R-SCR4 but not B5R-P189S viruses induced phosphorylation of A36R and actin tail formation. (C) B5R-SCR4 but not B5R-P189S virus recruited and activated Src. (D) Immunoblots of whole-cell lysates infected with various recombinant virus strains. B5R-SCR4 virus, but neither ΔB5R (a virus strain lacking the gene encoding B5R) nor B5R-P189S, induced phosphorylation of Src, cortactin, and A36R. Scale bars, 1 μm (B), 10 μm (C).

The coordinated regulation of multiple motors on the same cargo, including membranebound organelles, plays an important role in ensuring their delivery to the correct destination (25, 26). However, understanding the molecular basis by which microtubule motors recognize and bind their correct cargo will also be required to unravel the mechanisms that ensure their delivery to the right cellular location. Phosphorylation of components in motor complexes modulates their ability to bind cargoes (25, 26). Our results show that tyrosine phosphorylation of a cargo component can also be an important determinant regulating intracellular transport.

In addition, localized recruitment and activation of Src play an important role in regulating the switch between intracellular microtubule transport and actin-based motility at the plasma membrane (fig. S1). In uninfected cells, we envisage that local activation of Src, in addition to regulating turnover of focal adhesions, ensures coordinated microtubule-based delivery of membranes and their associated cargoes to areas of active actin polymerization at the leading edge of migrating cells.

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Materials and Methods

Fig. S1


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