Actin-based Motility Of Vaccinia Virus Research Articles

The amount of Nck rather than N-WASP correlates with the rate of actin-based motility of Vaccinia virus.

Vaccinia virus is a large double-stranded DNA virus and a close relative of Mpox and Variola virus, the causative agent of smallpox. During infection, Vaccinia hijacks its host's transport systems and promotes its spread into neighboring cells by recruiting a signaling network that stimulates actin polymerization. Over the years, Vaccinia has provided a powerful model to understand how signaling networks regulate actin polymerization. Nevertheless, we still lack important quantitative information about the system, including the precise number of viral and host molecules required to induce actin polymerization. Using quantitative fluorescence microscopy techniques, we have determined the number of viral and host signaling proteins accumulating on virions during their egress. Our analysis has uncovered two unexpected new aspects of this process: the number of viral proteins in the virion is not fixed and the velocity of virus movement depends on the level of a single adaptor within the signaling network.

The relative binding position of Nck and Grb2 adaptors impacts actin-based motility of Vaccinia virus.

Phosphotyrosine (pTyr) motifs in unstructured polypeptides orchestrate important cellular processes by engaging SH2-containing adaptors to assemble complex signalling networks. The concept of phase separation has recently changed our appreciation of multivalent networks, however, the role of pTyr motif positioning in their function remains to be explored. We have now investigated this parameter in the operation of the signalling cascade driving actin-based motility and spread of Vaccinia virus. This network involves two pTyr motifs in the viral protein A36 that recruit the adaptors Nck and Grb2 upstream of N-WASP and Arp2/3 complex-mediated actin polymerisation. Manipulating the position of pTyr motifs in A36 and the unrelated p14 from Orthoreovirus, we find that only specific spatial arrangements of Nck and Grb2 binding sites result in robust N-WASP recruitment, Arp2/3 complex driven actin polymerisation and viral spread. This suggests that the relative position of pTyr adaptor binding sites is optimised for signal output. This finding may explain why the relative positions of pTyr motifs are frequently conserved in proteins from widely different species. It also has important implications for regulation of physiological networks, including those undergoing phase transitions.

The rate of N-WASP exchange limits the extent of ARP2/3-complex-dependent actin-based motility

Understanding cell motility will require detailed knowledge not only of the localization of signalling networks regulating actin polymerization, but also of their dynamics. Unfortunately, many signalling networks are not amenable to such analysis, as they are frequently transient and dispersed. By contrast, the signalling pathways used by pathogens undergoing actin-based motility are highly localized and operate in a constitutive fashion. Taking advantage of this, we have analysed the dynamics of neuronal Wiskott-Aldrich syndrome protein (N-WASP), WASP-interacting protein (WIP), GRB2 and NCK, which are required to stimulate actin-related protein (ARP)2/3-complex-dependent actin-based motility of vaccinia virus, using fluorescence recovery after photobleaching. Here we show that all four proteins are rapidly exchanging, albeit at different rates, and that the turnover of N-WASP depends on its ability to stimulate ARP2/3-complex-mediated actin polymerization. Conversely, disruption of the interaction of N-WASP with GRB2 and/or the barbed ends of actin filaments increases its exchange rate and results in a faster rate of virus movement. We suggest that the exchange rate of N-WASP controls the rate of ARP2/3-complex-dependent actin-based motility by regulating the extent of actin polymerization by antagonizing filament capping.

Abl collaborates with Src family kinases to stimulate actin-based motility of vaccinia virus

Local activation of Src at the plasma membrane by extracellular vaccinia virus results in a signalling cascade that acts to stimulate actin polymerization beneath the virus to enhance its cell-to-cell spread. Initiation of this signalling cascade involves Src-mediated phosphorylation of tyrosine 112 and 132 of the viral membrane protein A36R. Here we show that recruitment of Src is dependent on its myristoylation and an interaction with A36R upstream of tyrosine 112 and 132. We further show that Src, Fyn and Yes have unique specificities towards these tyrosine residues. Using cell lines deficient in Src, Fyn and Yes, we demonstrate that multiple Src family members can stimulate vaccinia-induced actin polymerization and also uncover a role for Abl family kinases. Additionally, Abl and Arg are able to phosphorylate A36R in vitro and are recruited to vaccinia-induced actin tails. The ability of multiple families of tyrosine kinases to directly phosphorylate A36R ensures robust cell-to-cell spread of vaccinia virus will occur under a variety of cellular conditions.

Riding the Comet Tail

Vaccinia virus moves around infected cells propelled by comet-like actin tails. Newsome et al. (see the Perspective by Hall) have uncovered an outside-in signaling mechanism, induced by extracellular vaccinia virus particles, that is ultimately responsible for stimulating actin-based motility of vaccinia virus by locally recruiting and activating Src at the plasma membrane. The highly localized signaling circuit explains why actin tails are only formed at the plasma membrane and strengthens the hypothesis that actin-based motility of vaccinia mimics receptor kinase signaling at the leading edge of migrating cells. The authors also found a role for Src in regulating a switch from microtubule-based to actin-based motility at the leading edge of cells. T. P. Newsome, N. Scaplehorn, M. Way, Src mediates a switch from microtubule- to actin-based motility of vaccinia virus. Science 306 , 124-129 (2004). [Abstract] [Full Text] A. Hall, Src launches vaccinia. Science 306 , 65-67 (2004). [Summary] [Full Text]

SRC mediates a switch from microtubule- to actin-based motility of vaccinia virus.

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.

A role for VASP in RhoA-Diaphanous signalling to actin dynamics and SRF activity.

Vasodilator-stimulated phosphoprotein (VASP) is involved in multiple actin-mediated processes, including regulation of serum response factor (SRF) activity. We used the SRF transcriptional assay to define functional domains in VASP and to show that they coincide with those required for F-actin accumulation, as determined by a quantitative FACS assay. We identified inactive VASP mutants that can interfere both with F-actin assembly and with SRF activation by wild-type VASP. These VASP mutants also inhibit actin-based motility of Vaccinia virus and Shigella flexneri. VASP-induced F-actin accumulation and SRF activation require both functional Rho and its effector mDia, and conversely, mDia-mediated SRF activation is critically dependent on functional VASP. VASP and mDia also associate physically in vivo. These findings show that VASP and mDia function cooperatively downstream of Rho to control F-actin assembly and SRF activity.

The WH1 and EVH1 Domains of WASP and Ena/VASP Family Members Bind Distinct Sequence Motifs

A complex of N-WASP and WASP-interacting protein (WIP) plays an important role in actin-based motility of vaccinia virus and the formation of filopodia [1, 2]. WIP is also required to maintain the integrity of the actin cytoskeleton in T and B lymphocytes and is essential for T cell activation [3, 4]. However, in contrast to many other N-WASP binding proteins, WIP does not stimulate the ability of N-WASP to activate the Arp2/3 complex [2]. Although the WASP homology 1 (WH1) domain of N-WASP interacts directly with WIP [2, 5, 6], we still lack the exact nature of its binding site. We have now identified and characterized the N-WASP WH1 binding motif in WIP in vitro and in vivo using Shigella and vaccinia systems. The WH1 domain, which is predicted to have a similar structural fold to the Ena/VASP homology 1 (EVH1) domain [7, 8], binds to a sequence motif in WIP (ESRFYFHPISD) that is very different from the EVH1 proline-rich DL/FPPPP ligand [9–11]. Interaction of the WH1 domain of N-WASP with WIP is dependent on the two highly conserved phenylalanine residues in the motif. The WH1 binding motif we have identified is conserved in WIP, CR16, WICH, and yeast verprolin.

Grb2 and Nck Act Cooperatively to Promote Actin-Based Motility of Vaccinia Virus

The Wiskott-Aldrich syndrome protein family member N-WASP is a key integrator of the multiple signalling pathways that regulate actin polymerization via the Arp2/3 complex [1-4]. Our previous studies have shown that N-WASP is required for the actin-based motility of vaccinia virus and is recruited via Nck and WIP [5, 6]. We now show that Grb2 is an additional component of the vaccinia actin tail-forming complex. Recruitment of Nck and Grb2 to viral particles requires phosphorylation of tyrosine residues 112 and 132 of A36R, the vaccinia actin tail nucleator, respectively. The presence of Grb2 on the virus is also dependent on the polyproline-rich region of N-WASP. The Grb2 pathway alone is therefore unable to nucleate actin tails, as its recruitment requires the prior recruitment of N-WASP by Nck. However, Grb2 does play an important role in actin-based motility of vaccinia, as in its absence, the mean number of actin tails per cell is reduced 2.6-fold. Thus, both Nck and Grb2 act in a cooperative manner to stabilize and/or activate the vaccinia actin-nucleating complex. We suggest that such cooperativity between “primary” and “secondary” adaptor proteins is likely to be a general feature of receptor-mediated signalling.

Kinesin-dependent movement on microtubules precedes actin-based motility of vaccinia virus.

Vaccinia virus, a close relative of the causative agent of smallpox, exploits actin polymerization to enhance its cell-to-cell spread. We show that actin-based motility of vaccinia is initiated only at the plasma membrane and remains associated with it. There must therefore be another form of cytoplasmic viral transport, from the cell centre, where the virus replicates, to the periphery. Video analysis reveals that GFP-labelled intracellular enveloped virus particles (IEVs) move from their perinuclear site of assembly to the plasma membrane on microtubules. We show that the viral membrane protein A36R, which is essential for actin-based motility of vaccinia, is also involved in microtubule-mediated movement of IEVs. We further show that conventional kinesin is recruited to IEVs via the light chain TPR repeats and is required for microtubule-based motility of the virus. Vaccinia thus sequentially exploits the microtubule and actin cytoskeletons to enhance its cell-to-cell spread.

A complex of N-WASP and WIP integrates signalling cascades that lead to actin polymerization.

Wiskott-Aldrich syndrome protein (WASP) and N-WASP have emerged as key proteins connecting signalling cascades to actin polymerization. Here we show that the amino-terminal WH1 domain, and not the polyproline-rich region, of N-WASP is responsible for its recruitment to sites of actin polymerization during Cdc42-independent, actin-based motility of vaccinia virus. Recruitment of N-WASP to vaccinia is mediated by WASP-interacting protein (WIP), whereas in Shigella WIP is recruited by N-WASP. Our observations show that vaccinia and Shigella activate the Arp2/3 complex to achieve actin-based motility, by mimicking either the SH2/SH3-containing adaptor or Cdc42 signalling pathways to recruit the N-WASP-WIP complex. We propose that the N-WASP-WIP complex has a pivotal function in integrating signalling cascades that lead to actin polymerization.

Actin-based motility of vaccinia virus mimics receptor tyrosine kinase signalling.

Studies of the actin-based motility of the intracellular pathogens Listeria monocytogenes and Shigella flexneri have provided important insight into the events occurring at the leading edges of motile cells. Like the bacteria Listeria and Shigella, vaccinia virus, a relative of the causative agent of smallpox, uses actin-based motility to spread between cells. In contrast to Listeria or Shigella, the actin-based motility of vaccinia is dependent on an unknown phosphotyrosine protein, but the underlying mechanism remains obscure. Here we show that phosphorylation of tyrosine 112 in the viral protein A36R by Src-family kinases is essential for the actin-based motility of vaccinia. Tyrosine phosphorylation of A36R results in a direct interaction with the adaptor protein Nck and the recruitment of the Ena/VASP family member N-WASP to the site of actin assembly. We also show that Nck and N-WASP are essential for the actin-based motility of vaccinia virus. We suggest that vaccinia virus spreads by mimicking the signalling pathways that are normally involved in actin polymerization at the plasma membrane.

Viruses, microorganisms and scientists meet the nuclear pore. Leysin, VD, Switzerland, February 26-March 1, 1998.

The modular architecture of the nuclear pore complex (NPC) consists of a membrane‐embedded basic framework (the ‘spoke complex’) made up of eight multidomain spokes with two rings on each face (Figure 1A). The ring facing the cytoplasm is decorated with eight ∼50 nm fibrils extending into the cytosol, and the nuclear ring is capped with a basket‐like assembly of eight thin, 50–100 nm filaments joined distally by a 30–40 nm diameter terminal ring. The center of the basic framework harbors the ‘gated transport channel’, which is involved in signal‐mediated bidirectional transport of macromolecules and is plugged to the central ‘transporter’, whose ultimate structure and functional role in mediated nuclear transport remains to be established. N.Pante and U.Aebi (Basel) reported their approaches to the functional roles of the different NPC components and to dissection of the steps of nuclear import and export at the NPC by direct visualization of gold‐labeled proteins and RNAs moving in or out of the cell nucleus through the NPC (Figure 1B). Figure 1. ( A ) Current consensus model of the membrane‐bound NPC. Its major structural components include the basic framework, the central plug or gated channel, the cytoplasmic and nuclear rings, and the cytoplasmic fibrils and nuclear basket. ( B ) Visualization of the different steps of nuclear import of an NLS‐bearing protein through the NPC. At the bottom are selected examples of cross‐sectioned NPCs with associated nucleoplasmin–gold particles (8 nm diameter), depicted at different stages of transport; at the top is a schematic diagram of the distinct steps visualized by electron microscopy. In the first step of transport, the NLS protein to be imported associates with the NLS receptor (importins α and β) (1). This step takes place in the cytoplasm and does not require physical interaction with any NPC component. In a second step, this ‘targeting complex’ docks …

Actin-based motility of vaccinia virus.

The role of the cytoskeleton during viral infection is poorly understood. Here we show, using a combination of mutant and drug studies, that the intracellular enveloped form of vaccinia virus is capable of inducing the formation of actin tails that are strikingly similar to those seen in Listeria, Shigella and Rickettsia infections. Analysis using video microscopy reveals that single viral particles are propelled in vivo on the tip of actin tails, at a speed of 2.8 mumol min-1. On contact with the cell surface, virus particles extend outwards on actin projections at a similar rate, to contact and infect neighboring cells. Given the similarities between the motility of vaccinia virus and bacterial pathogens, we suggest that intracellular pathogens have developed a common mechanism to exploit the actin cytoskeleton as a means to facilitate their direct spread between cells.