key: cord-1049279-ayz8zl37
authors: Aliyu, Isah Abubakar; Kumurya, Abdulhadi Sale; Bala, Jamilu Abubakar; Yahaya, Hassan; Saidu, Hayatu
title: Proteomes, kinases and signalling pathways in virus‐induced filopodia, as potential antiviral therapeutics targets
date: 2020-12-12
journal: Rev Med Virol
DOI: 10.1002/rmv.2202
sha: 9cbf56509e3ab665941b91fe50c2c639f54cbb5a
doc_id: 1049279
cord_uid: ayz8zl37

Filopodia are thin finger‐like protrusions at the surface of cells that are internally occupied with bundles of tightly parallel actin filaments. They play significant roles in cellular physiological processes, such as adhesion to extracellular matrix, guidance towards chemo‐attractants and in wound healing. Filopodia were recently reported to play important roles in viral infection including initial viral attachment to host cells, cell surfing, viral trafficking, internalization, budding, virus release and spread to other cells in a form that would avoid the host immune system. The detailed virus‐host protein interactions underlying most of these processes remain to be elucidated. This review will describe some reported virus‐host protein interactions on filopodia with the aim of identifying potential new anti‐virus therapeutic targets. Exploring this research area may lead to the development of novel classes of anti‐viral therapeutics that can block signalling pathways used by the virus to trigger filopodia formation. Successful compounds would inhibit initial virus attachment, formation of filopodia, expression of putative virus binding protein, extracellular virus trafficking, and budding.

pseudopodia like structures inside which tight bundles of filamentous actin are arranged in parallel (extensively reviewed by Mattila and Lappalainen. 1 Both structures are generated when the end of the barbed rapidly growing filamentous actin is towards the cell membrane. These extension of filaments adjust the terminal end of the cell which subsequently results in cell motility. 2, 3 Filopodia are sometimes found rooted or protruding from lamellipodial actin network. [4] [5] [6] They play important functions like adherence to outer cell matrix, wound healing, recognition and response to chemical stimulus, neuron functions and embryogenesis. 7, 8 Recent findings showed increasing significance of these structures in viral infection; viruses were associated with filopodia in many cells, although, how these interactions contribute to infection remains poorly understood. 9 Viral binding to filopodia shows rapid surfing (highly ordered lateral movement) in the direction of cell membrane before internalization, disruption of this movement significantly reduced viral infection. 9 Smith, et al. 10 noted that virus can rearrange cell cytoskeleton proteins; these follow cellular signals which subsequently result in filopodia formation. Upon virus attachment, reorganization of cytoskeleton plays an important role in virus adherence to the cytoplasmic membrane for internalization. 11, 12 Viruses can travel along pre-formed filopodia after attachment in search for internalization receptors. 13 Moreover, it was recently suggested that viruses activate the formation of filopodia to achieved enhanced infection of host cells. 14, 15 Endocytosis in most viruses occurs only after the virus has accessed and formed virus-internalization receptor complex on the filopodia. 16 Despite extensive studies on filopodia and its proteomes, the biological functions of filopodia as it relates to virus infection, the mechanism with which virus induced their formation, the mechanisms of enhanced infection upon interaction with filopodia, and filopodial proteins involved in virus-filopodia interaction are still poorly elucidated. Here, we summarize the current understanding concerning the role of filopodia in viral infection, mechanism of virus induced filopodia, the signalling pathways and proteins involve in virus induced filopodia.

To study filopodia-viral interaction there is a need to first review some of the important sets of proteins that are actively involved in the generation of filopodia in different types of cells and organism. In this review our discussion will be limited to well-studied proteins that are directly involved in filopodia stimulation, generation and maintenance. Most of these proteins regulate actin cytoskeleton and are localized to regulate filopodia formation.

Myosins are very important protein family in filopodia biology. They are motor proteins which bind to actin, and play crucial role in cell motility, they are reported to play important role in vesicle trafficking and are also important in the formation of actin protrusions. 17 They demonstrate a fine directed movement along actin filament. Myosin-X is one of the most important members of the myosin family protein that strongly promotes formation of filopodia. 17 The exact role they play in filopodia formation is not fully understood. Myosin-X a member of unconventional group of myosin, consists structurally of three pH domains; a coiled-coil domain known to facilitate dimerization, Sequence enriched with Pro, Glu, Ser, and Thr domain involved in binding to microtubules, known as myosin tail homology 4 and the last domain, which are in addition to the major domain called actin motor domain three IQ motifs. 18 It has also been observed that overexpression of myosin-X triggers formation of filopodia in many cell types. Furthermore, this protein was observed to be localized to filopodia tips. 17 Other proteins associated to myosin family like myosin VIIa, VIIb and XV are essential in microvilli formation, a structure similar to filopodia. 19 Myosin-X moves along the length of filopodia shaft toward filament barbed end, and then uses retrograde actin flow to slide down, demonstrating the motor activity of the protein. 17, 18 Myosin-X interaction with other proteins different from filamentous actin has also been reported, these includes integrins, vasodilator-stimulated phosphoprotein (VASP), netrin receptors, microtubules and phosphatidylinositol phosphates. [20] [21] [22] [23] More information is still needed to fully elucidate the functions of myosin-X in the formation and function of filopodia. It was however reported that myosin-X transports important filopodial proteins to the dense tip of filopodia during active filopodia formation; these include enabled/vasodilator stimulated phosphoprotein (ENA/VASP) and integrins. And the motor domain of this protein is involved in the initiation of filopodia. 23 Furthermore, lateral movement of myosin-X at the periphery of cells was observed, this indicated the movement of barbed end of filamentous actin and the subsequent convergence of actin filament that results in filopodia formation. 24 

Integrins are heterodimeric molecular receptors which are very important in cell migration. 25 Integrins play crucial roles in the organization of actin by transducing the signals which activate regulatory pathways involved in this process. 26 Integrins are conveyed actively to the tip of filopodia by myosin-X during filopodia formation. 21 But whether the binding of integrin to myosin-X is required to regulate filopodia formation is not very clear.

Induction of lamellipodia nucleation which is an important event in filopodia formation was reported to be Rac1-dependent and was mediated by integrin-filopodia-adhesion. 27 Thus, Arjonan et al. 28 proposed that, though integrins are useful in filopodia dynamics, myosin-X can still induce the formation of filopodia independent of integrin, though they acknowledged the contribution of this protein in the function and stability of filopodia and lamellipodia especially in migrating cells.

Formins are group of proteins characterized by having a conserved actin polymerizing formin homology2 domain, that is important in filopodia formation; they comprise about 15 Ras homologue (Rho) guanosine triphosphatase (GTPase) effector proteins. 29 Formins are important in many cellular processes, including cytokinesis, cell polarity and migration. 30 

The ENA/VASP-family are relatively large multifunctional actinbinding proteins that contribute significantly to filopodia formation in mammals and other organisms. 33 ENA/VASP were localized in mammalian cells at leading edge of filopodia and are useful in cellcell contact. 34, 35 They were thought to function in removal of capping protein during filopodia formation. However, findings with total internal reflection fluorescence microscopic studies and biochemical investigation have shown that, these proteins do not uncap barbed ends of actin filament, but rather demonstrate strong anti-capping activity during active rearrangement of actin in filopodia initiation. 36 Furthermore, ENA/VASP play important roles in anti-branching of actin, F-actin bundling, and enhancement of filament elongation, apart from inhibition of actin capping, which were important events in filopodia dynamics. 33, 37 Functions and the role of this protein in filopodia formation need to be further elucidated, in vivo relevance of some of these studied activities also need to be established, as well as total description of ENA/VASP protein role in the formation of filopodia and its function.

Rho GTPases belong to a protein family of (Mr∼21,000) signalling G proteins, and a subgroup of Ras protein superfamily, they are functionally involved in regulation of actin cytoskelaton and cell morphology. For this review, cell division control protein 42 homologue (Cdc42), Ras-related C3 botulinum toxin substrate 1(Rac1) and

Ras homologue family member A (RhoA) will be discussed, which are the best studied mammalian Rho GTPases. 38 Ras homologue family member A One of the most important functions of Rho protein has been observed in structural changes that lead to filopodia and lamellipodia formation, where Rho activates lamellipodia and filopodia formation in fibroblasts and other eukaryotic cells. 39 Differential equations were used by Sakumura, et al. 40 to develop a mathematical model for the activity of Rhos and their relation to cell motility. 40 The model includes RhoA, Cdc42 and Rac protein; in this model, actin de-polymerization and the extension of lamellipodia is correlated to Rac proteins, while blocking of actin de-polymerization and filopodia elongation was encouraged by Cdc42 and RhoA was significant in actin retraction. 40 

This is yet another member of Rho family GTPase, that is an important and potent filopodia stimulator. 46 They are reported to induce filopodia formation independent of other small GTPase and Cdc42, but reported to induce filopodia formation in association with Dia2. 45 actin and membrane tabulation were induced by IRSp53, which is seen to directly facilitate formation of filopodia; IRSp52 was also reported to play part in engaging actin-regulatory proteins to the lamellipodia and filopodia. 49 

Fascin is an evolutionary conserved acting bundling protein known to regulate filopodia formation. 50 Expression of Fascin-1 in cells triggered cellular migration in vitro. 51, 52 Fascin also plays significant roles in generation and maintenance of filopodial tight F-actin bundles. Fascin has also been demonstrated as the most significant actin filamentbundling proteins, which are directly linked to filopodia formation. 53 Fascin generates parallel and stiff filamentous actin bundles; even though they are not efficient preformed filament bundlers, they are implicated in bundling of loosely linked and polymerized actin filaments. 54 GTPases, Vimentin S39 and S56, stathmin S16 and S25 which are the phosphorylation target site for PARK1/2 kinases were also down regulated during severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) infection in Vero cells. 60 Interaction of SARS-CoV-2 Nsp-7 with RhoA has been implicated in the down regulation of these kinases during the virus infection. 61 On the other hand, strong up-regulation was observed of casein kinase 2 (CK2) signalling pathway, this is evident by strong phosphorylation of the CK2 target proteins, like motor domain of myosin IIa. 62 Furthermore, SARS-CoV-2 Nsp2 protein interacts directly with a sub-unit protein of actin assembly-inducing WASH complex protein called strumpellin, strongly supporting the actin structural reorganization that leads to filopodia formation. 60 

The interaction of virus with the cellular filamentous actin that results in viral particles surfing, demonstrated a process that is actin motor-driven. The role of actin in this process has been experimentally proven by the addition of cytochalasin D, a blocker of barbed end actin filament. After its addition in viral infection experiment, viral surfing was inhibited, suggesting the significance of actin in viral surfing. 9 Lehmann, et al. 9 reported that treatment of the cell with cytochalasin D or sodium azide resulted in complete loss of directional motility, and diffuse random movement toward the cell body.

This indicates that surfing might be an energy-dependent processes which is controlled primarily by actin cytoskeleton. Furthermore, myosin motors were involved in actin filament movement on filopodia and lamellipodia 63 by retrograde F-actin flow mechanism. 64 Research implicates different members of myosin in this important cellular event. Myosin II, a plus-end motor protein localized to lamellipodium and retraction fibres, 65 Virus surfing along lamellum of fibroblast cells was blocked in similar experiments, which ascertained that viral surfing over the surface of this cellular protrusion is also facilitated by the myosin IIactin machineries. 9 Host cell infection by vaccinia virus can be inhibited by treatment with blebbistatin. 14 A study shows diffuse perinuclear redistribution of myosin II in a cell treated with blebbistatin. 9 From the highlighted studies it can be seen that viral motility on the filopodia and lamellipodia are promoted by myosin II.

Myosin II also affects retrograde filamentous actin flow in filopodia, concluding that the major ATPase implicated in virus surfing is myosin II and qualifying myosin II activities as a potential virus therapeutic target with broader consequences.

Myosin-X plays vital roles in the transportation of Marburg virus on host cell filopodia before subsequent internalization. Schudt et al. reported that even though the signalling, adaptor and motor protein that mediate the transportation of nucleocapsids of Marburg virus (MARV NC) in the cell cytoplasm is not fully elucidated, the velocity of the propelled MARV on different surfaces of the cell and on the filopodia, implicated differential regulation of actin-motor proteins. 67 Myosin-V was good candidate, looking at the speed of the virus on filopodia, because the speed of 200 to 1,000 nm/s was associated with plus end directed myosin-V activity, and the virus was propelled at similar speed. 68 It was further suggested that minus end-directed myosin-VI is involved in MARV surfing, for the fact that myosin-VI also uses speeds of 300-400 nm/s to transport its cargo. 69 Myosin-X mediates slower MAR virus movement (84 ± 36 nm/s). 17 Taken together Myosin-X, myosin-V and myosin VI, were crucially important for virus surfing. Attenuation of these proteins negatively affected or completely blocked virus surfing, and subsequently affected infection and hence, may serve as potential anti-virus drug or therapeutic targets.

Virus surfing has been reported in many viruses and it is an important process required for virus infection. Virus surf filopodia in the search for an internalization hot-spot or virus receptor/co-receptor, which upon encounter, forms a virus-host receptor complex, this complex was observed to be transported via actin cuticle and become internalized. This was demonstrated in an assay involving MLV, where modified MLV containing viral capsid poly-protein precursor (Gag) labelled with yellow fluorescence protein and MLV capsid containing envelope glycoprotein of ALV were fused with ALV receptor Tva (forming virus-receptor complex) on the surface of filopodia, and surf along HEK 239 cells before internalization. 70 Filopodia surfing was also reported in human immunodeficiency virus (HIV) upon encounter with the HIV receptors C-X-C chemokine receptor type 4 (CXCR4) and cluster of differentiation 4. This was further demonstrated in particles bearing the VSV envelope protein on the filopodia of rat fibroblasts. 9 Furthermore, progressive surfing along filopodia toward cytoplasmic membrane was observed in Vesicular stomatitits virus G protein-containing viruses, this continued until the particle disappeared from the microscopic plane of confocal microscopy, indicating virus internalization. 9 In another demonstration, colocalisation of clathrin with the viruses within fifteen minutes after exposure was reported. Recruitment of clathrin to the surfing viruses occurs as soon as the virus particle reaches the plasma membrane.

Interestingly, this movement continued as the clathrin is recruited.

These findings showed that viruses move along filopodia to reach cell surface for endocytic hot spots. 9 Internalization of particles after surfing was also reported in human papillomavirus 31 (HPV31) on A431 HKs and human epidermal keratinocyte line HKs cells. In these cells, HPV31 travelled along filopodia towards cytoplasmic ALIYU ET AL.

-5 of 9 membrane, indicating the role of filopodia surfing in facilitated uptake of the HPV31 from extracellular matrix. 10 Furthermore, it has been reported that formation of filopodia in a cell facilitate enhanced virus infection, this was achieved via one of the several ways among which is, filopodial retraction, in which a distal viral particle is transferred to the cell body via filopodia for internalization. Or by a mechanism called lateral curling; here the virus particle which just attached to the filopodia tip is quickly and immediately internalized by filopodia bending backward to the cell body and the particle is internalized. 10 In this way virus particles are internalized even more quickly than in a normal viral particle surfing.

Another form of viral motility on filopodia was observed in endocytic viruses like Semliki Forest virus and VSV, where the virus is transported directly to clathrin coated pits by inducing directed and fast viral trafficking along filopodia to reach the cell body for entry into cells, in an efficient infectious pathway. 9 Taken together, it is obvious that virus surfing can be a promising anti-virus therapeutic target, with potential to block initial virus infection or reinfection.

Viruses transport along filopodia during the attachment that result in virus internalization has been reported in many enveloped viruses, 9, 14 and non-enveloped viruses. 10, 71 The particle motility on filopodia is defined as positive when the movement is directed to the cell cytoplasm, and as negative when the particles is moving away from the cell cytoplasm to filopodia tip. Lehmann, et al. 9 reported initial random movement of MLV particles, which later changed to directional surfing toward cytoplasm at various speed. Upon virus attachment to the filopodia, particles were observed to rapidly move along the length of filopodia toward the cell body, and more than 90% of the particles were internalized shortly after this event.

In a study by Mercer and Helenius, 14 Furthermore, filopodia tip was recently reported to be the point through which MARV budding takes place. 73, 74 MARV is released into the surrounding environment via filopodia. 73, 75 Similarly, MARV NCs were reported to bud out from the tip and the side of the filopodia in addition to the surfing through the interior of filopodia. 73, 74 MARV utilized this strategy to spread to the neighbouring cell without been affected by the hostile host environment immune system and proteases. 67 Release of other virus via filopodia is well documented. Virus egress via filopodia has been reported in retroviruses. 9, 76 Furthermore, the budding of HIV-1 particles from filopodia has also been reported in infected dendritic cells. 76 

One of the promising cellular events not fully explore for virus-host interaction is filopodia-viral interaction; there is a paucity of data and the published data mostly concentrate on imaging studies. Filopodia play a significant role in viral infection; they facilitate initial viral attachment to cells, they serve as a transportation medium for the virus to reach the cell surface, they are involved in viral trafficking, cell surfing and virus budding, egress and spread.

Filopodia enhance virus infection by many physical strategies including lateral curling and filopodia retraction.

Exploring filopodia proteomics and signalling dynamics may potentially lead to the development of a novel class of viral therapeutics that can work, for example, to block some signal pathways that the virus uses to trigger filopodia formation. Such a compound could target inhibition or reduction of initial attachment of virus particles to the cell. Novel classes of therapeutics might also target blockage, expression or activation of some important proteins expressed upon filopodia formation, which are used by the virus for infection. They may inhibit filopodia formation, viral transport to cell surface, subsequent internalization and productive infection. If formation of filopodia is inhibited, internalized virus particles cannot bud out or be released to infect other cells.

Filopodia: molecular architecture and cellular functions

Cellular motility driven by assembly and disassembly of actin filaments

The many faces of actin: matching assembly factors with cellular structures

Filament arrangements in negatively stained cultured cells: the organization of actin

Nerve growth cone lamellipodia contain two populations of actin filaments that differ in organization and polarity

Mechanism of filopodia initiation by reorganization of a dendritic network

Filopodia: the fingers that do the walking

The making of filopodia

Actin-and myosin-driven movement of viruses along filopodia precedes their entry into cells

Virus activated filopodia promote human papillomavirus type 31 uptake from the extracellular matrix

Single-particle tracking of murine polyoma virus-like particles on live cells and artificial membranes

Local actin polymerization and dynamin recruitment in SV40-induced internalization of caveolae

Virus-induced Abl and Fyn kinase signals permit coxsackievirus entry through epithelial tight junctions

Vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells

Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 envelope glycoprotein gB induces the integrin-dependent focal adhesion kinase-Src-phosphatidylinositol 3-kinase-rho GTPase signal pathways and cytoskeletal rearrangements

Endocytic traffic in polarized epithelial cells: role of the actin and microtubule cytoskeleton

Myosin-X is an unconventional myosin that undergoes intrafilopodial motility

Myosin-X, a novel myosin with pleckstrin homology domains, associates with regions of dynamic actin

Myosin-X: a molecular motor at the cell's fingertips

Myosin X transports Mena/VASP to the tip of filopodia

Myosin-X provides a motor-based link between integrins and the cytoskeleton

A microtubule-binding myosin required for nuclear anchoring and spindle assembly

Myosin X regulates netrin receptors and functions in axonal path-finding

The motor activity of myosin-X promotes actin fiber convergence at the cell periphery to initiate filopodia formation

Extracellular matrix and integrin signalling: the shape of things to come

Adhesion signaling-crosstalk between integrins, Src and Rho

Lamellipodia nucleation by filopodia depends on integrin occupancy and downstream Rac1 signaling

Filopodia and adhesion in cancer cell motility

Mechanism and function of formins in the control of actin assembly

Staying in shape with formins

Novel roles of formin mDia2 in lamellipodia and filopodia formation in motile cells

Disruption of the diaphanous-related Formin< i> Drf1</i> gene encoding mDia1 reveals a role for Drf3 as an effector for Cdc42

Ena/VASP proteins: regulators of the actin cytoskeleton and cell migration

The 46/50 kDa phosphoprotein VASP purified from human platelets is a novel protein associated with actin filaments and focal contacts

Mena is required for neurulation and commissure formation

Ena/VASP proteins enhance actin polymerization in the presence of barbed end capping proteins

Borisy GG Ena/VASP proteins have an anti-capping independent function in filopodia formation

Rho GTPases and actin dynamics in membrane protrusions and vesicle trafficking

Rho GTPases and the actin cytoskeleton

A molecular model for axon guidance based on cross talk between rho GTPases

Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia

The interaction between N-WASP and the Arp2/3 complex links Cdc42-dependent signals to actin assembly

Integration of multiple signals through cooperative regulation of the N-WASP-Arp2/3 complex

Protein complexes regulating Arp2/3-mediated actin assembly

The Rho family GTPase Rif induces filopodia through mDia2

The novel Rho-family GTPase rif regulates coordinated actin-based membrane rearrangements

IRSp53: crossing the road of membrane and actin dynamics in the formation of membrane protrusions

Cdc42 induces filopodia by promoting the formation of an IRSp53: mena complex

Missing-in-metastasis and IRSp53 deform PI (4, 5) P2-rich membranes by an inverse BAR domain-like mechanism

Role of fascin in filopodial protrusion

Fascin, an actin-bundling protein, modulates colonic epithelial cell invasiveness and Differentiation in Vitro

Fascin, an actinbundling protein, induces membrane protrusions and increases cell motility of epithelial cells

Evidence for fascin cross-links between the actin filaments in coelomocyte filopodia

Fascins, and their roles in cell structure and function

Intrinsic dynamic behavior of fascin in filopodia

Arp2/3 complex is bound and activated by two WASP proteins

Actin and Rho GTPases in herpesvirus biology

Cross-talk between Rac1 and Cdc42 GTPases regulates formation of filopodia required for dengue virus type-2 entry into HMEC-1 cells

The global phosphorylation landscape of SARS-CoV-2 infection

A SARS-CoV-2 protein interaction map reveals targets for 923 drug repurposing

A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug-Repurposing

Two distinct actin networks drive the protrusion of migrating cells

Cytoskeletal remodeling during growth conetarget interactions

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Dissecting temporal and spatial control of cytokinesis with a myosin II Inhibitor

Live-cell imaging of Marburg virus-infected cells uncovers actin-dependent transport of nucleocapsids over long distances

Brain myosin-V is a two-headed unconventional myosin with motor activity

Myosin at work: motor adaptations for a variety of cellular functions

A receptor for subgroup A Rous sarcoma virus is related to the low density lipoprotein receptor

Filopodia and viruses: an analysis of membrane processes in entry mechanisms

Specificity of blebbistatin, an inhibitor of myosin II

Budding of Marburgvirus is associated with filopodia

Electron tomography reveals the steps in filovirus budding

Cryo-electron tomography of Marburg virus particles and their morphogenesis within infected cells

Retroviruses can establish filopodial bridges for efficient cell-to-cell transmission

Mobilization of HIV spread by diaphanous 2 dependent filopodia in infected dendritic cells

HIV-1 Nef inhibits ruffles, induces filopodia, and modulates migration of infected lymphocytes

Simultaneous cell-to-cell transmission of human immunodeficiency virus to multiple targets through polysynapses

Cell-to-cell contact facilitates HIV transmission from lymphocytes to astrocytes via CXCR4

Annexin II as a dengue virus serotype 2 interacting protein mediating virus interaction on Vero cells

Proteomes, kinases and signalling pathways in virus-induced filopodia, as potential antiviral therapeutics targets