An excursus – virus entry-induced signalling

Receptor interaction is not only required for virus binding to the host cell but also to induce a cascade of cellular signalling events that finally lead to the uptake of the virus particle. By

reason of their surface localisation it is most likely that integrins are involved in WNV replication in particular via virus internalisation mediated by signal transduction.

Little is known about cellular signalling events induced by Flavivirus and, in particular, WNV infection. Activation of the Rho GTPases, Rac1 and Cdc42 upon virus binding results in actin cytoskeleton reorganisation, and formation of filopodia, both of which have been shown to be crucial in DENV-2 entry [521]. Rho GTPases, a subgroup of the Ras superfamily, control the organisation of the actin cytoskeleton. Activation leads to (i) bundling of actin filaments to form stress fibres which are associated with integrin focal adhesion complexes and (ii) actin polymerisation to form filopodia [295]. Another member of the Ras superfamily, Rab 5, an important regulator of membrane trafficking has been implicated in the transport of WNV and DENV to early endosomes [256]. Filopodia are thin plasma protrusions containing tightly parallel organised bundles of actin filaments. Their possible function in viral entry is the transport of virus-receptor-complexes to the cell body where viral particles are subsequently internalised [309]. Filopodia are especially rich in lipid rafts, cholesterol-rich membrane microdomains that act as signalling platforms due to high concentrations of cellular receptors and lipid raft-associated signalling molecules [131, 158]. The significance of lipid rafts as sites of virus entry has been described for DENV and WNV [315, 398]. Phosphatidylinositol 3-kinase (PI3-K) activation and downstream Akt phosphorylation as viral mechanisms to prevent early apoptotic cell death, observed for JEV and DENV-2 in neuroblastoma mouse cells, was suggested to be dependent on lipid rafts [265]. Recently, Scherbik et al. [423]

showed that WNV infection, at early stages, leads to a rapid and sustained Ca²⁺ influx presumably by triggering the opening of Ca²⁺ channels. This results in cleavage of caspase 3, an apoptotic regulator, and activation of several kinases, such as FAK, which constitute a mechanism to prolong cell survival. Increased Ca²⁺ levels had no effect on virus entry and, therefore, were suggested to be involved in the virus-induced rearrangement of ER mem-branes. Contrary to Chu and Ng [99], Scherbik and co-workers, op. cit., showed that FAK activation was not a result of integrin-triggered outside-in signalling of infected cells [423].

Rather it was effected by other integrin independent processes, e.g. mediated by G-protein coupled receptors, growth factor receptors, cytokines or increased levels of cytosolic Ca²⁺. As a result, other downstream targets are recruited, e.g. Src kinase family [427].

Figure 47 illustrates that integrins in particular are involved in many cellular pathways with diverse functions in the cell cycle (see also 2.7.2). Therewith most of the above described processes, implicated in Flavivirus internalisation or induced upon binding, are linked to integrin-mediated signalling cascades.

Figure 47. Integrin signalling. Graphical presentation of some important signal transduction pathways that affect the cytoskeleton re-organisation, cell proliferation, differentiation, mi-gration and survival/apoptosis (see also 2.7.3). The major submembraneous linkers between integrins and pathways are collated close to the cytoplasmic tails. Important key players (Ras, RhoA, Rac, PI3-K and Cdc42) are highlighted in red. Integrins synergise with other cell surface receptors including growth factor receptors to activate and to coordinate signal-ling pathways. Modified from Cell Signalsignal-ling Technology [78].

Research aimed to specify the signalling processes, induced by virus binding, that ultimately result in virus internalisation would significantly help understanding the first steps in WNV entry and to identify the molecules involved. It has been described in State of Knowledge (2.7.3) that the cytoplasmic domains of integrins, especially those of the β subunits, interact with adaptor proteins crucial for transmitting signals and thereby can initiate endocytosis. The β cytoplasmic domains have also been implicated in the cross-talk with those of other integrins [38, 375]. In this context, to specify the function of the cytoplasmic tail in WNV

infection as to its involvement in WNV endocytosis, the β3-deficient MEFs of this study were transfected with cDNA encoding the β3 subunit devoid of its cytoplasmic domain (4.4.3.3.4).

By this way the conserved membrane-proximal motif NPXY and membrane-distal motif NXXY (where X denotes any amino acid) which normally recruit adaptor proteins to the cellular membrane are deleted. Similar experiments were conducted to investigate the function of integrins in FMDV entry [349]. It was demonstrated that the complete cytoplas-mic domain of the β subunit is not required for endocytosis of FMDV. The reason that the construct used in this study was not expressed properly as it was not recognised by specific antibodies may be associated with the extent of its deletion. Other studies report successful expression of β3 subunit truncation products that still retain the membrane proximal region of the cytoplasmic domain [349, 494]. This conserved region consisting of six residues (KLLITI) controls the integrin’s affinity to ligands and participates in the regulation of signal transduction [188, 214, 421]. In the absence of interactions it is located within the cell membrane and becomes exposed upon activation.

The increasing number of viruses that have been found to use integrins for cell entry suggests a more important role than merely constituting docking sites for the virus. There is growing evidence that integrins comply with different functions beyond virus binding, e.g. in entry by initiating endocytosis, re-arrangement and modification of the cytoskeleton, by activation of signalling pathways or in ultimately providing a more stable environment for the virus by down-regulating the host-immune response. Echovirus 1 and coxsackievirus A9 enter cells via integrin-mediated endocytosis regulated by a dynamin-dependent mechanism [199, 374].

Adenovirus interaction with αv integrins, resulting in endocytosis, was reported to induce signals through the FAK pathway involving PI3-K and GTP-binding proteins [273, 274].

DENV binding to the integrin αvβ3 has been shown to induce actin cytoskeleton rearrange-ment [523]. Upon binding integrin αvβ3 routes herpes simplex virus (HSV) to lipid rafts and dynamin-2-dependent acidic compartments [174]. It is not clear, however, whether integrins trigger HSV endocytosis or rather modify the cell surface to preclude virus entry. There is currently no evidence that HSV directly interacts with integrin αvβ3 [173], similar to findings of WNV binding experiments in this study (5.3.1.3). The authors postulated integrins to rather affect remodelling of the cell surface or signalling activities that in consequence suppress other pathways [174].

Integrins are not only the main activators in linking extracellular stimuli to the cytoskeleton, inducing actin rearrangement and in regulating the cell cycle. Most remarkable, in terms of viral entry, is their ability to guide the trafficking of other signalling receptors, in particular

the growth factor receptors EGFR and VEGFR (vascular endothelial growth factor receptor), and cellular structures, such as cholesterol-rich membrane microdomains [75]. Moreover they influence the manner by which the growth factor receptors respond to their ligands [75, 492].

For instance, a key function of intgrin αvβ3 is to suppress trafficking of receptors that promote cell migration. This occurs by modulating Rho GTPase signalling and by altering the recycling of receptors such as EGFR [74] and VEGFR [399]. In case the function of integrin αvβ3 is disturbed other integrins and receptor tyrosine kinases are increasingly recycled to the plasma membrane [74]. Since integrins cluster within lipid rafts which constitute docking sites for many signalling molecules, integrins are implicated in coordinated signalling, i.e. the regulation of the intensity of multiple signalling cascades and membrane traffics [20, 172, 376, 442]. This important feature is again addressed in 5.4.