The role of the actin cytoskeleton in HSV1 infection

Signalling to and exploitation of the host-cell actin cytoskeleton is pivotal for many viruses to promote their entry into non-phagocytic host cells. HSV1 induces its uptake through stimulation of a unique combination of signalling pathways and engages host-cell receptors and cofactors activating cellular regulators upstream of actin polymerization. We studied the role of a dynamic actin cytoskeleton and its perturbation in HSV1 entry in a comparative approach in different epithelial cell lines (Devadas et al., 2014; Koithan et al., in preparation-a).

In confocal life-cell imaging we could show that HSV1 particles bind to actin-rich protrusions followed by surfing of viral particles towards the cell body in HeLa, HEp-2, and PtK2 but not Vero cells. After initial contact with HSPG on finger-like protrusions (Herold et al., 1991;

Lycke et al., 1991) (Oh et al., 2010), HSV1 particles hijack a host-cell machinery for ligand transport and surf towards the cell body (Burckhardt and Greber, 2009; Dixit et al., 2008;

Koithan et al., in preparation-a; Lidke et al., 2005; Oh et al., 2010). This transport of viral particles along filopodia has been described for different enveloped and unenveloped viruses and is thought to enrich particle on the cell surface especially in wounded epithelial cells and direct viral particles to endocytosis hot spots or lipid rafts (Burckhardt and Greber, 2009; Dixit et al., 2008; Koithan et al., in preparation-a; Lidke et al., 2005; Oh et al., 2010). HSV1 particles showed either a random diffuse movement or a slow or fast directed unidirectional transport. This bipolar speed distribution was also observed in other viruses (Lehmann et al., 2005; Schelhaas et al., 2008) and is reminiscent of actin retrograde flow (Burckhardt and Greber, 2009). The clustering of the murine leukemia virus receptor mCAT-1 and the adenovirus receptor CAR potentially combined with additional signaling events provide a cue to link the receptors to the actin cytoskeleton and provide a “switch” to between random diffuse motions to directional transport (Burckhardt et al., 2011; Lehmann et al., 2005).

However, only a dimerization of receptors by epidermal growth factor (EGF) was sufficient to induce transport of the EGF using single molecule tracking (Lidke et al., 2005). Oh et al.

suggested that the binding of HSV1 glycoprotein gB to HSPG provides the cue for retrograde

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transport (Oh et al., 2010). However, since HSPG is also bound by gC it remains unclear which HSV1 glycoprotein is required. In addition, fusion-deficient HSV1 particles lacking gH were still surfing on finger-like protrusions (personal communication with Yuan Zhao and own observations). However, it remains unclear whether HSPG binding is sufficient or if a clustering of HSPG with other attachment factors is required for HSV1 surfing.

Furthermore, HSV1 triggered a transient global induction of finger-like protrusions in HeLa and HEp-2 cells and membrane ruffles in Vero and HEp-2 cells 15-30 min PI while fusion-deficient HSV1 particles lacking gH or gB did not induce any changes of the cell morphology.

HSV1 is known to induce the formation of finger-like protrusions, lamellipodia and ruffles in different cell lines (Clement et al., 2006; Dixit et al., 2008; Hoppe et al., 2006; Oh et al., 2010;

Petermann et al., 2009). In CHO-nectin1 cells and corneal fibroblasts HSV1 were engulfed by plasma membrane protrusions and internalized in big vacuoles into the cell. This phagocytosis-like uptake was clathrin independent while actin dynamics and dynamin played a central role (Clement et al., 2006; Oh et al., 2010). The massive actin rearrangements facilitated the passage of HSV1 particles through the cortical actin and were induced by activated RhoA and Cdc42 GTPase. In MDCKII, HaCat cells or primary human keratinocytes the Rho GTPases Rac1 and Cdc42 but not RhoA were induced (Hoppe et al., 2006;

Petermann et al., 2009). Even though the HSV1 gene expression was perturbed by overexpression of dominant negative or constitutively active Rac1 the internalization of HSV1 was not perturbed by siRNA knockdown of either Cdc42 or Rac1, or in Rac1 knockout primary keratinocytes (Petermann et al., 2009). A systematic study of the HSV1 entry pathways in HeLa, Vero, PtK2 and HEp-2 cells showed that HSV1 gene expression was dependent on the actin modulating proteins p21-activated kinase, protein kinase C and Rac1 but other hall mark proteins of macropinocytosis or endocytosis were not required (Devadas et al., 2014 ). Similar to the results of Petermann et al., the gene expression is strongly reduced by perturbation of Rac1 by the inhibitor EHT-1864 (Onesto et al., 2008), while HSV1 internalization per se was not affected. Even though EHT-1864 might also affect MT stability in higher concentration, the overall organization of the MT network was not affected at concentrations that inhibited HSV1 gene expression.

The dynamic changes of the actin cytoskeleton could not be observed with fusion incompetent HSV1 particles lacking gB or gH (Koithan et al., in preparation-a). This lack of Rho GTPase activation could be due to the absent interaction between either of the glycoproteins with a cellular interaction partner (Arii et al., 2010a; Gianni et al., 2010a; Gianni et al., 2010b; Gianni et al., 2013; Satoh et al., 2008), the fusion with the plasma membrane (Holm et al., 2012) or the block of the release of tegument kinases into the cytoplasm. We cannot exclude that the lack of gH and gL is necessary to engage and cluster cellular

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receptors and induce signaling to the actin cytoskeleton. However, fusion of HSV1 particles with the plasma membrane releases the tegument kinases US3 and UL13 into the cytosol which might result in the necessary signaling to pass the actin cortex. Further, Holm et al.

reported that the fusion of small virus like particles with the plasma membrane induced extensive intracellular signaling events (Holm et al., 2012).

In summary, HSV1 induces cell-type specific Rho GTPases and preferably binds to cells with plasma membrane extensions (Burckhardt and Greber, 2009; Hoppe et al., 2006; Koithan et al., in preparation-a) however the functional role of Rho GTPase signalling remains unclear.

Even though a dynamic actin cytoskeleton is not required for internalization it may facilitate HSV1 passage through the dense meshwork of the cortical meshwork or increase the rate of macropinocytosis (Devadas et al., 2014 ; Mercer and Helenius, 2009; Mercer and Helenius, 2012).

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