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US3 is a serine/threonine kinase conserved throughout the alphaherpesvirus subfamily. This viral kinase is responsible for dramatic changes in the actin cytoskeleton of the host cell, consisting of stress fiber disassembly and the formation of actin-based protrusions. These protrusions are associated with enhanced viral cell-to-cell spread and are dependent on US3-mediated phosphorylation and activation of p21 activated kinases (PAKs). The general aim of this study was to obtain better insights in the mechanism and biological consequences of pseudorabies virus (PRV) US3-mediated actin rearrangements, which was investigated through three specific studies. The first chapter gives an introduction on PRV, along with its viral structure and replication cycle. An overview of PRV-induced Aujeszky’s disease and its economic impact is given, followed by reasons to use PRV as a model organism to study alphaherpesvirus biology. US3 is elaborately introduced, along with its functions, followed by a thorough section concerning the actin cytoskeleton, where the most important actin filament based structures are highlighted. Furthermore, Rho GTPase signaling, with special attention for the RhoA- and PAK-pathway and their downstream effectors, including the WASP superfamily and ADF/cofilin family is reviewed. Finally, the current knowledge on alphaherpesvirus interactions with the actin cytoskeleton and Rho GTPases during viral entry and egress is described. The second chapter describes the aims of this study. In the third chapter, we showed that the US3 kinase of PRV affects the RhoA signaling pathway to mediate stress fiber breakdown and protrusion formation. To induce these cytoskeletal rearrangements, US3 was described earlier to phosphorylate and activate p21 activated kinases (PAKs), which are able to counteract RhoA signaling and vice versa. RhoA phosphorylation on serine residue 188 (S188) is an important RhoA inactivation mechanism through its sequestration to the cytoplasm by RhoGDI. We demonstrated a US3-mediated RhoA S188 phosphorylation following PRV infection as well as transfection of US3 in ST cells. Furthermore, co-expression of US3 with non-phosphorylatable S188A RhoA in ST cells caused a suppression on US3-induced actin rearrangements compared to co-expression of wild type RhoA with US3, underscoring the importance of US3-triggered RhoA S188 phosphorylation for these rearrangements. Kinase assays indicated the US3 probably does not directly phosphorylate RhoA S188, but may activate a cellular kinase that phosphorylates RhoA instead. Cellular protein kinase A (PKA) has been reported earlier to phosphorylate RhoA at S188 and the HSV-1 and VZV US3 orthologs have been found earlier to trigger PKA activation. Here, we found indications that PRV US3 may also trigger PKA activation, since detection of phosphorylated PKA substrates on Western blot showed substantially increased protein bands following inoculation with WT PRV compared to US3null PRV. Importantly, treatment with PKA inhibitor PKI during infection abrogated PRV-induced RhoA phosphorylation, indicating that PRV US3 indirectly triggers RhoA phosphorylation on S188 via PKA, a process that is involved in US3-mediated actin rearrangements. In the fourth chapter, we investigated how PRV US3-mediated effects on actin-controling cell signaling translates to downstream actin regulators. A central player in actin dynamics is cofilin, which is activated through dephosphorylation on serine residue 3 (S3). ST cells infected with WT PRV showed decreased phospho-S3 cofilin levels, while this was not the case for US3null PRV and mock-infected cells. Further supporting US3-mediated cofilin dephosphorylation, transfection of ST cells with US3 and consecutive staining with phospho-S3 cofilin antibody demonstrated suppressed phospho-S3 cofilin levels in transfected cells. The kinase activity of US3 was required to suppress phosphorylation of cofilin, as infection with kinase-deficient D223A Be PRV was not able to suppress phospho-S3 cofilin levels, in contrast to WT Be PRV. Unexpectedly, we found that infection with both US3null and D223A PRV led to increased phospho-S3 cofilin levels compared to mock-infected cells, which could indicate currently unknown biological consequences of viral infection and might be interesting to further investigate. Overexpression of US3 together with constitutively inactive S3D or constitutively active S3A cofilin mutants allowed us to demonstrate the involvement of cofilin dephosphorylation in PRV US3-induced actin rearrangements, as phosphomimetic S3D cofilin suppressed US3-induced actin rearrangements in ST cells, while wild type or S3A cofilin did not. Interestingly, group I PAKs are involved in US3-induced cofilin dephosphorylation, as treatment with group I PAK inhibitor IPA-3 restored the phospho-S3 cofilin signal in PRV-infected cells, while leaving mock-infected cells unaffected, supporting recent evidence for a signaling axis that connects PAK activation to cofilin dephosphorylation. In conclusion, we report that PRV US3 leads to S3 dephosphorylation (activation) of cofilin, which contributes to US3-mediated actin rearrangements. In the fifth chapter, we further investigated the biological consequences of US3-mediated actin rearrangements. LifeAct is a small peptide capable to bind filamentous (F)-actin in a non-disturbing, low-affinity, highly specific manner and thereby represents a suitable marker to (quantitatively) determine changes in F-actin in living cells. Using flow cytometric analysis of LifeAct transduced ST cells, we found that infection with WT PRV induces F-actin disassembly from 2 hours post inoculation (hpi), becoming more pronounced at later timepoints until the end of our observations at 6 hpi, while this was not the case for infection with US3null PRV. Furthermore, the kinase activity of US3 was required for F-actin disassembly, as infection with kinase-deficient D223A Be PRV could not induce decreased F-actin levels. As F-actin disassembly promotes viral entry in HIV, we wanted to investigate whether US3 also contributes to nuclear delivery of viral genomes. Therefore, qPCR assays were performed on nuclear fractions of infected cells, revealing that US3null virus DNA reached the nucleus significantly less efficiently in comparison to WT virus DNA, indeed showing a role for US3 during viral genome delivery to the nucleus. Addition of cytochalasin D not only significantly increased viral genome delivery of US3null PRV, but also increased viral genome delivery of WT PRV, showing that actin depolymerization in fact overcompensates for the lack of US3, pointing towards a beneficial effect of F-actin breakdown during viral genome delivery to the nucleus. Taken together, the PRV US3 kinase induces F-actin disassembly and plays a role in viral genome delivery to the nucleus. As our findings show that actin depolymerization leads to increased viral genome delivery to the nucleus, our data suggest that US3-induced F-actin disassembly plays a role during virus entry in host cells. The sixth chapter contains a general discussion on the results that were obtained in this thesis. Conclusion: Previous work from our research group showed that PRV US3 phosphorylates and activates PAKs, downstream effectors of Rho GTPases Rac1/Cdc42, which plays a central role in US3-mediated actin stress fiber breakdown and protrusion formation. Here, we found that, in addition, US3 also regulates actin stress fiber breakdown and protrusion formation through phosphorylation of RhoA on serine 188, which leads to RhoA inactivation through RhoGDI. We also found that downstream US3 signalization leads, via a PAK-dependent mechanism, to cofilin dephosphorylation and activation, which is important for US3-induced actin rearrangements. In addition, we have shown that US3 induces a kinase dependent disassembly of filamentous (F)-actin and plays a previously uncharacterized role in nuclear delivery of viral genomes during entry. Treatment with actin depolymerizing drug cytochalasin D increased nuclear delivery of PRV, supporting a role for F-actin disassembly during viral entry. |
