Abstract
The orthopoxvirus vaccinia virus (VACV) interacts with both actin and microtubule cytoskeletons in order to generate and spread progeny virions. Here, we present evidence demonstrating the involvement of PAK1 (p21-activated kinase 1) in the dissemination of VACV. Although PAK1 activation has previously been associated with optimal VACV entry via macropinocytosis, its absence does not affect the production of intracellular mature virions (IMVs) and extracellular enveloped virions (EEVs). Our data demonstrate that low-multiplicity infection of PAK1-/- MEFs leads to a reduction in plaque size followed by decreased production of both IMVs and EEVs, strongly suggesting that virus spread was impaired in the absence of PAK1. Confocal and scanning electron microscopy showed a substantial reduction in the amount of VACV-induced actin tails in PAK1-/- MEFs, but no significant alteration in the total amount of cell-associated enveloped virions (CEVs). Furthermore, the decreased VACV dissemination in PAK1-/- cells was correlated with the absence of phosphorylated ARPC1 (Thr21), a downstream target of PAK1 and a key regulatory subunit of the ARP2/3 complex, which is necessary for the formation of actin tails and viral spread. We conclude that PAK1, besides its role in virus entry, also plays a relevant role in VACV dissemination.
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Acknowledgments
The authors are grateful to Dr. Jonathan Chernoff (Tumor Cell Biology Program, Fox Chase Cancer Center, Philadelphia, USA), who kindly provided us with wild-type (WT) and pak1 knockout (PAK1-/-) mouse embryonic fibroblasts (MEFs). We also thank Dr. B. Moss (NIAID, Bethesda, MD, USA) for providing us with VACV F13L-GFP and antibody against viral protein L1R, and Dr. C. Jungwirth (Universität Würzburg, Germany), who provided us with VACV WR. We are indebted to Gisele C. dos Santos, Paula Marinho, João R. Santos and Alice A. Torres (from Viruses Laboratory) for excellent technical support. We are also grateful to Stephanie Lamb, Jia Liu, Masmudur Rahman, Dorothy Smith and Sherin Smallwood (from G.M.’s laboratory) for helpful discussions and assistance. This work was supported by grants from The Minas Gerais State’s Foundation for Research Support (FAPEMIG), Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES), and the National Council for Scientific and Technological Development (CNPq). L.G.A and J.D.A were recipients of pre-doctoral fellowships from CNPq. J.D.A. was also a recipient of a fellowship from the International Program for doctoral training (PDSE/CAPES). F.L.B.M was the recipient of a Scientific Initiation Fellowship from CNPq. C.A.B, E.G.K, F.G.F. and G.B.M. are recipients of research fellowships from CNPq. G.M.’s laboratory is supported by NIH grant R01 AI080607. Confocal microscopy and scanning electron microscopy were performed at the Institute of Biological Sciences’ Center for Electron Microscopy (CEMEL) and at the UFMG’s Microscopy Center (CM-UFMG), respectively.
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This study was funded by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and National Institutes of Health (NIH) (grant number R01 AI080607).
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The authors declare that they have no conflict of interest.
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Luciana G. Andrade and Jonas D. Albarnaz contributed equally to this study.
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Andrade, L.G., Albarnaz, J.D., Mügge, F.L.B. et al. Vaccinia virus dissemination requires p21-activated kinase 1. Arch Virol 161, 2991–3002 (2016). https://doi.org/10.1007/s00705-016-2996-3
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DOI: https://doi.org/10.1007/s00705-016-2996-3