Viruses have evolved mechanisms to manipulate microtubules (MTs) for the efficient realization of their replication programs. Studying the mechanisms of replication of mouse polyomavirus (MPyV), we observed previously that in the late phase of infection, a considerable amount of the main structural protein, VP1, remains in the cytoplasm associated with hyperacetylated microtubules. VP1-microtubule interactions resulted in blocking the cell cycle in the G2/M phase. We are interested in the mechanism leading to microtubule hyperacetylation and stabilization and the roles of tubulin acetyltransferase 1 (αTAT1) and deacetylase histone deacetylase 6 (HDAC6) and VP1 in this mechanism. Therefore, HDAC6 inhibition assays, αTAT1 knock out cell infections, in situ cell fractionation, and confocal and TIRF microscopy were used. The experiments revealed that the direct interaction of isolated microtubules and VP1 results in MT stabilization and a restriction of their dynamics. VP1 leads to an increase in polymerized tubulin in cells, thus favoring αTAT1 activity. The acetylation status of MTs did not affect MPyV infection. However, the stabilization of MTs by VP1 in the late phase of infection may compensate for the previously described cytoskeleton destabilization by MPyV early gene products and is important for the observed inhibition of the G2→M transition of infected cells to prolong the S phase.

Department of Genetics and Microbiology Faculty of Science Charles University BIOCEV 25250 Vestec Czech Republic

Faculty of Mathematics and Physics Charles University 12844 Prague Czech Republic

Institute of Biotechnology of the Czech Academy of Sciences BIOCEV 25250 Vestec Czech Republic

Zobrazit více v PubMed

Barouch D.H., Harrison S.C. Interactions among the major and minor coat proteins of polyomavirus. J. Virol. 1994;68:3982–3989. doi: 10.1128/JVI.68.6.3982-3989.1994. PubMed DOI PMC

Soldatova I., Prilepskaja T., Abrahamyan L., Forstová J., Huerfano S. Interaction of the Mouse Polyomavirus Capsid Proteins with Importins Is Required for Efficient Import of Viral DNA into the Cell Nucleus. Viruses. 2018;10:165. doi: 10.3390/v10040165. PubMed DOI PMC

Horníková L., Fraiberk M., Man P., Janovec V., Forstová J. VP 1, the major capsid protein of the mouse polyomavirus, binds microtubules, promotes their acetylation and blocks the host cell cycle. FEBS J. 2017;284:301–323. doi: 10.1111/febs.13977. PubMed DOI

Salunke D.M., Caspar D.L., Garcea R.L. Self-assembly of purified polyomavirus capsid protein VP1. Cell. 1986;46:895–904. doi: 10.1016/0092-8674(86)90071-1. PubMed DOI

O’Hara S.D., Stehle T., Garcea R.L. Glycan receptors of the Polyomaviridae: Structure, function, and pathogenesis. Curr. Opin. Virol. 2014;7:73–78. doi: 10.1016/j.coviro.2014.05.004. PubMed DOI

Bird G., O’Donnell M., Moroianu J., Garcea R.L. Possible Role for Cellular Karyopherins in Regulating Polyomavirus and Papillomavirus Capsid Assembly. J. Virol. 2008;82:9848–9857. doi: 10.1128/JVI.01221-08. PubMed DOI PMC

Palková Z., Španielová H., Gottifredi V., Hollanderová D., Forstová J., Amati P. The polyomavirus major capsid protein VP1 interacts with the nuclear matrix regulatory protein YY1. FEBS Lett. 2000;467:359–364. doi: 10.1016/S0014-5793(00)01170-4. PubMed DOI

Carbone M., Reale A., Di Sauro A., Sthandier O., Garcia M.-I., Maione R., Caiafa P., Amati P. PARP-1 Interaction with VP1 Capsid Protein Regulates Polyomavirus Early Gene Expression. J. Mol. Boil. 2006;363:773–785. doi: 10.1016/j.jmb.2006.05.077. PubMed DOI

Garcia M.-I., Perez M., Caruso M., Sthandier O., Ferreira R., Cermola M., Macchia C., Amati P. A Mutation in the DE Loop of the VP1 Protein That Prevents Polyomavirus Transcription and Replication. Virology. 2000;272:293–301. doi: 10.1006/viro.2000.0351. PubMed DOI

Chromy L.R., Pipas J.M., Garcea R.L. Chaperone-mediated in vitro assembly of Polyomavirus capsids. Proc. Natl. Acad. Sci. USA. 2003;100:10477–10482. doi: 10.1073/pnas.1832245100. PubMed DOI PMC

Soppina V., Herbstman J.F., Skiniotis G., Verhey K.J. Luminal Localization of α-tubulin K40 Acetylation by Cryo-EM Analysis of Fab-Labeled Microtubules. PLoS ONE. 2012;7:e48204. doi: 10.1371/journal.pone.0048204. PubMed DOI PMC

Xu Z., Schaedel L., Portran D., Aguilar A., Gaillard J., Marinkovich M.P., Thery M., Nachury M.V. Microtubules acquire resistance from mechanical breakage through intralumenal acetylation. Science. 2017;356:328–332. doi: 10.1126/science.aai8764. PubMed DOI PMC

Portran D., Schaedel L., Xu Z., Théry M., Nachury M.V. Tubulin acetylation protects long-lived microtubules against mechanical ageing. Nat. Cell Biol. 2017;19:391–398. doi: 10.1038/ncb3481. PubMed DOI PMC

Eshun-Wilson L., Zhang R., Portran D., Nachury M.V., Toso D.B., Löhr T., Vendruscolo M., Bonomi M., Fraser J.S., Nogales E. Effects of α-tubulin acetylation on microtubule structure and stability. Proc. Natl. Acad. Sci. USA. 2019;116:10366–10371. doi: 10.1073/pnas.1900441116. PubMed DOI PMC

Akella J.S., Wloga D., Kim J., Starostina N.G., Lyons-Abbott S., Morrissette N.S., Dougan S.T., Kipreos E.T., Gaertig J. MEC-17 is an alpha-tubulin acetyltransferase. Nature. 2010;467:218–222. doi: 10.1038/nature09324. PubMed DOI PMC

Kalebic N., Sorrentino S., Perlas E., Bolasco G., Martinez C., Heppenstall P.A. αTAT1 is the major α-tubulin acetyltransferase in mice. Nat. Commun. 2013;4:1962. doi: 10.1038/ncomms2962. PubMed DOI

Shida T., Cueva J.G., Xu Z., Goodman M.B., Nachury M.V. The major alpha-tubulin K40 acetyltransferase alphaTAT1 promotes rapid ciliogenesis and efficient mechanosensation. Proc. Natl. Acad. Sci. USA. 2010;107:21517–21522. doi: 10.1073/pnas.1013728107. PubMed DOI PMC

Hubbert C., Guardiola A., Shao R., Kawaguchi Y., Ito A., Nixon A., Yoshida M., Wang X.-F., Yao T.-P. HDAC6 is a microtubule-associated deacetylase. Nature. 2002;417:455–458. doi: 10.1038/417455a. PubMed DOI

Miyake Y., Keusch J.J., Wang L., Saito M., Hess D., Wang X., Melancon B.J., Helquist P., Gut H., Matthias P. Structural insights into HDAC6 tubulin deacetylation and its selective inhibition. Nat. Methods. 2016;12:748–754. doi: 10.1038/nchembio.2140. PubMed DOI

Chen L., Fluck M.M. Kinetic Analysis of the Steps of the Polyomavirus Lytic Cycle. J. Virol. 2001;75:8368–8379. doi: 10.1128/JVI.75.18.8368-8379.2001. PubMed DOI PMC

Dahl J., You J., Benjamin T. Induction and Utilization of an ATM Signaling Pathway by Polyomavirus. J. Virol. 2005;79:13007–13017. doi: 10.1128/JVI.79.20.13007-13017.2005. PubMed DOI PMC

Dailey L., Basilico C. Sequences in the polyomavirus DNA regulatory region involved in viral DNA replication and early gene expression. J. Virol. 1985;54:739–749. doi: 10.1128/JVI.54.3.739-749.1985. PubMed DOI PMC

Aguilar A., Becker L., Tedeschi T., Heller S., Iomini C., Nachury M.V. α-Tubulin K40 acetylation is required for contact inhibition of proliferation and cell–substrate adhesion. Mol. Boil. Cell. 2014;25:1854–1866. doi: 10.1091/mbc.e13-10-0609. PubMed DOI PMC

Horníková L., Zila V., Španielová H., Forstová J. Mouse Polyomavirus: Propagation, Purification, Quantification, and Storage. Curr. Protoc. Microbiol. 2015;38 doi: 10.1002/9780471729259.mc14f01s38. PubMed DOI

Boura E., Liebl D., Špíšek R., Fric J., Marek M., Štokrová J., Holan V., Forstová J. Polyomavirus EGFP-pseudocapsids: Analysis of model particles for introduction of proteins and peptides into mammalian cells. FEBS Lett. 2005;579:6549–6558. doi: 10.1016/j.febslet.2005.10.062. PubMed DOI

Tolstov Y.L., Pastrana D.V., Feng H., Becker J.C., Jenkins F.J., Moschos S., Chang Y., Buck C.B., Moore P.S. Human Merkel cell polyomavirus infection II. MCV is a common human infection that can be detected by conformational capsid epitope immunoassays. Int. J. Cancer. 2009;125:1250–1256. doi: 10.1002/ijc.24509. PubMed DOI PMC

Horníková L., Man P., Forstová J. Blue native protein electrophoresis for studies of mouse polyomavirus morphogenesis and interactions between the major capsid protein VP1 and cellular proteins. J. Virol. Methods. 2011;178:229–234. doi: 10.1016/j.jviromet.2011.08.019. PubMed DOI

Forstová J., Krauzewicz N., Wallace S., Street A.J., Dilworth S.M., Beard S., Griffin E.B. Cooperation of structural proteins during late events in the life cycle of polyomavirus. J. Virol. 1993;67:1405–1413. doi: 10.1128/JVI.67.3.1405-1413.1993. PubMed DOI PMC

Dilworth S.M., Griffin B.E. Monoclonal antibodies against polyoma virus tumor antigens. Proc. Natl. Acad. Sci. USA. 1982;79:1059–1063. doi: 10.1073/pnas.79.4.1059. PubMed DOI PMC

Staufenbiel M., Deppert W. Preparation of nuclear matrices from cultured cells: Subfractionation of nuclei in situ. J. Cell Boil. 1984;98:1886–1894. doi: 10.1083/jcb.98.5.1886. PubMed DOI PMC

Sennepin A.D., Charpentier S., Normand T., Sarré C., Legrand A., Mollet L.M. Multiple reprobing of Western blots after inactivation of peroxidase activity by its substrate, hydrogen peroxide. Anal. Biochem. 2009;393:129–131. doi: 10.1016/j.ab.2009.06.004. PubMed DOI

Minotti A.M., Barlow S.B., Cabral F. Resistance to antimitotic drugs in Chinese hamster ovary cells correlates with changes in the level of polymerized tubulin. J. Boil. Chem. 1991;266:3987–3994. PubMed

Gell C., Friel C.T., Borgonovo B., Drechsel D.N., Hyman A.A., Howard J. Purification of Tubulin from Porcine Brain. Adv. Struct. Saf. Stud. 2011;777:15–28. PubMed

Castoldi M., Popov A.V. Purification of brain tubulin through two cycles of polymerization–depolymerization in a high-molarity buffer. Protein Expr. Purif. 2003;32:83–88. doi: 10.1016/S1046-5928(03)00218-3. PubMed DOI

Hyman A., Drechsel D., Kellogg U., Salser S., Sawin K., Steffen P., Wordeman L., Mitchison T. Preparation of modified tubulins. Methods Enzym. 1991;196:478–485. PubMed

Nitzsche B., Bormuth V., Bräuer C., Howard J., Ionov L., Kerssemakers J., Korten T., LeDuc C., Ruhnow F., Diez S. Studying Kinesin Motors by Optical 3D-Nanometry in Gliding Motility Assays. Methods Cell Biol. 2010;95:247–271. PubMed

Braun M., Lansky Z., Fink G., Ruhnow F., Diez S., Janson M.E. Adaptive braking by Ase1 prevents overlapping microtubules from sliding completely apart. Nature. 2011;13:1259–1264. doi: 10.1038/ncb2323. PubMed DOI

Coombes C., Yamamoto A., McClellan M., Reid T.A., Plooster M., Luxton G.G., Alper J., Howard J., Gardner M.K. Mechanism of microtubule lumen entry for the α-tubulin acetyltransferase enzyme αTAT1. Proc. Natl. Acad. Sci. USA. 2016;113:E7176–E7184. doi: 10.1073/pnas.1605397113. PubMed DOI PMC

Lin W., Hata T., Kasamatsu H. Subcellular distribution of viral structural proteins during simian virus 40 infection. J. Virol. 1984;50:363–371. doi: 10.1128/JVI.50.2.363-371.1984. PubMed DOI PMC

Erickson K.D., Bouchet-Marquis C., Heiser K., Szomolanyi-Tsuda E., Mishra R., Lamothe B., Hoenger A., Garcea R.L. Virion Assembly Factories in the Nucleus of Polyomavirus-Infected Cells. PLOS Pathog. 2012;8 doi: 10.1371/journal.ppat.1002630. PubMed DOI PMC

Montross L., Watkins S., Moreland R.B., Mamon H., Caspar D.L., Garcea R.L. Nuclear assembly of polyomavirus capsids in insect cells expressing the major capsid protein VP1. J. Virol. 1991;65:4991–4998. doi: 10.1128/JVI.65.9.4991-4998.1991. PubMed DOI PMC

Zhernov I. Results Show That EGFP Does Not Bind to Microtubules. Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, Vestec, Czech Republic; Faculty of Mathematics and Physics, Charles University; Prague, Czech Republic: 2020.

Garcea R.L., Ballmer-Hofer K., Benjamin T.L. Virion assembly defect of polyomavirus hr-t mutants: Underphosphorylation of major capsid protein VP1 before viral DNA encapsidation. J. Virol. 1985;54:311–316. doi: 10.1128/JVI.54.2.311-316.1985. PubMed DOI PMC

Haggarty S.J., Koeller K.M., Wong J.C., Grozinger C.M., Schreiber S.L. Domain-selective small-molecule inhibitor of histone deacetylase 6 (HDAC6)-mediated tubulin deacetylation. Proc. Natl. Acad. Sci. USA. 2003;100:4389–4394. doi: 10.1073/pnas.0430973100. PubMed DOI PMC

Seigneurin-Berny D., Verdel A., Curtet S., Lemercier C., Garin J., Rousseaux S., Khochbin S. Identification of Components of the Murine Histone Deacetylase 6 Complex: Link between Acetylation and Ubiquitination Signaling Pathways. Mol. Cell. Boil. 2001;21:8035–8044. doi: 10.1128/MCB.21.23.8035-8044.2001. PubMed DOI PMC

Kovacs J.J., Murphy P.J., Gaillard S., Zhao X., Wu J.-T., Nicchitta C.V., Yoshida M., Toft D.O., Pratt W.B., Yao T.-P. HDAC6 Regulates Hsp90 Acetylation and Chaperone-Dependent Activation of Glucocorticoid Receptor. Mol. Cell. 2005;18:601–607. doi: 10.1016/j.molcel.2005.04.021. PubMed DOI

Zhang L., Liu S., Liu N., Zhang Y., Liu M., Li D., Seto E., Yao T.-P., Shui W., Zhou J. Proteomic identification and functional characterization of MYH9, Hsc70, and DNAJA1 as novel substrates of HDAC6 deacetylase activity. Protein Cell. 2015;6:42–54. doi: 10.1007/s13238-014-0102-8. PubMed DOI PMC

Naghavi M.H., Walsh D. Microtubule Regulation and Function during Virus Infection. J. Virol. 2017;91 doi: 10.1128/JVI.00538-17. PubMed DOI PMC

Zila V., Difato F., Klimova L., Huerfano S., Forstová J. Involvement of Microtubular Network and Its Motors in Productive Endocytic Trafficking of Mouse Polyomavirus. PLoS ONE. 2014;9 doi: 10.1371/journal.pone.0096922. PubMed DOI PMC

Sanjuan N., Porrás A., Otero J. Microtubule-dependent intracellular transport of murine polyomavirus. Virology. 2003;313:105–116. doi: 10.1016/S0042-6822(03)00309-X. PubMed DOI

Eash S., Atwood W.J. Involvement of Cytoskeletal Components in BK Virus Infectious Entry. J. Virol. 2005;79:11734–11741. doi: 10.1128/JVI.79.18.11734-11741.2005. PubMed DOI PMC

Zheng K., Jiang Y., He Z., Kitazato K., Wang Y. Cellular defence or viral assist: The dilemma of HDAC6. J. Gen. Virol. 2017;98:322–337. doi: 10.1099/jgv.0.000679. PubMed DOI

Wenzel E.D., Speidell A., Flowers S.A., Wu C., Avdoshina V., Mocchetti I. Histone deacetylase 6 inhibition rescues axonal transport impairments and prevents the neurotoxicity of HIV-1 envelope protein gp120. Cell Death Dis. 2019;10:1–15. doi: 10.1038/s41419-019-1920-7. PubMed DOI PMC

Husain M., Harrod K. Influenza A virus-induced caspase-3 cleaves the histone deacetylase 6 in infected epithelial cells. FEBS Lett. 2009;583:2517–2520. doi: 10.1016/j.febslet.2009.07.005. PubMed DOI

Avdoshina V., Caragher S., Wenzel E.D., Taraballi F., Mocchetti I., Harry G.J. The viral protein gp120 decreases the acetylation of neuronal tubulin: Potential mechanism of neurotoxicity. J. Neurochem. 2017;141:606–613. doi: 10.1111/jnc.14015. PubMed DOI PMC

Sabo Y., Walsh D., Barry D.S., Tinaztepe S., Santos K.D.L., Goff S.P., Gundersen G.G., Naghavi M.H. HIV-1 induces the formation of stable microtubules to enhance early infection. Cell Host Microbe. 2013;14:535–546. doi: 10.1016/j.chom.2013.10.012. PubMed DOI PMC

Warren J.C., Rutkowski A., Cassimeris L. Infection with Replication-deficient Adenovirus Induces Changes in the Dynamic Instability of Host Cell Microtubules. Mol. Boil. Cell. 2006;17:3557–3568. doi: 10.1091/mbc.e05-09-0850. PubMed DOI PMC

Hyde J.L., Gillespie L.K., MacKenzie J. Mouse Norovirus 1 Utilizes the Cytoskeleton Network To Establish Localization of the Replication Complex Proximal to the Microtubule Organizing Center. J. Virol. 2012;86:4110–4122. doi: 10.1128/JVI.05784-11. PubMed DOI PMC

Rathje L.-S.Z., Nordgren N., Pettersson T., Rönnlund D., Widengren J., Aspenström P., Gad A. Oncogenes induce a vimentin filament collapse mediated by HDAC6 that is linked to cell stiffness. Proc. Natl. Acad. Sci. USA. 2014;111:1515–1520. doi: 10.1073/pnas.1300238111. PubMed DOI PMC

Knight L.M., Stakaityte G., Wood J.J., Abdul-Sada H., Griffiths D.A., Howell G.J., Wheat R., Blair G.E., Steven N.M., Macdonald A., et al. Merkel cell polyomavirus small T antigen mediates microtubule destabilization to promote cell motility and migration. J. Virol. 2015;89:35–47. doi: 10.1128/JVI.02317-14. PubMed DOI PMC

Dompierre J.P., Godin J.D., Charrin B.C., Cordelières F.P., King S.J., Humbert S., Saudou F. Histone deacetylase 6 inhibition compensates for the transport deficit in Huntington’s disease by increasing tubulin acetylation. J. Neurosci. 2007;27:3571–3583. doi: 10.1523/JNEUROSCI.0037-07.2007. PubMed DOI PMC

Boyault C., Gilquin B., Zhang Y., Rybin V., Garman E.F., Meyer-Klaucke W., Matthias P., Müller C.W., Khochbin S. HDAC6–p97/VCP controlled polyubiquitin chain turnover. EMBO J. 2006;25:3357–3366. doi: 10.1038/sj.emboj.7601210. PubMed DOI PMC

Li Y., Shin D., Kwon S.H. Histone deacetylase 6 plays a role as a distinct regulator of diverse cellular processes. FEBS J. 2013;280:775–793. doi: 10.1111/febs.12079. PubMed DOI

Fiskus W., Ren Y., Mohapatra A., Bali P., Mandawat A., Rao R., Herger B., Yang Y., Atadja P., Wu J., et al. Hydroxamic acid analogue histone deacetylase inhibitors attenuate estrogen receptor-alpha levels and transcriptional activity: A result of hyperacetylation and inhibition of chaperone function of heat shock protein 90. Clin. Cancer Res. 2007;13:4882–4890. doi: 10.1158/1078-0432.CCR-06-3093. PubMed DOI

Lu C.-Y., Chang Y.-C., Hua C.-H., Chuang C., Huang S.-H., Kung S.-H., Hour M.-J., Lin C.-W. Tubacin, an HDAC6 Selective Inhibitor, Reduces the Replication of the Japanese Encephalitis Virus via the Decrease of Viral RNA Synthesis. Int. J. Mol. Sci. 2017;18:954. doi: 10.3390/ijms18050954. PubMed DOI PMC

Bolen J.B., Anders D.G., Trempy J., Consigli A.R. Differences in the subpopulations of the structural proteins of polyoma virions and capsids: Biological functions of the multiple VP1 species. J. Virol. 1981;37:80–91. doi: 10.1128/JVI.37.1.80-91.1981. PubMed DOI PMC

Mouse polyomavirus infection induces lamin reorganisation

Microtubules in Polyomavirus Infection