The Autophagy Receptor SQSTM1/p62 Is a Restriction Factor of HCMV Infection
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plasmids
2.2. Cell Culture
2.3. Generation of Autophagy Receptor Knockout (ko) HFFs Using CRISPR/Cas9-Mediated Genome Editing
2.4. Generation of HFF GFP-SQSTM1
2.5. Viruses and BAC-Mutagenesis
2.6. Quantitative PCR Analysis
2.7. IE1 Assay
2.8. Immunofluorescence
2.9. Western Blot
2.10. Coimmunoprecipitation Experiments (CoIP)
2.11. MS Analysis of Interaction Partners and Phosphosites
2.12. Statistical Analysis
3. Results
3.1. CRISPR/Cas9 Knockout of p62 Leads to Moderately Enhanced Viral Genome Replication and Enhanced Progeny Release
3.2. p62 Interacts with a Number of Early HCMV Proteins
3.3. Impact of p62 on the Steady-State Levels of Viral Proteins
3.4. The Lack of p62 Has No Impact on the Induction of Autophagy in HCMV-Infected Cells
3.5. The Phosphorylation Status of p62 Is Altered Only at Serine 272 in HCMV-Infected Cells
3.6. Generation of Recombinant HCMV Strains, Expressing Different p62 Versions
3.7. p62 Specifically Interacts with Nuclear Pore Proteins in HCMV-Infected Cells
3.8. S272 Phosphorylation of p62 Shifts the Localization of the Receptor toward the Cytoplasm
3.9. Lack of S272 Phosphorylation of p62 Leads to Its Proteasomal Degradation in Infected Cells
3.10. Lack of p62-S272 Phosphorylation Leads to Enhanced HCMV Protein Expression, Genome Replication, and Progeny Release
4. Discussion
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Paludan, S.R.; Pradeu, T.; Masters, S.L.; Mogensen, T.H. Constitutive immune mechanisms: Mediators of host defence and immune regulation. Nat. Rev. Immunol. 2021, 21, 137–150. [Google Scholar] [CrossRef] [PubMed]
- Schilling, E.M.; Scherer, M.; Stamminger, T. Intrinsic Immune Mechanisms Restricting Human Cytomegalovirus Replication. Viruses 2021, 13, 179. [Google Scholar] [CrossRef] [PubMed]
- Chun, Y.; Kim, J. Autophagy: An Essential Degradation Program for Cellular Homeostasis and Life. Cells 2018, 7, 278. [Google Scholar] [CrossRef]
- Parzych, K.R.; Klionsky, D.J. An overview of autophagy: Morphology, mechanism, and regulation. Antioxid. Redox Signal 2014, 20, 460–473. [Google Scholar] [CrossRef]
- Proulx, J.; Borgmann, K.; Park, I.W. Role of Virally-Encoded Deubiquitinating Enzymes in Regulation of the Virus Life Cycle. Int. J. Mol. Sci. 2021, 22, 4438. [Google Scholar] [CrossRef]
- Dong, X.; Levine, B. Autophagy and viruses: Adversaries or allies? J. Innate Immun. 2013, 5, 480–493. [Google Scholar] [CrossRef] [PubMed]
- Paul, P.; Munz, C. Autophagy and Mammalian Viruses: Roles in Immune Response, Viral Replication, and Beyond. Adv. Virus Res. 2016, 95, 149–195. [Google Scholar] [CrossRef]
- Richetta, C.; Faure, M. Autophagy in antiviral innate immunity. Cell Microbiol. 2013, 15, 368–376. [Google Scholar] [CrossRef]
- Vargas, J.N.S.; Hamasaki, M.; Kawabata, T.; Youle, R.J.; Yoshimori, T. The mechanisms and roles of selective autophagy in mammals. Nat. Rev. Mol. Cell Biol. 2023, 24, 167–185. [Google Scholar] [CrossRef]
- Gubas, A.; Dikic, I. A guide to the regulation of selective autophagy receptors. FEBS J. 2022, 289, 75–89. [Google Scholar] [CrossRef]
- Chu, Y.; Yang, X. SUMO E3 ligase activity of TRIM proteins. Oncogene 2011, 30, 1108–1116. [Google Scholar] [CrossRef] [PubMed]
- Rogov, V.; Dotsch, V.; Johansen, T.; Kirkin, V. Interactions between autophagy receptors and ubiquitin-like proteins form the molecular basis for selective autophagy. Mol. Cell 2014, 53, 167–178. [Google Scholar] [CrossRef] [PubMed]
- Sun, D.; Wu, R.; Zheng, J.; Li, P.; Yu, L. Polyubiquitin chain-induced p62 phase separation drives autophagic cargo segregation. Cell Res. 2018, 28, 405–415. [Google Scholar] [CrossRef]
- Khaminets, A.; Behl, C.; Dikic, I. Ubiquitin-Dependent And Independent Signals In Selective Autophagy. Trends Cell Biol. 2016, 26, 6–16. [Google Scholar] [CrossRef] [PubMed]
- Kirkin, V.; Rogov, V.V. A Diversity of Selective Autophagy Receptors Determines the Specificity of the Autophagy Pathway. Mol. Cell 2019, 76, 268–285. [Google Scholar] [CrossRef]
- Chaumorcel, M.; Souquere, S.; Pierron, G.; Codogno, P.; Esclatine, A. Human cytomegalovirus controls a new autophagy-dependent cellular antiviral defense mechanism. Autophagy 2008, 4, 46–53. [Google Scholar] [CrossRef]
- Chaumorcel, M.; Lussignol, M.; Mouna, L.; Cavignac, Y.; Fahie, K.; Cotte-Laffitte, J.; Geballe, A.; Brune, W.; Beau, I.; Codogno, P.; et al. The human cytomegalovirus protein TRS1 inhibits autophagy via its interaction with Beclin 1. J. Virol. 2012, 86, 2571–2584. [Google Scholar] [CrossRef]
- Belzile, J.P.; Sabalza, M.; Craig, M.; Clark, E.; Morello, C.S.; Spector, D.H. Trehalose, an mTOR-Independent Inducer of Autophagy, Inhibits Human Cytomegalovirus Infection in Multiple Cell Types. J. Virol. 2015, 90, 1259–1277. [Google Scholar] [CrossRef]
- Clark, A.E.; Sabalza, M.; Gordts, P.; Spector, D.H. Human Cytomegalovirus Replication Is Inhibited by the Autophagy-Inducing Compounds Trehalose and SMER28 through Distinctively Different Mechanisms. J. Virol. 2018, 92, e02015-17. [Google Scholar] [CrossRef]
- Zimmermann, C.; Krämer, N.; Krauter, S.; Strand, D.; Sehn, E.; Wolfrum, U.; Freiwald, A.; Butter, F.; Plachter, B. Autophagy interferes with human cytomegalovirus genome replication, morphogenesis, and progeny release. Autophagy 2020, 13, 779–795. [Google Scholar] [CrossRef]
- König, P.; Svrlanska, A.; Read, C.; Feichtinger, S.; Stamminger, T. The Autophagy-Initiating Protein Kinase ULK1 Phosphorylates Human Cytomegalovirus Tegument Protein pp28 and Regulates Efficient Virus Release. J. Virol. 2021, 5, e02346-20. [Google Scholar] [CrossRef] [PubMed]
- Taisne, C.; Lussignol, M.; Hernandez, E.; Moris, A.; Mouna, L.; Esclatine, A. Human cytomegalovirus hijacks the autophagic machinery and LC3 homologs in order to optimize cytoplasmic envelopment of mature infectious particles. Sci. Rep. 2019, 9, 4560. [Google Scholar] [CrossRef] [PubMed]
- Stolz, A.; Ernst, A.; Dikic, I. Cargo recognition and trafficking in selective autophagy. Nat. Cell Biol. 2014, 16, 495–501. [Google Scholar] [CrossRef]
- Galluzzi, L.; Baehrecke, E.H.; Ballabio, A.; Boya, P.; Bravo-San Pedro, J.M.; Cecconi, F.; Choi, A.M.; Chu, C.T.; Codogno, P.; Colombo, M.I.; et al. Molecular definitions of autophagy and related processes. EMBO J. 2017, 36, 1811–1836. [Google Scholar] [CrossRef]
- Pankiv, S.; Clausen, T.H.; Lamark, T.; Brech, A.; Bruun, J.A.; Outzen, H.; Overvatn, A.; Bjorkoy, G.; Johansen, T. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J. Biol. Chem. 2007, 282, 24131–24145. [Google Scholar] [CrossRef]
- Bjorkoy, G.; Lamark, T.; Brech, A.; Outzen, H.; Perander, M.; Overvatn, A.; Stenmark, H.; Johansen, T. p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death. J. Cell Biol. 2005, 171, 603–614. [Google Scholar] [CrossRef] [PubMed]
- Mohamud, Y.; Qu, J.; Xue, Y.C.; Liu, H.; Deng, H.; Luo, H. CALCOCO2/NDP52 and SQSTM1/p62 differentially regulate coxsackievirus B3 propagation. Cell Death Differ. 2019, 26, 1062–1076. [Google Scholar] [CrossRef]
- Shi, J.; Fung, G.; Piesik, P.; Zhang, J.; Luo, H. Dominant-negative function of the C-terminal fragments of NBR1 and SQSTM1 generated during enteroviral infection. Cell Death Differ. 2014, 21, 1432–1441. [Google Scholar] [CrossRef]
- Sagnier, S.; Daussy, C.F.; Borel, S.; Robert-Hebmann, V.; Faure, M.; Blanchet, F.P.; Beaumelle, B.; Biard-Piechaczyk, M.; Espert, L. Autophagy restricts HIV-1 infection by selectively degrading Tat in CD4+ T lymphocytes. J. Virol. 2015, 89, 615–625. [Google Scholar] [CrossRef]
- Ghosh, A.K.; Su, Y.P.; Forman, M.; Keyes, R.F.; Smith, B.C.; Hu, X.; Ferrer, M.; Arav-Boger, R. Harnessing the Noncanonical Keap1-Nrf2 Pathway for Human Cytomegalovirus Control. J. Virol. 2023, 97, e0016023. [Google Scholar] [CrossRef]
- Schweiger, L.; Lelieveld-Fast, L.A.; Mikulicic, S.; Strunk, J.; Freitag, K.; Tenzer, S.; Clement, A.M.; Florin, L. HPV16 Induces Formation of Virus-p62-PML Hybrid Bodies to Enable Infection. Viruses 2022, 14, 1478. [Google Scholar] [CrossRef] [PubMed]
- Hennig, P.; Fenini, G.; Di Filippo, M.; Karakaya, T.; Beer, H.D. The Pathways Underlying the Multiple Roles of p62 in Inflammation and Cancer. Biomedicines 2021, 9, 707. [Google Scholar] [CrossRef] [PubMed]
- Moscat, J.; Karin, M.; Diaz-Meco, M.T. p62 in Cancer: Signaling Adaptor Beyond Autophagy. Cell 2016, 167, 606–609. [Google Scholar] [CrossRef] [PubMed]
- Ning, S.; Wang, L. The Multifunctional Protein p62 and Its Mechanistic Roles in Cancers. Curr. Cancer Drug Targets 2019, 19, 468–478. [Google Scholar] [CrossRef] [PubMed]
- Babu, J.R.; Geetha, T.; Wooten, M.W. Sequestosome 1/p62 shuttles polyubiquitinated tau for proteasomal degradation. J. Neurochem. 2005, 94, 192–203. [Google Scholar] [CrossRef]
- Seibenhener, M.L.; Babu, J.R.; Geetha, T.; Wong, H.C.; Krishna, N.R.; Wooten, M.W. Sequestosome 1/p62 is a polyubiquitin chain binding protein involved in ubiquitin proteasome degradation. Mol. Cell Biol. 2004, 24, 8055–8068. [Google Scholar] [CrossRef]
- Lobb, I.T.; Morin, P.; Martin, K.; Thoms, H.C.; Wills, J.C.; Lleshi, X.; Olsen, K.C.F.; Duncan, R.R.; Stark, L.A. A Role for the Autophagic Receptor, SQSTM1/p62, in Trafficking NF-kappaB/RelA to Nucleolar Aggresomes. Mol. Cancer Res. 2021, 19, 274–287. [Google Scholar] [CrossRef]
- Pankiv, S.; Alemu, E.A.; Brech, A.; Bruun, J.A.; Lamark, T.; Overvatn, A.; Bjorkoy, G.; Johansen, T. FYCO1 is a Rab7 effector that binds to LC3 and PI3P to mediate microtubule plus end-directed vesicle transport. J. Cell Biol. 2010, 188, 253–269. [Google Scholar] [CrossRef]
- Haeussler, M.; Schonig, K.; Eckert, H.; Eschstruth, A.; Mianne, J.; Renaud, J.B.; Schneider-Maunoury, S.; Shkumatava, A.; Teboul, L.; Kent, J.; et al. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol. 2016, 17, 148. [Google Scholar] [CrossRef]
- Concordet, J.P.; Haeussler, M. CRISPOR: Intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Res. 2018, 46, W242–W245. [Google Scholar] [CrossRef]
- Gonzalez-Perez, A.C.; Stempel, M.; Wyler, E.; Urban, C.; Piras, A.; Hennig, T.; Ganskih, S.; Wei, Y.; Heim, A.; Landthaler, M.; et al. The Zinc Finger Antiviral Protein ZAP Restricts Human Cytomegalovirus and Selectively Binds and Destabilizes Viral UL4/UL5 Transcripts. mBio 2021, 12. [Google Scholar] [CrossRef] [PubMed]
- Schilling, E.M.; Scherer, M.; Reuter, N.; Schweininger, J.; Muller, Y.A.; Stamminger, T. The Human Cytomegalovirus IE1 Protein Antagonizes PML Nuclear Body-Mediated Intrinsic Immunity via the Inhibition of PML De Novo SUMOylation. J. Virol. 2017, 91. [Google Scholar] [CrossRef] [PubMed]
- Yu, D.; Smith, G.A.; Enquist, L.W.; Shenk, T. Construction of a self-excisable bacterial artificial chromosome containing the human cytomegalovirus genome and mutagenesis of the diploid TRL/IRL13 gene. J. Virol. 2002, 76, 2316–2328. [Google Scholar] [CrossRef]
- Sinzger, C.; Hahn, G.; Digel, M.; Katona, R.; Sampaio, K.L.; Messerle, M.; Hengel, H.; Koszinowski, U.; Brune, W.; Adler, B. Cloning and sequencing of a highly productive, endotheliotropic virus strain derived from human cytomegalovirus TB40/E. J. Gen. Virol. 2008, 89, 359–368. [Google Scholar] [CrossRef] [PubMed]
- Warming, S.; Costantino, N.D.L.C.; Jenkins, N.A.; Copeland, N.G. Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res. 2005, 33, e36. [Google Scholar] [CrossRef]
- Arlt, H.; Lang, D.; Gebert, S.; Stamminger, T. Identification of binding sites for the 86-kilodalton IE2 protein of human cytomegalovirus within an IE2-responsive viral early promoter. J. Virol. 1994, 68, 4117–4125. [Google Scholar] [CrossRef]
- Cherrington, J.M.; Khoury, E.L.; Mocarski, E.S. Human cytomegalovirus ie2 negatively regulates alpha gene expression via a short target sequence near the transcription start site. J. Virol. 1991, 65, 887–896. [Google Scholar] [CrossRef]
- Zimmermann, C.; Büscher, N.; Krauter, S.; Kramer, N.; Wolfrum, U.; Sehn, E.; Tenzer, S.; Plachter, B. The Abundant Tegument Protein pUL25 of Human Cytomegalovirus Prevents Proteasomal Degradation of pUL26 and Supports Its Suppression of ISGylation. J. Virol. 2018, 92. [Google Scholar] [CrossRef]
- Krämer, N.; Schütz, M.; Mato, U.G.; Herhaus, L.; Marschall, M.; Zimmermann, C. Recombinant Human Cytomegalovirus Expressing an Analog-Sensitive Kinase pUL97 as Novel Tool for Functional Analyses. Viruses 2022, 14, 2285. [Google Scholar] [CrossRef]
- Fleckenstein, B.; Muller, I.; Collins, J. Cloning of the complete human cytomegalovirus genome in cosmids. Gene 1982, 18, 39–46. [Google Scholar] [CrossRef]
- Plachter, B.; Britt, W.J.; Vornhagen, R.; Stamminger, T.; Jahn, G. Analysis of proteins encoded by IE-regions 1 and 2 of human cytomegalovirus using monoclonal antibodies generated against recombinant antigens. Virology 1993, 193, 642–652. [Google Scholar] [CrossRef] [PubMed]
- Herhaus, L.; Bhaskara, R.M.; Lystad, A.H.; Gestal-Mato, U.; Covarrubias-Pinto, A.; Bonn, F.; Simonsen, A.; Hummer, G.; Dikic, I. TBK1-mediated phosphorylation of LC3C and GABARAP-L2 controls autophagosome shedding by ATG4 protease. EMBO Rep. 2020, 21, e48317. [Google Scholar] [CrossRef] [PubMed]
- Afifi, A. Viral Interaction of the Human Cytomegalovirus with Components of the Autophagy Machinery. Master of Science. Master’s Thesis, Johannes Gutenberg Universität Mainz, Mainz, Germany, 2021. [Google Scholar]
- Chen, M.; Meng, Q.; Qin, Y.; Liang, P.; Tan, P.; He, L.; Zhou, Y.; Chen, Y.; Huang, J.; Wang, R.F.; et al. TRIM14 Inhibits cGAS Degradation Mediated by Selective Autophagy Receptor p62 to Promote Innate Immune Responses. Mol. Cell 2016, 64, 105–119. [Google Scholar] [CrossRef]
- Prabakaran, T.; Bodda, C.; Krapp, C.; Zhang, B.C.; Christensen, M.H.; Sun, C.; Reinert, L.; Cai, Y.; Jensen, S.B.; Skouboe, M.K.; et al. Attenuation of cGAS-STING signaling is mediated by a p62/SQSTM1-dependent autophagy pathway activated by TBK1. EMBO J. 2018, 37, e97858. [Google Scholar] [CrossRef]
- Szklarczyk, D.; Kirsch, R.; Koutrouli, M.; Nastou, K.; Mehryary, F.; Hachilif, R.; Gable, A.L.; Fang, T.; Doncheva, N.T.; Pyysalo, S.; et al. The STRING database in 2023: Protein-protein association networks and functional enrichment analyses for any sequenced genome of interest. Nucleic Acids Res. 2023, 51, D638–D646. [Google Scholar] [CrossRef] [PubMed]
- Pankiv, S.; Lamark, T.; Bruun, J.A.; Overvatn, A.; Bjorkoy, G.; Johansen, T. Nucleocytoplasmic shuttling of p62/SQSTM1 and its role in recruitment of nuclear polyubiquitinated proteins to promyelocytic leukemia bodies. J. Biol. Chem. 2010, 285, 5941–5953. [Google Scholar] [CrossRef]
- Thakar, K.; Karaca, S.; Port, S.A.; Urlaub, H.; Kehlenbach, R.H. Identification of CRM1-dependent Nuclear Export Cargos Using Quantitative Mass Spectrometry. Mol. Cell Proteom. 2013, 12, 664–678. [Google Scholar] [CrossRef]
- Funakoshi, T.; Clever, M.; Watanabe, A.; Imamoto, N. Localization of Pom121 to the inner nuclear membrane is required for an early step of interphase nuclear pore complex assembly. Mol. Biol. Cell 2011, 22, 1058–1069. [Google Scholar] [CrossRef] [PubMed]
- Doucet, C.M.; Talamas, J.A.; Hetzer, M.W. Cell cycle-dependent differences in nuclear pore complex assembly in metazoa. Cell 2010, 141, 1030–1041. [Google Scholar] [CrossRef]
- Chen, T.; Tu, S.; Ding, L.; Jin, M.; Chen, H.; Zhou, H. The role of autophagy in viral infections. J. Biomed. Sci. 2023, 30, 5. [Google Scholar] [CrossRef]
- Jassey, A.; Jackson, W.T. Viruses and autophagy: Bend, but don’t break. Nat. Rev. Microbiol. 2024, 22, 309–321. [Google Scholar] [CrossRef] [PubMed]
- Liu, W.J.; Ye, L.; Huang, W.F.; Guo, L.J.; Xu, Z.G.; Wu, H.L.; Yang, C.; Liu, H.F. p62 links the autophagy pathway and the ubiqutin-proteasome system upon ubiquitinated protein degradation. Cell Mol. Biol. Lett. 2016, 21, 29. [Google Scholar] [CrossRef]
- Kumar, A.V.; Mills, J.; Lapierre, L.R. Selective Autophagy Receptor p62/SQSTM1, a Pivotal Player in Stress and Aging. Front. Cell Dev. Biol. 2022, 10, 793328. [Google Scholar] [CrossRef]
- Fan, L.; Yin, S.; Zhang, E.; Hu, H. Role of p62 in the regulation of cell death induction. Apoptosis 2018, 23, 187–193. [Google Scholar] [CrossRef] [PubMed]
- Orvedahl, A.; MacPherson, S.; Sumpter, R., Jr.; Talloczy, Z.; Zou, Z.; Levine, B. Autophagy protects against Sindbis virus infection of the central nervous system. Cell Host Microbe 2010, 7, 115–127. [Google Scholar] [CrossRef] [PubMed]
- Sanz, L.; Sanchez, P.; Lallena, M.J.; Diaz-Meco, M.T.; Moscat, J. The interaction of p62 with RIP links the atypical PKCs to NF-kappaB activation. EMBO J. 1999, 18, 3044–3053. [Google Scholar] [CrossRef] [PubMed]
- Matsumoto, G.; Wada, K.; Okuno, M.; Kurosawa, M.; Nukina, N. Serine 403 phosphorylation of p62/SQSTM1 regulates selective autophagic clearance of ubiquitinated proteins. Mol. Cell 2011, 44, 279–289. [Google Scholar] [CrossRef]
- Lim, J.; Lachenmayer, M.L.; Wu, S.; Liu, W.; Kundu, M.; Wang, R.; Komatsu, M.; Oh, Y.J.; Zhao, Y.; Yue, Z. Proteotoxic stress induces phosphorylation of p62/SQSTM1 by ULK1 to regulate selective autophagic clearance of protein aggregates. PLoS Genet. 2015, 11, e1004987. [Google Scholar] [CrossRef]
Protein | Site | PEP Value | Localization Probability |
---|---|---|---|
p62 | S262 | 6.58 × 10−180 | 0.999972 |
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Krämer, N.; Mato, U.G.; Krauter, S.; Büscher, N.; Afifi, A.; Herhaus, L.; Florin, L.; Plachter, B.; Zimmermann, C. The Autophagy Receptor SQSTM1/p62 Is a Restriction Factor of HCMV Infection. Viruses 2024, 16, 1440. https://doi.org/10.3390/v16091440
Krämer N, Mato UG, Krauter S, Büscher N, Afifi A, Herhaus L, Florin L, Plachter B, Zimmermann C. The Autophagy Receptor SQSTM1/p62 Is a Restriction Factor of HCMV Infection. Viruses. 2024; 16(9):1440. https://doi.org/10.3390/v16091440
Chicago/Turabian StyleKrämer, Nadine, Uxía Gestal Mato, Steffi Krauter, Nicole Büscher, Ahmad Afifi, Lina Herhaus, Luise Florin, Bodo Plachter, and Christine Zimmermann. 2024. "The Autophagy Receptor SQSTM1/p62 Is a Restriction Factor of HCMV Infection" Viruses 16, no. 9: 1440. https://doi.org/10.3390/v16091440