Discovery of a Novel Antiviral Effect of the Restriction Factor SPOC1 against Human Cytomegalovirus
<p>Increasing HCMV doses are able to antagonize SPOC1-mediated repression of IE1 and IE2 expression. (<b>A</b>) The 24 hpi lysates of HCMV-infected control fibroblasts (HFF/Ctrl) and fibroblasts expressing SPOC1 (HFF/SPOC1) infected with HCMV TB40/E at MOIs of 0.01 (lanes 1 and 2), 1 (lanes 3 and 4) and 3 (lanes 5 and 6) were investigated by Western blotting. Expression levels of viral immediate-early proteins IE1 and IE2, β-actin and SPOC1 were analyzed. (<b>B</b>) Quantification of IE1 and IE2 signal intensities normalized to β-actin levels in HFF/SPOC1 relative to normalized IE1 and IE2 levels in HFF/Ctrl of three independent experiments. Statistical analysis was performed using Student’s <span class="html-italic">t</span>-test (one sample, two-tailed); **** <span class="html-italic">p</span> < 0.0001, * <span class="html-italic">p</span> < 0.05, <span class="html-italic">n.s.</span> = not significant.</p> "> Figure 2
<p>SPOC1 expression negatively affects HCMV DNA replication and viral particle release. HFF/Ctrl and HFF/SPOC1 were infected in triplicate with HCMV strain TB40/E at an MOI of 1 or 3. (<b>A</b>) At 96 hpi, supernatants were analyzed for viral genome equivalents via qPCR. (<b>B</b>) At 96 hpi, intracellular DNA was isolated and HCMV genome equivalents were quantified via qPCR and normalized to albumin copy numbers. One out of three independent experiments is shown. Statistical analysis was performed using Student’s <span class="html-italic">t</span>-test (unpaired, two-tailed); *** <span class="html-italic">p</span> < 0.001, **** <span class="html-italic">p</span> < 0.0001. (<b>C</b>) Experimental set-up: Doxycycline (Dox)-inducible HFF/SPOC1 cells were either treated with Dox 24 h prior to or 24 h post infection with AD169 at MOI 0.1. (<b>D</b>) Dox-inducible HFF/SPOC1 was infected with AD169, at MOI 0.1, in triplicate. The infected cells were either left untreated or treated with Dox 24 h prior to or 24 h post infection (<b>C</b>). At 96 hpi, the supernatant was harvested and analyzed for viral genome equivalents via qPCR. One out of two experiments is shown. Statistical analysis was performed utilizing the one sample <span class="html-italic">t</span>-test; * <span class="html-italic">p</span> < 0.05.</p> "> Figure 3
<p>SPOC1 leads to lower expression levels of viral early and late proteins. (<b>A</b>) Lysates of mock-infected (m) or TB40/E- (MOI of 3) or AD169-infected (MOI of 3 and 1) HFF/Ctrl and HFF/SPOC1 cells were analyzed at 24 to 72 hpi by separation on a 10 % polyacrylamide gel followed by Western blot detection of indicated proteins. Expression kinetics of viral immediate-early protein IE1, viral early protein pUL44 and viral late proteins pp28 and MCP were investigated. Asterisks (*) highlight the protein bands that are attenuated upon SPOC1 expression. (<b>B</b>) Quantification of IE1 levels in HFF/SPOC1 normalized to β-actin are depicted as fold change of the normalized IE1 level of HFF/Ctrl (indicated by dashed line at y = 1). (<b>C</b>) Quantification of early and late viral proteins of HFF/SPOC1 normalized to β-actin are depicted as fold change of the regarding normalized protein levels of HFF/Ctrl (indicated by dashed line at y = 1).</p> "> Figure 4
<p>Impaired transcription of viral early and late genes in HFF/SPOC1 cells at late times in the replicative cycle. HFF/Ctrl and HFF/SPOC1 cells were infected with AD169 at an MOI of 1. (<b>A</b>) The 24 hpi IE1 and IE2 protein levels were analyzed via SDS PAGE and Western blotting. The relative intensity values were normalized to β-actin. Quantification of three independent experiments is shown. Statistical analysis was performed using Student’s <span class="html-italic">t</span>-test (one sample, two-tailed); <span class="html-italic">n.s.</span> = not significant. (<b>B</b>) IE1, IE2 and US3 transcript levels normalized to GAPDH were evaluated at 8 hpi using qPCR. Shown are the mean values of triplicates of one out of two experiments. Statistical analysis was performed using Student’s <span class="html-italic">t</span>-test (one sample, two-tailed). (<b>C</b>) At 48 and 72 hpi, total cellular RNA was isolated, cDNA was synthesized and viral mRNA levels of two immediate-early, early and late genes were quantified via qPCR, respectively. Shown are the mean values of triplicates of one out of three experiments. Statistical analysis was performed with Student’s <span class="html-italic">t</span>-test (unpaired, two-tailed); ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001, **** <span class="html-italic">p</span> < 0.0001.</p> "> Figure 5
<p>SPOC1 localization during the time course of HCMV infection. (<b>A</b>) HFF/mCherry-SPOC1 cells were infected with AD169 at MOI 1 and fixed at the indicated time points. An antibody against pUL44 was used in combination with the secondary antibody Alexa-488. DAPI staining was used to visualize the nucleus. (<b>B</b>) At 72 hpi, AD169-infected HFF/SPOC1 (MOI of 1) was treated with F-Ara-EdU prior to fixation at 96 hpi. Samples were stained for SPOC1 (secondary antibody: Alexa-488) as well as with an antibody against pUL44 in combination with the Alexa-647 antibody. Click chemistry was performed to visualize viral DNA (modified from [<a href="#B15-viruses-16-00363" class="html-bibr">15</a>]).</p> "> Figure 6
<p>SPOC1 co-localizes with PRC2 components close to viral DNA. HFF/SPOC1 was infected with AD169 at an MOI of 1. At 96 hpi, the cells were fixed and treated with antibodies directed against SPOC1, pUL44 and either (<b>A</b>) EZH2 or (<b>B</b>) SUZ12. As secondary antibodies, Alexa-488 (SPOC1) and a combination of either mouse or rabbit Alexa-555 and mouse or rabbit Alexa-647 were used. DAPI signals visualize the nucleus (modified from [<a href="#B15-viruses-16-00363" class="html-bibr">15</a>]). (<b>C</b>,<b>D</b>) At 72 hpi, EdC was added to SPOC1-expressing cells prior to fixation at 96 hpi. The same antibodies against EZH2 (<b>C</b>) or SUZ12 (<b>D</b>) were used as for (<b>A</b>) and (<b>B</b>); viral DNA was visualized by click chemistry. Nuclei were visualized by DAPI staining. Merge images were created from the two images above.</p> "> Figure 7
<p>Interaction between SPOC1 deletion mutants and EZH2. (<b>A</b>) Schematic representation of the generated FLAG-SPOC1 deletion mutants with indicated amino acid sequence. (<b>B</b>) HEK293T cells were co-transfected with an empty control plasmid or FLAG-SPOC1 deletion mutants together with an EZH2-expressing plasmid. FLAG-SPOC1 was precipitated, and lysate controls as well as immuno-precipitated (IP) samples were analyzed via Western blotting. SPOC1 was visualized using an anti-FLAG antibody. CoIP = Co-Immunoprecipitaton.</p> "> Figure 8
<p>Co-localization of SPOC1 with RNA pol II S5P. HFF/SPOC1 was infected with AD169 (MOI 1) and fixed at 96 hpi. Indirect immunostaining was performed using antibodies against SPOC1 and (<b>A</b>) 4H8 antibody detecting specifically Ser5 phosphorylated RNA pol II (modified from [<a href="#B15-viruses-16-00363" class="html-bibr">15</a>]) or (<b>B</b>) 8WG16 antibody to mark general RNA Pol II localization. DAPI was used to stain cell nuclei.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plasmids and Cloning
2.2. Cells and Transfection
2.3. Infection and Virus Stock Titration
2.4. Western Blotting and Quantification
2.5. Replication and Release Assays
2.6. RNA Isolation and Quantitative SYBR Green Reverse Transcription-PCR (qRT-PCR)
2.7. Indirect Immunofluorescence
2.8. Labelling of Viral DNA
2.9. Co-Immunoprecipitation
3. Results
3.1. HCMV Overcomes SPOC1-Mediated Immediate-Early Repression with Increasing Viral Doses
3.2. HCMV DNA Replication and Viral Particle Release Are Strongly Restricted upon SPOC1 Expression
3.3. SPOC1 Leads to Reduced Early and Late Viral Protein Expression
3.4. SPOC1 Blocks Transcription of Viral Early and Late Genes
3.5. SPOC1 Localizes in Close Proximity to Viral DNA during Later Time Points of the Replicative Cycle
3.6. SPOC1 Interacts and co-Localizes with PRC2 Adjacent to Viral DNA
3.7. Co-Localization of SPOC1 and RNA Pol II during Late Stages of Infection
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Schilling, E.M.; Scherer, M.; Stamminger, T. Intrinsic Immune Mechanisms Restricting Human Cytomegalovirus Replication. Viruses 2021, 13, 179. [Google Scholar] [CrossRef] [PubMed]
- Schreiner, S.; Kinkley, S.; Bürck, C.; Mund, A.; Wimmer, P.; Schubert, T.; Groitl, P.; Will, H.; Dobner, T. SPOC1-mediated antiviral host cell response is antagonized early in human adenovirus type 5 infection. PLoS Pathog. 2013, 9, e1003775. [Google Scholar] [CrossRef] [PubMed]
- Hofmann, S.; Dehn, S.; Businger, R.; Bolduan, S.; Schneider, M.; Debyser, Z.; Brack-Werner, R.; Schindler, M. Dual role of the chromatin-binding factor PHF13 in the pre- and post-integration phases of HIV-1 replication. Open Biol. 2017, 7, 170115. [Google Scholar] [CrossRef] [PubMed]
- Reichel, A.; Stilp, A.C.; Scherer, M.; Reuter, N.; Lukassen, S.; Kasmapour, B.; Schreiner, S.; Cicin-Sain, L.; Winterpacht, A.; Stamminger, T. Chromatin-Remodeling Factor SPOC1 Acts as a Cellular Restriction Factor against Human Cytomegalovirus by Repressing the Major Immediate Early Promoter. J. Virol. 2018, 92. [Google Scholar] [CrossRef] [PubMed]
- Mohrmann, G.; Hengstler, J.G.; Hofmann, T.G.; Endele, S.U.; Lee, B.; Stelzer, C.; Zabel, B.; Brieger, J.; Hasenclever, D.; Tanner, B.; et al. SPOC1, a novel PHD-finger protein: Association with residual disease and survival in ovarian cancer. Int. J. Cancer 2005, 116, 547–554. [Google Scholar] [CrossRef] [PubMed]
- Bördlein, A.; Scherthan, H.; Nelkenbrecher, C.; Molter, T.; Bösl, M.R.; Dippold, C.; Birke, K.; Kinkley, S.; Staege, H.; Will, H.; et al. SPOC1 (PHF13) is required for spermatogonial stem cell differentiation and sustained spermatogenesis. J. Cell Sci. 2011, 124, 3137–3148. [Google Scholar] [CrossRef]
- Mund, A.; Schubert, T.; Staege, H.; Kinkley, S.; Reumann, K.; Kriegs, M.; Fritsch, L.; Battisti, V.; Ait-Si-Ali, S.; Hoffbeck, A.S.; et al. SPOC1 modulates DNA repair by regulating key determinants of chromatin compaction and DNA damage response. Nucleic Acids Res. 2012, 40, 11363–11379. [Google Scholar] [CrossRef]
- Kinkley, S.; Staege, H.; Mohrmann, G.; Rohaly, G.; Schaub, T.; Kremmer, E.; Winterpacht, A.; Will, H. SPOC1: A novel PHD-containing protein modulating chromatin structure and mitotic chromosome condensation. J. Cell Sci. 2009, 122, 2946–2956. [Google Scholar] [CrossRef]
- Fuchs, A.; Torroba, M.; Kinkley, S. PHF13: A new player involved in RNA polymerase II transcriptional regulation and co-transcriptional splicing. Transcription 2017, 8, 106–112. [Google Scholar] [CrossRef]
- Chung, H.R.; Xu, C.; Fuchs, A.; Mund, A.; Lange, M.; Staege, H.; Schubert, T.; Bian, C.; Dunkel, I.; Eberharter, A.; et al. PHF13 is a molecular reader and transcriptional co-regulator of H3K4me2/3. eLife 2016, 5, e10607. [Google Scholar] [CrossRef]
- Griffiths, P.D. Burden of disease associated with human cytomegalovirus and prospects for elimination by universal immunisation. Lancet. Infect. Dis. 2012, 12, 790–798. [Google Scholar] [CrossRef] [PubMed]
- Landolfo, S.; Gariglio, M.; Gribaudo, G.; Lembo, D. The human cytomegalovirus. Pharmacol. Ther. 2003, 98, 269–297. [Google Scholar] [CrossRef] [PubMed]
- Nelson, J.A.; Gnann, J.W., Jr.; Ghazal, P. Regulation and tissue-specific expression of human cytomegalovirus. Curr. Top. Microbiol. Immunol. 1990, 154, 75–100. [Google Scholar] [CrossRef] [PubMed]
- Hofmann, H.; Flöss, S.; Stamminger, T. Covalent modification of the transactivator protein IE2-p86 of human cytomegalovirus by conjugation to the ubiquitin-homologous proteins SUMO-1 and hSMT3b. J. Virol. 2000, 74, 2510–2524. [Google Scholar] [CrossRef] [PubMed]
- Reichel, A. Role of the Chromatin Remodeling Factor SPOC1 for Human Cytomegalovirus Replication. Ph.D. Thesis, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany, 2018. [Google Scholar]
- 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]
- Hobom, U.; Brune, W.; Messerle, M.; Hahn, G.; Koszinowski, U.H. Fast screening procedures for random transposon libraries of cloned herpesvirus genomes: Mutational analysis of human cytomegalovirus envelope glycoprotein genes. J. Virol. 2000, 74, 7720–7729. [Google Scholar] [CrossRef] [PubMed]
- Scherer, M.; Otto, V.; Stump, J.D.; Klingl, S.; Müller, R.; Reuter, N.; Muller, Y.A.; Sticht, H.; Stamminger, T. Characterization of Recombinant Human Cytomegaloviruses Encoding IE1 Mutants L174P and 1-382 Reveals that Viral Targeting of PML Bodies Perturbs both Intrinsic and Innate Immune Responses. J. Virol. 2016, 90, 1190–1205. [Google Scholar] [CrossRef]
- Andreoni, M.; Faircloth, M.; Vugler, L.; Britt, W.J. A rapid microneutralization assay for the measurement of neutralizing antibody reactive with human cytomegalovirus. J. Virol. Methods 1989, 23, 157–167. [Google Scholar] [CrossRef]
- Bryson Waldo, F.; Tomana, M.; Britt, W.; Julian, B.; Mestecky, J. Non-Specific Mesangial Staining with Antibodies against Cytomegalovirus in Immunoglobulin-A Nephropathy. Lancet 1989, 333, 129–131. [Google Scholar] [CrossRef]
- Sanchez, V.; Sztul, E.; Britt, W.J. Human cytomegalovirus pp28 (UL99) localizes to a cytoplasmic compartment which overlaps the endoplasmic reticulum-golgi-intermediate compartment. J. Virol. 2000, 74, 3842–3851. [Google Scholar] [CrossRef]
- Schilling, E.M.; Scherer, M.; Rothemund, F.; Stamminger, T. Functional regulation of the structure-specific endonuclease FEN1 by the human cytomegalovirus protein IE1 suggests a role for the re-initiation of stalled viral replication forks. PLoS Pathog. 2021, 17, e1009460. [Google Scholar] [CrossRef] [PubMed]
- Stilp, A.C.; Scherer, M.; König, P.; Fürstberger, A.; Kestler, H.A.; Stamminger, T. The chromatin remodeling protein ATRX positively regulates IRF3-dependent type I interferon production and interferon-induced gene expression. PLoS Pathog. 2022, 18, e1010748. [Google Scholar] [CrossRef]
- White, E.A.; Clark, C.L.; Sanchez, V.; Spector, D.H. Small internal deletions in the human cytomegalovirus IE2 gene result in nonviable recombinant viruses with differential defects in viral gene expression. J. Virol. 2004, 78, 1817–1830. [Google Scholar] [CrossRef]
- Ravichandran, S.; Kim, Y.E.; Bansal, V.; Ghosh, A.; Hur, J.; Subramani, V.K.; Pradhan, S.; Lee, M.K.; Kim, K.K.; Ahn, J.H. Genome-wide analysis of regulatory G-quadruplexes affecting gene expression in human cytomegalovirus. PLoS Pathog. 2018, 14, e1007334. [Google Scholar] [CrossRef]
- Rauwel, B.; Jang, S.M.; Cassano, M.; Kapopoulou, A.; Barde, I.; Trono, D. Release of human cytomegalovirus from latency by a KAP1/TRIM28 phosphorylation switch. eLife 2015, 4, e06068. [Google Scholar] [CrossRef] [PubMed]
- Fu, Y.Z.; Su, S.; Gao, Y.Q.; Wang, P.P.; Huang, Z.F.; Hu, M.M.; Luo, W.W.; Li, S.; Luo, M.H.; Wang, Y.Y.; et al. Human Cytomegalovirus Tegument Protein UL82 Inhibits STING-Mediated Signaling to Evade Antiviral Immunity. Cell Host Microbe 2017, 21, 231–243. [Google Scholar] [CrossRef]
- Rothemund, F.; Scherer, M.; Schilling, E.M.; Schweininger, J.; Muller, Y.A.; Stamminger, T. Cross-Species Analysis of Innate Immune Antagonism by Cytomegalovirus IE1 Protein. Viruses 2022, 14, 1626. [Google Scholar] [CrossRef] [PubMed]
- Neef, A.B.; Luedtke, N.W. Dynamic metabolic labeling of DNA in vivo with arabinosyl nucleosides. Proc. Natl. Acad. Sci. USA 2011, 108, 20404–20409. [Google Scholar] [CrossRef]
- Tavalai, N.; Papior, P.; Rechter, S.; Leis, M.; Stamminger, T. Evidence for a role of the cellular ND10 protein PML in mediating intrinsic immunity against human cytomegalovirus infections. J. Virol. 2006, 80, 8006–8018. [Google Scholar] [CrossRef]
- Wagenknecht, N.; Reuter, N.; Scherer, M.; Reichel, A.; Müller, R.; Stamminger, T. Contribution of the Major ND10 Proteins PML, hDaxx and Sp100 to the Regulation of Human Cytomegalovirus Latency and Lytic Replication in the Monocytic Cell Line THP-1. Viruses 2015, 7, 2884–2907. [Google Scholar] [CrossRef]
- Stamminger, T.; Puchtler, E.; Fleckenstein, B. Discordant expression of the immediate-early 1 and 2 gene regions of human cytomegalovirus at early times after infection involves posttranscriptional processing events. J. Virol. 1991, 65, 2273–2282. [Google Scholar] [CrossRef]
- Rozman, B.; Nachshon, A.; Levi Samia, R.; Lavi, M.; Schwartz, M.; Stern-Ginossar, N. Temporal dynamics of HCMV gene expression in lytic and latent infections. Cell Rep. 2022, 39, 110653. [Google Scholar] [CrossRef]
- Thrower, A.R.; Bullock, G.C.; Bissell, J.E.; Stinski, M.F. Regulation of a human cytomegalovirus immediate-early gene (US3) by a silencer-enhancer combination. J. Virol. 1996, 70, 91–100. [Google Scholar] [CrossRef]
- Cao, R.; Wang, L.; Wang, H.; Xia, L.; Erdjument-Bromage, H.; Tempst, P.; Jones, R.S.; Zhang, Y. Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science 2002, 298, 1039–1043. [Google Scholar] [CrossRef]
- Abraham, C.G.; Kulesza, C.A. Polycomb repressive complex 2 silences human cytomegalovirus transcription in quiescent infection models. J. Virol. 2013, 87, 13193–13205. [Google Scholar] [CrossRef] [PubMed]
- Svrlanska, A.; Reichel, A.; Schilling, E.M.; Scherer, M.; Stamminger, T.; Reuter, N. A Noncanonical Function of Polycomb Repressive Complexes Promotes Human Cytomegalovirus Lytic DNA Replication and Serves as a Novel Cellular Target for Antiviral Intervention. J. Virol. 2019, 93, e02143-18. [Google Scholar] [CrossRef] [PubMed]
- Tamrakar, S.; Kapasi, A.J.; Spector, D.H. Human cytomegalovirus infection induces specific hyperphosphorylation of the carboxyl-terminal domain of the large subunit of RNA polymerase II that is associated with changes in the abundance, activity, and localization of cdk9 and cdk7. J. Virol. 2005, 79, 15477–15493. [Google Scholar] [CrossRef] [PubMed]
- Ertl, P.F.; Powell, K.L. Physical and functional interaction of human cytomegalovirus DNA polymerase and its accessory protein (ICP36) expressed in insect cells. J. Virol. 1992, 66, 4126–4133. [Google Scholar] [CrossRef] [PubMed]
- Isomura, H.; Stinski, M.F.; Kudoh, A.; Nakayama, S.; Iwahori, S.; Sato, Y.; Tsurumi, T. The late promoter of the human cytomegalovirus viral DNA polymerase processivity factor has an impact on delayed early and late viral gene products but not on viral DNA synthesis. J. Virol. 2007, 81, 6197–6206. [Google Scholar] [CrossRef] [PubMed]
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
Kuderna, A.K.; Reichel, A.; Tillmanns, J.; Class, M.; Scherer, M.; Stamminger, T. Discovery of a Novel Antiviral Effect of the Restriction Factor SPOC1 against Human Cytomegalovirus. Viruses 2024, 16, 363. https://doi.org/10.3390/v16030363
Kuderna AK, Reichel A, Tillmanns J, Class M, Scherer M, Stamminger T. Discovery of a Novel Antiviral Effect of the Restriction Factor SPOC1 against Human Cytomegalovirus. Viruses. 2024; 16(3):363. https://doi.org/10.3390/v16030363
Chicago/Turabian StyleKuderna, Anna K., Anna Reichel, Julia Tillmanns, Maja Class, Myriam Scherer, and Thomas Stamminger. 2024. "Discovery of a Novel Antiviral Effect of the Restriction Factor SPOC1 against Human Cytomegalovirus" Viruses 16, no. 3: 363. https://doi.org/10.3390/v16030363