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PLOS ONE  2014 

Analysis of Human Cytomegalovirus-Encoded SUMO Targets and Temporal Regulation of SUMOylation of the Immediate-Early Proteins IE1 and IE2 during Infection

DOI: 10.1371/journal.pone.0103308

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Abstract:

Post-translational modification of proteins by members of the small ubiquitin-like modifier (SUMO) is involved in diverse cellular functions. Many viral proteins are SUMO targets and also interact with the cellular SUMOylation system. During human cytomegalovirus (HCMV) infection, the immediate-early (IE) proteins IE1 and IE2 are covalently modified by SUMO. IE2 SUMOylation promotes its transactivation activity, whereas the role of IE1 SUMOylation is not clear. We performed in silico, genome-wide analysis to identify possible SUMOylation sites in HCMV-encoded proteins and evaluated their modification using the E. coli SUMOylation system and in vitro assays. We found that only IE1 and IE2 are substantially modified by SUMO in E. coli, although US34A was also identified as a possible SUMO target in vitro. We also found that SUMOylation of IE1 and IE2 is temporally regulated during viral infection. Levels of SUMO-modified form of IE1 were increased during the early phase of infection, but decreased in the late phase when IE2 and its SUMO-modified forms were expressed at high levels. IE2 expression inhibited IE1 SUMOylation in cotransfection assays. As in IE2 SUMOylation, PIAS1, a SUMO E3 ligase, interacted with IE1 and enhanced IE1 SUMOylation. In in vitro assays, an IE2 fragment that lacked covalent and non-covalent SUMO attachment sites, but was sufficient for PIAS1 binding, effectively inhibited PIAS1-mediated SUMOylation of IE1, indicating that IE2 expression negatively regulates IE1 SUMOylation. We also found that the IE2-mediated downregulation of IE1 SUMOylation correlates with the IE1 activity to repress the promoter containing the interferon stimulated response elements. Taken together, our data demonstrate that IE1 and IE2 are the main viral SUMO targets in HCMV infection and that temporal regulation of their SUMOylation may be important in the progression of this infection.

References

[1]  Hay RT (2005) SUMO: a history of modification. Mol Cell 18: 1–12. doi: 10.1016/j.molcel.2005.03.012
[2]  Gareau JR, Lima CD (2010) The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition. Nat Rev Mol Cell Biol 11: 861–871. doi: 10.1038/nrm3011
[3]  Kahyo T, Nishida T, Yasuda H (2001) Involvement of PIAS1 in the sumoylation of tumor suppressor p53. Mol Cell 8: 713–718. doi: 10.1016/s1097-2765(01)00349-5
[4]  Pichler A, Gast A, Seeler JS, Dejean A, Melchior F (2002) The nucleoporin RanBP2 has SUMO1 E3 ligase activity. Cell 108: 109–120. doi: 10.1016/s0092-8674(01)00633-x
[5]  Kagey MH, Melhuish TA, Wotton D (2003) The polycomb protein Pc2 is a SUMO E3. Cell 113: 127–137 118: Sternsdorf T et al. Sumo….[PMID: 12676099]Related Articles, Links. doi: 10.1016/s0092-8674(03)00159-4
[6]  Bernier-Villamor V, Sampson DA, Matunis MJ, Lima CD (2002) Structural basis for E2-mediated SUMO conjugation revealed by a complex between ubiquitin-conjugating enzyme Ubc9 and RanGAP1. Cell 108: 345–356. doi: 10.1016/s0092-8674(02)00630-x
[7]  Lin D, Tatham MH, Yu B, Kim S, Hay RT, et al. (2002) Identification of a substrate recognition site on ubc9. J Biol Chem 277: 21740–21748. doi: 10.1074/jbc.m108418200
[8]  Rodriguez MS, Dargemont C, Hay RT (2001) SUMO-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting. J Biol Chem 276: 12654–12659. doi: 10.1074/jbc.m009476200
[9]  Sampson DA, Wang M, Matunis MJ (2001) The small ubiquitin-like modifier-1 (SUMO-1) consensus sequence mediates Ubc9 binding and is essential for SUMO-1 modification. J Biol Chem 276: 21664–21669. doi: 10.1074/jbc.m100006200
[10]  Hay RT (2007) SUMO-specific proteases: a twist in the tail. Trends Cell Biol 17: 370–376. doi: 10.1016/j.tcb.2007.08.002
[11]  Yeh ET (2009) SUMOylation and De-SUMOylation: wrestling with life's processes. J Biol Chem 284: 8223–8227. doi: 10.1074/jbc.r800050200
[12]  Hickey CM, Wilson NR, Hochstrasser M (2012) Function and regulation of SUMO proteases. Nat Rev Mol Cell Biol 13: 755–766. doi: 10.1038/nrm3478
[13]  Minty A, Dumont X, Kaghad M, Caput D (2000) Covalent modification of p73alpha by SUMO-1. Two-hybrid screening with p73 identifies novel SUMO-1-interacting proteins and a SUMO-1 interaction motif. J Biol Chem 275: 36316–36323. doi: 10.1074/jbc.m004293200
[14]  Song J, Durrin LK, Wilkinson TA, Krontiris TG, Chen Y (2004) Identification of a SUMO-binding motif that recognizes SUMO-modified proteins. Proc Natl Acad Sci U S A 101: 14373–14378. doi: 10.1073/pnas.0403498101
[15]  Hannich JT, Lewis A, Kroetz MB, Li SJ, Heide H, et al. (2005) Defining the SUMO-modified proteome by multiple approaches in Saccharomyces cerevisiae. J Biol Chem 280: 4102–4110. doi: 10.1074/jbc.m413209200
[16]  Kerscher O (2007) SUMO junction-what's your function? New insights through SUMO-interacting motifs. EMBO Rep 8: 550–555. doi: 10.1038/sj.embor.7400980
[17]  Everett RD, Boutell C, Hale BG (2013) Interplay between viruses and host sumoylation pathways. Nat Rev Microbiol 11: 400–411. doi: 10.1038/nrmicro3015
[18]  Wimmer P, Schreiner S, Dobner T (2012) Human pathogens and the host cell SUMOylation system. J Virol 86: 642–654. doi: 10.1128/jvi.06227-11
[19]  Mocarski ES, Shenk T, Griffiths PD, Pass RF (2013) Cytomegaloviruses, p. 1960–2014. In D. MKnipe, P. MHowley, J. ICohen, D. EGriffin, R. ALamb, M. AMartin, V. RRacaniello, and BRoizman (ed.), Fields virology. Lippincott Williams & Wilkins. Philadelphia, PA.
[20]  Hofmann H, Floss S, Stamminger T (2000) Covalent modification of the transactivator protein IE2-p86 of human cytomegalovirus by conjugation to the ubiquitin-homologous proteins SUMO-1 and hSMT3b. J Virol 74: 2510–2524. doi: 10.1128/jvi.74.6.2510-2524.2000
[21]  Ahn JH, Xu Y, Jang WJ, Matunis MJ, Hayward GS (2001) Evaluation of interactions of human cytomegalovirus immediate-early IE2 regulatory protein with small ubiquitin-like modifiers and their conjugation enzyme Ubc9. J Virol 75: 3859–3872. doi: 10.1128/jvi.75.8.3859-3872.2001
[22]  Barrasa MI, Harel N, Yu Y, Alwine JC (2003) Strain variations in single amino acids of the 86-kilodalton human cytomegalovirus major immediate-early protein (IE2) affect its functional and biochemical properties: implications of dynamic protein conformation. J Virol 77: 4760–4772. doi: 10.1128/jvi.77.8.4760-4772.2003
[23]  Lee JM, Kang HJ, Lee HR, Choi CY, Jang WJ, et al. (2003) PIAS1 enhances SUMO-1 modification and the transactivation activity of the major immediate-early IE2 protein of human cytomegalovirus. FEBS Lett 555: 322–328. doi: 10.1016/s0014-5793(03)01268-7
[24]  Berndt A, Hofmann-Winkler H, Tavalai N, Hahn G, Stamminger T (2009) Importance of covalent and noncovalent SUMO interactions with the major human cytomegalovirus transactivator IE2p86 for viral infection. J Virol 83: 12881–12894. doi: 10.1128/jvi.01525-09
[25]  Lee HR, Ahn JH (2004) Sumoylation of the major immediate-early IE2 protein of human cytomegalovirus Towne strain is not required for virus growth in cultured human fibroblasts. J Gen Virol 85: 2149–2154. doi: 10.1099/vir.0.79954-0
[26]  Kim ET, Kim YE, Huh YH, Ahn JH (2010) Role of noncovalent SUMO binding by the human cytomegalovirus IE2 transactivator in lytic growth. J Virol 84: 8111–8123. doi: 10.1128/jvi.00459-10
[27]  Greaves RF, Mocarski ES (1998) Defective growth correlates with reduced accumulation of a viral DNA replication protein after low-multiplicity infection by a human cytomegalovirus ie1 mutant. J Virol 72: 366–379.
[28]  Mocarski ES, Kemble GW, Lyle JM, Greaves RF (1996) A deletion mutant in the human cytomegalovirus gene encoding IE1(491aa) is replication defective due to a failure in autoregulation. Proc Natl Acad Sci U S A 93: 11321–11326. doi: 10.1073/pnas.93.21.11321
[29]  Ahn JH, Hayward GS (1997) The major immediate-early proteins IE1 and IE2 of human cytomegalovirus colocalize with and disrupt PML-associated nuclear bodies at very early times in infected permissive cells. J Virol 71: 4599–4613.
[30]  Ahn JH, Brignole EJ 3rd, Hayward GS (1998) Disruption of PML subnuclear domains by the acidic IE1 protein of human cytomegalovirus is mediated through interaction with PML and may modulate a RING finger-dependent cryptic transactivator function of PML. Mol Cell Biol 18: 4899–4913.
[31]  Korioth F, Maul GG, Plachter B, Stamminger T, Frey J (1996) The nuclear domain 10 (ND10) is disrupted by the human cytomegalovirus gene product IE1. Exp Cell Res 229: 155–158. doi: 10.1006/excr.1996.0353
[32]  Wilkinson GW, Kelly C, Sinclair JH, Rickards C (1998) Disruption of PML-associated nuclear bodies mediated by the human cytomegalovirus major immediate early gene product. J Gen Virol 79 (Pt 5) 1233–1245.
[33]  Kim YE, Lee JH, Kim ET, Shin HJ, Gu SY, et al. (2011) Human cytomegalovirus infection causes degradation of Sp100 proteins that suppress viral gene expression. J Virol 85: 11928–11937. doi: 10.1128/jvi.00758-11
[34]  Tavalai N, Adler M, Scherer M, Riedl Y, Stamminger T (2011) Evidence for a Dual Antiviral Role of the Major Nuclear Domain 10 Component Sp100 during the Immediate-Early and Late Phases of the Human Cytomegalovirus Replication Cycle. J Virol 85: 9447–9458. doi: 10.1128/jvi.00870-11
[35]  Tavalai N, Papior P, Rechter S, Leis M, Stamminger T (2006) Evidence for a role of the cellular ND10 protein PML in mediating intrinsic immunity against human cytomegalovirus infections. J Virol 80: 8006–8018. doi: 10.1128/jvi.00743-06
[36]  Paulus C, Krauss S, Nevels M (2006) A human cytomegalovirus antagonist of type I IFN-dependent signal transducer and activator of transcription signaling. Proc Natl Acad Sci U S A 103: 3840–3845. doi: 10.1073/pnas.0600007103
[37]  Huh YH, Kim YE, Kim ET, Park JJ, Song MJ, et al. (2008) Binding STAT2 by the acidic domain of human cytomegalovirus IE1 promotes viral growth and is negatively regulated by SUMO. J Virol 82: 10444–10454. doi: 10.1128/jvi.00833-08
[38]  Krauss S, Kaps J, Czech N, Paulus C, Nevels M (2009) Physical requirements and functional consequences of complex formation between the cytomegalovirus IE1 protein and human STAT2. J Virol 83: 12854–12870. doi: 10.1128/jvi.01164-09
[39]  Xu Y, Ahn JH, Cheng M, apRhys CM, Chiou CJ, et al. (2001) Proteasome-independent disruption of PML oncogenic domains (PODs), but not covalent modification by SUMO-1, is required for human cytomegalovirus immediate-early protein IE1 to inhibit PML-mediated transcriptional repression. J Virol 75: 10683–10695. doi: 10.1128/jvi.75.22.10683-10695.2001
[40]  Spengler ML, Kurapatwinski K, Black AR, Azizkhan-Clifford J (2002) SUMO-1 modification of human cytomegalovirus IE1/IE72. J Virol 76: 2990–2996. doi: 10.1128/jvi.76.6.2990-2996.2002
[41]  Nevels M, Brune W, Shenk T (2004) SUMOylation of the human cytomegalovirus 72-kilodalton IE1 protein facilitates expression of the 86-kilodalton IE2 protein and promotes viral replication. J Virol 78: 7803–7812. doi: 10.1128/jvi.78.14.7803-7812.2004
[42]  Lee HR, Kim DJ, Lee JM, Choi CY, Ahn BY, et al. (2004) Ability of the human cytomegalovirus IE1 protein to modulate sumoylation of PML correlates with its functional activities in transcriptional regulation and infectivity in cultured fibroblast cells. J Virol 78: 6527–6542. doi: 10.1128/jvi.78.12.6527-6542.2004
[43]  Shin HJ, Kim YE, Kim ET, Ahn JH (2012) The chromatin-tethering domain of human cytomegalovirus immediate-early (IE) 1 mediates associations of IE1, PML and STAT2 with mitotic chromosomes, but is not essential for viral replication. J Gen Virol 93: 716–721. doi: 10.1099/vir.0.037986-0
[44]  Sadanari H, Yamada R, Ohnishi K, Matsubara K, Tanaka J (2005) SUMO-1 modification of the major immediate-early (IE) 1 and 2 proteins of human cytomegalovirus is regulated by different mechanisms and modulates the intracellular localization of the IE1, but not IE2, protein. Arch Virol 150: 1763–1782. doi: 10.1007/s00705-005-0559-0
[45]  Green S, Issemann I, Sheer E (1988) A versatile in vivo and in vitro eukaryotic expression vector for protein engineering. Nucleic Acids Res 16: 369. doi: 10.1093/nar/16.1.369
[46]  Turner DL, Weintraub H (1994) Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate. Genes Dev 8: 1434–1447. doi: 10.1101/gad.8.12.1434
[47]  Kang H, Kim ET, Lee HR, Park JJ, Go YY, et al. (2006) Inhibition of SUMO-independent PML oligomerization by the human cytomegalovirus IE1 protein. J Gen Virol 87: 2181–2190. doi: 10.1099/vir.0.81787-0
[48]  Uchimura Y, Nakamura M, Sugasawa K, Nakao M, Saitoh H (2004) Overproduction of eukaryotic SUMO-1- and SUMO-2-conjugated proteins in Escherichia coli. Anal Biochem 331: 204–206. doi: 10.1016/s0003-2697(04)00378-1
[49]  Zhu J, Liao G, Shan L, Zhang J, Chen MR, et al. (2009) Protein array identification of substrates of the Epstein-Barr virus protein kinase BGLF4. J Virol 83: 5219–5231. doi: 10.1128/jvi.02378-08
[50]  Murphy E, Yu D, Grimwood J, Schmutz J, Dickson M, et al. (2003) Coding potential of laboratory and clinical strains of human cytomegalovirus. Proc Natl Acad Sci U S A 100: 14976–14981. doi: 10.1073/pnas.2136652100
[51]  Marchini A, Liu H, Zhu H (2001) Human cytomegalovirus with IE-2 (UL122) deleted fails to express early lytic genes. J Virol 75: 1870–1878. doi: 10.1128/jvi.75.4.1870-1878.2001
[52]  Ren J, Gao X, Jin C, Zhu M, Wang X, et al. (2009) Systematic study of protein sumoylation: Development of a site-specific predictor of SUMOsp 2.0. Proteomics 9: 3409–3412. doi: 10.1002/pmic.200800646
[53]  O'Connor CM, Shenk T (2011) Human cytomegalovirus pUS27 G protein-coupled receptor homologue is required for efficient spread by the extracellular route but not for direct cell-to-cell spread. J Virol 85: 3700–3707. doi: 10.1128/jvi.02442-10
[54]  Margulies BJ, Gibson W (2007) The chemokine receptor homologue encoded by US27 of human cytomegalovirus is heavily glycosylated and is present in infected human foreskin fibroblasts and enveloped virus particles. Virus Res 123: 57–71. doi: 10.1016/j.virusres.2006.08.003
[55]  Pizzorno MC, Mullen MA, Chang YN, Hayward GS (1991) The functionally active IE2 immediate-early regulatory protein of human cytomegalovirus is an 80-kilodalton polypeptide that contains two distinct activator domains and a duplicated nuclear localization signal. J Virol 65: 3839–3852.
[56]  Sinigalia E, Alvisi G, Segre CV, Mercorelli B, Muratore G, et al. (2012) The human cytomegalovirus DNA polymerase processivity factor UL44 is modified by SUMO in a DNA-dependent manner. PLoS One 7: e49630. doi: 10.1371/journal.pone.0049630
[57]  Scherer M, Reuter N, Wagenknecht N, Otto V, Sticht H, et al. (2013) Small ubiquitin-related modifier (SUMO) pathway-mediated enhancement of human cytomegalovirus replication correlates with a recruitment of SUMO-1/3 proteins to viral replication compartments. J Gen Virol 94: 1373–1384. doi: 10.1099/vir.0.051078-0
[58]  Ahn JH, Jang WJ, Hayward GS (1999) The human cytomegalovirus IE2 and UL112-113 proteins accumulate in viral DNA replication compartments that initiate from the periphery of promyelocytic leukemia protein-associated nuclear bodies (PODs or ND10). J Virol 73: 10458–10471.
[59]  Hagemeier SR, Dickerson SJ, Meng Q, Yu X, Mertz JE, et al. (2010) Sumoylation of the Epstein-Barr virus BZLF1 protein inhibits its transcriptional activity and is regulated by the virus-encoded protein kinase. J Virol 84: 4383–4394. doi: 10.1128/jvi.02369-09
[60]  Li R, Wang L, Liao G, Guzzo CM, Matunis MJ, et al. (2012) SUMO binding by the Epstein-Barr virus protein kinase BGLF4 is crucial for BGLF4 function. J Virol 86: 5412–5421. doi: 10.1128/jvi.00314-12
[61]  Boggio R, Colombo R, Hay RT, Draetta GF, Chiocca S (2004) A mechanism for inhibiting the SUMO pathway. Mol Cell 16: 549–561. doi: 10.1016/j.molcel.2004.11.007
[62]  Boggio R, Passafaro A, Chiocca S (2007) Targeting SUMO E1 to ubiquitin ligases: a viral strategy to counteract sumoylation. J Biol Chem 282: 15376–15382. doi: 10.1074/jbc.m700889200
[63]  Heaton PR, Deyrieux AF, Bian XL, Wilson VG (2011) HPV E6 proteins target Ubc9, the SUMO conjugating enzyme. Virus Res 158: 199–208. doi: 10.1016/j.virusres.2011.04.001

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