Covalent modification by SUMO is required for efficient disruption of PML oncogenic domains by Kaposi's sarcoma-associated herpesvirus latent protein LANA2
The multifunctional Kaposi's sarcoma-associated herpesvirus (KSHV) latent protein latency-associated nuclear antigen 2 (LANA2) has a critical role in KSHV-induced B-cell malignancies. LANA2 increases the level of small ubiquitin-like modifier (SUMO)2-ubiquitin-modified PML and induces the disruption of PML oncogenic domains (PODs) by a process that requires a non-covalent SUMO interaction domain (SIM) in LANA2. We now demonstrate that LANA2 is covalently conjugated to SUMO1 and SUMO2 both in vitro and in latently KSHV-infected B-cells. We show that a LANA2 SIM mutant exhibits a slightly altered sumoylation pattern, which suggests that non-covalent SUMO interactions represent a mechanism for determining SUMO substrate recognition and modification. In addition, several lysine residues were mapped as SUMO conjugation sites. A sumoylation-deficient mutant shows impaired ability to induce disruption of PODs, which suggests that either directly bound or covalently conjugated SUMO moieties may act as a bridge for interaction between LANA2 and other SUMO-modified or SUMO-interacting proteins required for disruption of PODs.
Adamson, A. L. & Kenney, S.(2001). Epstein–Barr virus immediate-early protein BZLF1 is SUMO-1 modified and disrupts promyelocytic leukemia bodies. J Virol75, 2388–2399.[CrossRef][Google Scholar]
Boggio, R. & Chiocca, S.(2006). Viruses and sumoylation: recent highlights. Curr Opin Microbiol9, 430–436.[CrossRef][Google Scholar]
Campagna, M., Herranz, D., Garcia, M. A., Marcos-Villar, L., Gonzalez-Santamaria, J., Gallego, P., Gutierrez, S., Collado, M., Serrano, M. & other authors(2010). SIRT1 stabilizes PML promoting its sumoylation. Cell Death Differ in press. doi: 10.1038/cdd.2010.77.
[Google Scholar]
Chakrabarti, S. R., Sood, R., Nandi, S. & Nucifora, G.(2000). Posttranslational modification of TEL and TEL/AML1 by SUMO-1 and cell-cycle-dependent assembly into nuclear bodies. Proc Natl Acad Sci U S A97, 13281–13285.[CrossRef][Google Scholar]
Chang, P. C., Izumiya, Y., Wu, C. Y., Fitzgerald, L. D., Campbell, M., Ellison, T. J., Lam, K. S., Luciw, P. A. & Kung, H. J.(2010). Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a SUMO E3 ligase that is SIM-dependent and SUMO-2/3-specific. J Biol Chem285, 5266–5273.[CrossRef][Google Scholar]
Desterro, J. M., Rodriguez, M. S. & Hay, R. T.(1998). SUMO-1 modification of IκBα inhibits NF-κB activation. Mol Cell2, 233–239.[CrossRef][Google Scholar]
Eladad, S., Ye, T. Z., Hu, P., Leversha, M., Beresten, S., Matunis, M. J. & Ellis, N. A.(2005). Intra-nuclear trafficking of the BLM helicase to DNA damage-induced foci is regulated by SUMO modification. Hum Mol Genet14, 1351–1365.[CrossRef][Google Scholar]
Esteban, M., Garcia, M. A., Domingo-Gil, E., Arroyo, J., Nombela, C. & Rivas, C.(2003). The latency protein LANA2 from Kaposi's sarcoma-associated herpesvirus inhibits apoptosis induced by dsRNA-activated protein kinase but not RNase L activation. J Gen Virol84, 1463–1470.[CrossRef][Google Scholar]
Gill, G.(2004). SUMO and ubiquitin in the nucleus: different functions, similar mechanisms? Genes Dev18, 2046–2059.[CrossRef][Google Scholar]
Hoege, C., Pfander, B., Moldovan, G. L., Pyrowolakis, G. & Jentsch, S.(2002). RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature419, 135–141.[CrossRef][Google Scholar]
Johnson, E. S.(2004). Protein modification by SUMO. Annu Rev Biochem73, 355–382.[CrossRef][Google Scholar]
Joo, C. H., Shin, Y. C., Gack, M., Wu, L., Levy, D. & Jung, J. U.(2007). Inhibition of interferon regulatory factor 7 (IRF7)-mediated interferon signal transduction by the Kaposi's sarcoma-associated herpesvirus viral IRF homolog vIRF3. J Virol81, 8282–8292.[CrossRef][Google Scholar]
Kamitani, T., Kito, K., Nguyen, H. P., Wada, H., Fukuda-Kamitani, T. & Yeh, E. T.(1998). Identification of three major sentrinization sites in PML. J Biol Chem273, 26675–26682.[CrossRef][Google Scholar]
Kerscher, O.(2007). SUMO junction–what's your function? New insights through SUMO-interacting motifs. EMBO Rep8, 550–555.[CrossRef][Google Scholar]
Li, X., Luo, Y., Yu, L., Lin, Y., Luo, D., Zhang, H., He, Y., Kim, Y. O., Kim, Y. & other authors(2008). SENP1 mediates TNF-induced desumoylation and cytoplasmic translocation of HIPK1 to enhance ASK1-dependent apoptosis. Cell Death Differ15, 739–750.[CrossRef][Google Scholar]
Lin, X., Sun, B., Liang, M., Liang, Y. Y., Gast, A., Hildebrand, J., Brunicardi, F. C., Melchior, F. & Feng, X. H.(2003). Opposed regulation of corepressor CtBP by SUMOylation and PDZ binding. Mol Cell11, 1389–1396.[CrossRef][Google Scholar]
Lin, D. Y., Huang, Y. S., Jeng, J. C., Kuo, H. Y., Chang, C. C., Chao, T. T., Ho, C. C., Chen, Y. C., Lin, T. P. & other authors(2006). Role of SUMO-interacting motif in Daxx SUMO modification, subnuclear localization, and repression of sumoylated transcription factors. Mol Cell24, 341–354.[CrossRef][Google Scholar]
Lubyova, B. & Pitha, P. M.(2000). Characterization of a novel human herpesvirus 8-encoded protein, vIRF-3, that shows homology to viral and cellular interferon regulatory factors. J Virol74, 8194–8201.[CrossRef][Google Scholar]
Marcos-Villar, L., Lopitz-Otsoa, F., Gallego, P., Munoz-Fontela, C., Gonzalez-Santamaria, J., Campagna, M., Shou-Jiang, G., Rodriguez, M. S. & Rivas, C.(2009). Kaposi's sarcoma-associated herpesvirus protein LANA2 disrupts PML oncogenic domains and inhibits PML-mediated transcriptional repression of the survivin gene. J Virol83, 8849–8858.[CrossRef][Google Scholar]
Matunis, M. J., Zhang, X. D. & Ellis, N. A.(2006). SUMO: the glue that binds. Dev Cell11, 596–597.[CrossRef][Google Scholar]
Meluh, P. B. & Koshland, D.(1995). Evidence that the MIF2 gene of Saccharomyces cerevisiae encodes a centromere protein with homology to the mammalian centromere protein CENP-C. Mol Biol Cell6, 793–807.[CrossRef][Google Scholar]
Minty, A., Dumont, X., Kaghad, M. & Caput, D.(2000). Covalent modification of p73α by SUMO-1. Two-hybrid screening with p73 identifies novel SUMO-1-interacting proteins and a SUMO-1 interaction motif. J Biol Chem275, 36316–36323.[CrossRef][Google Scholar]
Rivas, C., Thlick, A. E., Parravicini, C., Moore, P. S. & Chang, Y.(2001). Kaposi's sarcoma-associated herpesvirus LANA2 is a B-cell-specific latent viral protein that inhibits p53. J Virol75, 429–438.[CrossRef][Google Scholar]
Rui, H. L., Fan, E., Zhou, H. M., Xu, Z., Zhang, Y. & Lin, S. C.(2002). SUMO-1 modification of the C-terminal KVEKVD of Axin is required for JNK activation but has no effect on Wnt signaling. J Biol Chem277, 42981–42986.[CrossRef][Google Scholar]
Seo, T., Park, J., Lim, C. & Choe, J.(2004). Inhibition of nuclear factor κB activity by viral interferon regulatory factor 3 of Kaposi's sarcoma-associated herpesvirus. Oncogene23, 6146–6155.[CrossRef][Google Scholar]
Shin, Y. C., Joo, C. H., Gack, M. U., Lee, H. R. & Jung, J. U.(2008). Kaposi's sarcoma-associated herpesvirus viral IFN regulatory factor 3 stabilizes hypoxia-inducible factor-1 alpha to induce vascular endothelial growth factor expression. Cancer Res68, 1751–1759.[CrossRef][Google Scholar]
Suzuki, T., Ichiyama, A., Saitoh, H., Kawakami, T., Omata, M., Chung, C. H., Kimura, M., Shimbara, N. & Tanaka, K.(1999). A new 30-kDa ubiquitin-related SUMO-1 hydrolase from bovine brain. J Biol Chem274, 31131–31134.[CrossRef][Google Scholar]
Takahashi, H., Hatakeyama, S., Saitoh, H. & Nakayama, K. I.(2005). Noncovalent SUMO-1 binding activity of thymine DNA glycosylase (TDG) is required for its SUMO-1 modification and colocalization with the promyelocytic leukemia protein. J Biol Chem280, 5611–5621.[CrossRef][Google Scholar]
Tatham, M. H., Jaffray, E., Vaughan, O. A., Desterro, J. M., Botting, C. H., Naismith, J. H. & Hay, R. T.(2001). Polymeric chains of SUMO-2 and SUMO-3 are conjugated to protein substrates by SAE1/SAE2 and Ubc9. J Biol Chem276, 35368–35374.[CrossRef][Google Scholar]
Ulrich, H. D.(2009). The SUMO system: an overview. Methods Mol Biol497, 3–16.
[Google Scholar]
Vertegaal, A. C., Andersen, J. S., Ogg, S. C., Hay, R. T., Mann, M. & Lamond, A. I.(2006). Distinct and overlapping sets of SUMO-1 and SUMO-2 target proteins revealed by quantitative proteomics. Mol Cell Proteomics5, 2298–2310.[CrossRef][Google Scholar]
Weger, S., Hammer, E. & Heilbronn, R.(2004). SUMO-1 modification regulates the protein stability of the large regulatory protein Rep78 of adeno associated virus type 2 (AAV-2). Virology330, 284–294.[CrossRef][Google Scholar]
Wies, E., Mori, Y., Hahn, A., Kremmer, E., Sturzl, M., Fleckenstein, B. & Neipel, F.(2008). The viral interferon-regulatory factor-3 is required for the survival of KSHV-infected primary effusion lymphoma cells. Blood111, 320–327.[CrossRef][Google Scholar]
Zhao, J.(2007). Sumoylation regulates diverse biological processes. Cell Mol Life Sci64, 3017–3033.[CrossRef][Google Scholar]
Zhu, J., Zhu, S., Guzzo, C. M., Ellis, N. A., Sung, K. S., Choi, C. Y. & Matunis, M. J.(2008). Small ubiquitin-related modifier (SUMO) binding determines substrate recognition and paralog-selective SUMO modification. J Biol Chem283, 29405–29415.[CrossRef][Google Scholar]
Covalent modification by SUMO is required for efficient disruption of PML oncogenic domains by Kaposi's sarcoma-associated herpesvirus latent protein LANA2
Journal of General Virology vol.
92 , part 1, pp. 188–194.
Supplementary Fig. S1. Modification of PML by
SUMO1
in vivoSupplementary Fig. S2. SUMO interaction domain
contributes to SUMOylation in LANA2
Supplementary Fig. S3. LANA2 protein stability
is unaffected by SUMOylation
Supplementary Fig. S4. SUMO conjugation to LANA2
contributes to the degradation of haemagglutinin–PML
Supplementary Table S1. Putative SUMO-binding
domains for LANA2
Supplementary Table S2. Oligonucleotides for
LANA2 mutagenesis