Regulation of EBNA1 Protein Stability by PLOD1 Lysine Hydroxylase

Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus that is causally associated with various malignancies and autoimmune disease. Epstein-Barr Nuclear Antigen 1 (EBNA1) is the viral-encoded DNA binding protein required for viral episome maintenance and DNA replication during latent infection in proliferating cells. EBNA1 is known to be a highly stable protein, but its mechanism of protein stability is not completely understood. Proteomic analysis of EBNA1 revealed interaction with Procollagen Lysine-2 Oxoglutarate 5 Dioxygenase (PLOD) family of proteins. Depletion of PLOD1 by shRNA or inhibition with small molecule inhibitors 2,-2’ dipyridyl resulted in the loss of EBNA1 protein levels, along with a selective growth inhibition of EBV-positive lymphoid cells. PLOD1 depletion also caused a loss of EBV episomes from latently infected cells and inhibited oriP-dependent DNA replication. We used mass spectrometry to identify EBNA1 peptides with lysine hydroxylation at K460 or K461. Mutation of K460 to alanine or arginine abrogates EBNA1-driven DNA replication of oriP, while K461 mutations enhanced replication. These findings suggest that PLOD1 is a novel post-translational regulator of EBNA1 protein stability and function in viral plasmid replication, episome maintenance and host cell survival. Importance EBNA1 is essential for EBV latent infection and implicated in viral pathogenesis. We found that EBNA1 interacts with PLOD family of lysine hydroxylases and that this interaction is required for EBNA1 protein stability and function in viral persistence during viral latent infection. Identification of PLOD1 regulation of EBNA1 protein stability provide new opportunity to target EBNA1 for degradation in EBV associated disease.


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Epstein-Barr virus (EBV) is a ubiquitous human g-herpesvirus that is causally associated with 19 various malignancies and autoimmune disease. Epstein-Barr Nuclear Antigen 1 (EBNA1) is the 20 viral-encoded DNA binding protein required for viral episome maintenance and DNA replication 21 during latent infection in proliferating cells. EBNA1 is known to be a highly stable protein, but its 22 mechanism of protein stability is not completely understood. Proteomic analysis of EBNA1

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EBNA1 is essential for EBV latent infection and implicated in viral pathogenesis. We found that 39 EBNA1 interacts with PLOD family of lysine hydroxylases and that this interaction is required for 40 EBNA1 protein stability and function in viral persistence during viral latent infection.

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Identification of PLOD1 regulation of EBNA1 protein stability provide new opportunity to target Introduction 45 Epstein-Barr Virus (EBV) is a human gammaherpesvirus that establishes life-long latent 46 infection in over 90% of the adult population world-wide [1,2]. EBV latent infection is a causal 47 agent for several cancers, including Burkitt Lymphoma (BL), Nasopharyngeal Carcinoma (NPC), 48 and post-transplant lymphoproliferative diseases (PTLD) [3][4][5]. EBV is also associated with 49 several autoimmune diseases, especially multiple sclerosis (MS) where viral proteins have been 50 implicated as the molecular mimic and trigger for auto-reactive antibodies and T-cells [6,7].

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Epstein-Barr Nuclear Antigen 1 (EBNA1) is the viral-encoded sequence-specific DNA-52 binding protein that binds to tandem repeats in the viral origin of plasmid replication (oriP) and is 53 required for viral episome maintenance and plasmid replication during latent infection in 54 proliferating cells [8,9]. EBNA1 can also modulate transcription of viral and host genes, and 55 interacts with host proteins that are implicated in viral oncogenesis, such as USP7 and CK2 [10-56 12]. EBNA1 is predominantly localized to the nucleus of infected cells, and is the most 57 consistently detected protein in EBV-associated tumors. EBNA1 is also known to have a 58 relatively long half-life (~20 hrs) in B-cells [13]. EBNA1 stabilization is partly dependent on a 59 central gly-ala repeat that resists proteolysis associated with MHC peptide presentation [14,15].

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However, EBNA1 interaction with other proteins and post-translational modifications may also 61 contribute to its stability [16] .

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The Procollagen-Lysine,2-Oxoglutarate 5-Dioxygenases (PLODs) are required for the 63 post-translational modification that allows collagen cross-links and maturation of extracellular

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FLAG-EBNA1 was expressed from stable oriP-containing episomes to enrich for cellular 82 proteins that bound to EBNA1 in the functional context of the oriP. We report here the 83 identification of PLOD1, 2, and 3 as proteins highly enriched in FLAG-EBNA1 fraction relative to 84 the FLAG-vector control ( Fig. 1A and B). We also identified the USP7, which has been well-85 characterized for its interaction with EBNA1, and P4HA2, a proline hydroxylase related to 86 PLODs (Fig. 1B). RNA analysis of PLODs revealed that two isoforms of PLOD1 (A and B) were 87 expressed at higher levels than PLOD2 or PLOD3 in EBV+ B-cell lines (Supplementary Fig. 88 S1). We therefore focused our efforts on characterization of PLOD1 with EBNA1 in these B-89 lymphocytes. Immunoprecipitation (IP) with endogenous EBNA1 in Raji and Mutu I Burkitt 90 lymphoma cell lines revealed selective enrichment of PLOD1 relative to IgG control (Fig. 1C).

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Similarly, reverse IP with PLOD1 in Raji and Mutu I cells revealed selective enrichment of 92 EBNA1 relative to IgG control (Fig. 1D). Interestingly, EBNA1 species precipitated in PLOD1 IP 93 had an additional EBNA1 reactive species (*) of slower mobility, suggesting potential EBNA1 94 post-translational modification when complexed with PLOD1.

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Inhibitor of PLOD1 leads to loss of EBNA1. To investigate the potential effects of PLOD1 on 97 EBNA1 protein expression, we first generated lentivirus expressing shRNA targeting PLOD1.

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We found that shRNA depletion of PLOD1 in Raji BL cells led to a significant loss of expression 99 of PLOD1, indicating that shRNA knock-down was working efficiently ( Fig. 2A, top panel). In 100 the same knock-down of PLOD1, we observed a reduction in EBNA1 protein, along with a 101 down-shift in EBNA1 mobility in SDS-PAGE Western blot ( Fig. 2A). We also observed a similar 102 change in EBNA2 and to a lesser extent that of LMP1, while cellular actin was not affected (

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(also known as 2,2 dipyridil and referred to here as 2-DP) has been reported to have selective 106 inhibition of PLOD1 [26]. We found that treatment of Raji and LCLs with 2-DP (100 µM) led to a 107 loss of EBNA1 and EBNA2 in both cell types, with less of an effect on LMP1 or cellular actin 108 ( Fig. 2B), thus phenocopying shRNA depletion of PLOD1. To determine if the loss of EBNA1 109 protein levels were partly due to proteosome degradation, we assayed the effects of 2-DP in 110 combination with proteosome inhibitor MG132 (Fig. 2C). We found that MG132 stabilized 111 EBNA1 protein in the presence of 2-DP, suggesting that 2-DP leads to proteosomal degradation 112 of EBNA1. EBNA1 protein can be destabilized by other small molecules, such as the HSP90 113 inhibitor 17-DMGA [27]. We found that 17-DMGA did not lead to the degradation of EBNA1 as 114 did 2-DP under these conditions. Since 2-DP has the potential to chelate iron and induce 115 hypoxia stress response, we compared the effects of 2-DP to treatment of CoCl2 a known 116 inducer of hypoxic stress response through stabilization of HIF1A (Fig. 2D). We found that 2-117 DP led to a loss of PLOD1 and EBNA1 in both Raji and LCL, and stabilized HIF1A modestly in 118 LCLs only. In contrast, CoCl2 stabilized HIF1A in both Raji and LCL, and reduced PLOD1 and 119 EBNA1 in LCL, but had only weak effects on PLOD1 and EBNA1 in Raji cells. These findings 120 suggest that 2-DP may inhibit PLOD1 through mechanisms distinct from HSP90 inhibition or hypoxia stress response, although there may be some cell-type dependent overlaps with these 122 pathways.

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Inhibition of PLOD1 selectively block EBV+ B cell survival.

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We next tested the effects of PLOD1 shRNA depletion and inhibition by 2-DP on EBV-

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PFGE analysis revealed that shPLOD1 depletion caused a significant loss of EBV episomal 140 DNA in each cell type ( Fig. 4A and B). The efficiency of shPLOD1 depletion was measured by 141 RT-qPCR and Western blot for each cell type (Supplementary Fig S2). EBV episome loss was

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EBNA1 DNA replication function, we assayed transient plasmid replication in HEK293 cells transfected with oriP-containing plasmids that also expressed FLAG-EBNA1. We also assayed 148 two different PLOD1 shRNAs, shPLOD1.a and shPLOD1.b for their ability to efficiently deplete 149 PLOD1. While shPLOD1.a and shPLOD1.b led to a modest reduction in PLOD1 protein at this 150 time point, the depletion on FLAG-EBNA1 expression was substantial (Fig. 5A). We then 151 assayed the effect of shPLOD1 on EBNA1-dependent DNA replication. We found that both 152 shPLOD1.a and shPLOD1.b substantially reduced oriP-dependent DNA replication, as 153 measured by DpnI resistance assay and Southern blot detection of oriP-containing plasmid 154 DNA ( Fig. 5B and C). These findings further support the role for PLOD1 in the stabilization of 155 EBNA1 protein levels, and its functional importance in for oriP-dependent DNA replication.

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Lysine hydroxylation of EBNA1. To investigate the possibility that EBNA1 may be subject to 158 post-translational modification through lysine hydroxylation, we performed LC-MS/MS analysis 159 of immunoprecipitated EBNA1. We identified one peptide with a mass/charge (m/z) shift 160 consistent with a single lysine hydroxylation ( Fig. 6A-C). The EBNA1 peptide aa 416-465 had 161 two potential lysine residues that could be hydroxylated, K460 and K461. PLOD1 typically 162 hydroxylates lysines that precede glycine. We therefore first tested whether mutations in K461 163 impacted EBNA1 function in oriP-dependent DNA replication ( Fig. 6D-F). We found that K461A 164 had a modest stimulatory effect, while K461R had no significant effect on oriP replication ( Fig.   165 6D-F). We next asked whether mutations in the neighboring K460 had any effects on oriP-DNA 166 replication ( Fig. 6G-I). We also included a mutation in K83A, which also has a PLOD1 167 consensus recognition site, and has been previously implicated in the PLOD1 interaction with 168 the EBNA1 N-terminus. All EBNA1 mutants were expressed at similar levels in HEK293T cells 169 (Fig. 6G). We found that K83A had a modest enhancement of oriP replication, while both 170 K460A and K460R reduced oriP replication >5-fold ( Fig. 6H and I). We also found that 171 mutations in both K460A and K461A bound to oriP similar to wild-type EBNA1 as measured by 172 ChIP assay, suggesting that these effects are not due to the disruption of EBNA1-DNA binding ( Supplementary Figs S3 and S4). Taken together, these findings indicate that EBNA1 can be 174 hydroxylated on either K460 or K461, and that mutations in K460 reduces EBNA1 replication 175 function, but not its ability to bind at oriP.

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EBNA1 is thought to be a highly stable protein in the nucleus of cells latently infected with EBV.

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Herein, we describe an EBNA1 interaction partner, PLOD1, that contributes to EBNA1 protein