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. 2019 Dec 13:10:2879.
doi: 10.3389/fmicb.2019.02879. eCollection 2019.

Epstein-Barr Virus Nuclear Antigen 1 Recruits Cyclophilin A to Facilitate the Replication of Viral DNA Genome

Shuyu Xin  1   2 , Shujuan Du  1   2 , Lingzhi Liu  1   2 , Yan Xie  1   2 , Lielian Zuo  1 , Jing Yang  1 , Jingjin Hu  2 , Wenxing Yue  1   2 , Jing Zhang  1   2 , Pengfei Cao  1   2 , Fanxiu Zhu  1   3 , Jianhong Lu  1   2
Affiliations

Epstein-Barr Virus Nuclear Antigen 1 Recruits Cyclophilin A to Facilitate the Replication of Viral DNA Genome

Shuyu Xin et al. Front Microbiol. .

Abstract

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1)-mediated DNA episomal genome replication and persistence are essential for the viral pathogenesis. Cyclophilin A (CYPA) is upregulated in EBV-associated nasopharyngeal carcinoma (NPC) with unknown roles. In the present approach, cytosolic CYPA was found to be bound with EBNA1 into the nucleus. The amino acid 376-459 of the EBNA1 domain was important for the binding. CYPA depletion attenuated and ectopic CYPA expression improved EBNA1 expression in EBV-positive cells. The loss of viral copy number was also accelerated by CYPA consumption in daughter cells during culture passages. Mechanistically, CYPA mediated the connection of EBNA1 with oriP (origin of EBV DNA replication) and subsequent oriP transcription, which is a key step for the initiation of EBV genome replication. Moreover, CYPA overexpression markedly antagonized the connection of EBNA1 to Ubiquitin-specific protease 7 (USP7), which is a strong host barrier with a role of inhibiting EBV genome replication. The PPIase activity of CYPA was required for the promotion of oriP transcription and antagonism with USP7. The results revealed a strategy that EBV recruited a host factor to counteract the host defense, thus facilitating its own latent genome replication. This study provides a new insight into EBV pathogenesis and potential virus-targeted therapeutics in EBV-associated NPC, in which CYPA is upregulated at all stages.

Keywords: Epstein-Barr virus nuclear antigen 1; cyclophilin A; latent genome; pathogenesis; persistence; replication.

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Figures

FIGURE 1
FIGURE 1
Detection of CYPA-EBNA1 binding by the BiMC and co-IP assays. (A) Detection of the EBNA1-CYPA interaction by the BiMC assay. HEK293T cells were transfected with the indicated plasmids, including CYPA-NY, EBNA1-CY, EBNA1ΔNLS-CY, and CYPB-NY following by BiMC analysis, and fluorescence was observed. Transfection of a single CYPA-NY plasmid did not lead to the production of fluorescence. Scale bar, 50 μm. (B) The proteins expressed from the plasmids used in the BiMC assay were detected by WB. (C) The detection of CYPA and EBNA1 in EBV-negative and EBV-positive HEK293 cells by IF assay. Scale bar, 50 μm. (D) Endogenous CYPA interacts with EBNA1 in HEK293 cells. The plasmid pCAGGS-Myc-EBNA1 was transfected into cells. An anti-Myc antibody was used for the pull-down the CYPA, and the WB assay was carried out for detection. (E) Endogenous CYPA interacts with EBNA1 in C2089 cells. EBNA1 was immune-precipitated with anti-CYPA. EBNA1 was detected by WB (F) Exogenous CYPA interacts with EBNA1. HEK293 cells were transiently transfected with Flag-CYPA alone or with Myc-EBNA1. Flag-CYPA was immune-precipitated with anti-Myc antibody. IgG was used as a negative control for the pull-down in the co-IP assay. Flag-CYPA was detected by WB. (G) Co-IP assay for comparison of the interactions of CYPA and CYPB with EBNA1. PCAGGS-Myc-EBNA1 and pCAGGS-Flag-CYPA or pCAGGS-Flag-CYPB were transfected in HEK293 cells. Myc-EBNA1 was immune-precipitated with anti-Flag antibody.
FIGURE 2
FIGURE 2
Identification of the EBNA1 domain required for binding to CYPA. (A) Diagram of the EBNA1 deletion mutants. The start and end amino acid residues for each fragment are indicated according to a previous report (Young and Murray, 2003). (B) Validation of the interaction between each mutant EBNA1 and CYPA by the co-IP assay. PCAGGS-Myc-EBNA1, pCAGGS-Myc-EBNA1 mutants, and pCAGGS-Flag-CYPA were transfected into HEK293 cells. Myc-EBNA1 was immune-precipitated with anti-FLAG antibody. Flag-CYPA and Myc-EBNA1 were detected by WB.
FIGURE 3
FIGURE 3
Effect of CYPA on EBNA1 expression and loss of viral copy numbers. (A) Designations of the CYPA siRNAs. Three siRNAs for CYPA and a control siRNA were designed and used for detection of the interference efficiency. HEK293 cells were transfected with one of the siRNAs, and a WB assay was performed at 48 h post-transfection for analysis of the CYPA protein level. (B,C) Effect of siCYPA on EBNA1 expression in EBV-positive cells. Two EBV-positive cell lines [C666-1 (B) and C2089 (C)] were used for siRNA transfection. At 48 h post-transfection, the cell lysates and RNA were subjected to the WB and RT-qPCR assays respectively, with siCYPA standarized to 1. (D,E) Effect of CYPA overexpression on EBNA1 expression at protein and mRNA levels in EBV-positive cells [C666-1 (D) and C2089 (E)]. (F) The effect of CYPA depletion on loss of EBV copy numbers during passages. EBV-positive C2089 was used for shRNA stable transfection and selection. Cell lines stably transfected with shRNA-CYPA or shRNA-NC were established for the experiment. Cell dispersal for each passage was performed at a ratio 1:2. The copy number of the C2089-shCYPA cells was decreased compared with the C2089-shNC, with passage 10th of C2089-shCYPA set to 1. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗ P < 0.001.
FIGURE 4
FIGURE 4
The effect of CYPA knockdown on EBNA1–mediated oriP transcription activity. (A) The detection of CYPA expression in stably transfected cell lines with shRNA-CYPA and shRNA-NC. (B) Restoration of EBNA1 protein expression in C2089-shCYPA cells transfected with wild-type CYPA expression plasmids. CYPA and EBNA1 proteins were detected by WB. (C) Effect of CYPA knockdown on EBNA1-oriP-mediated transcription activity in the luciferase reporter assay. The EBNA1 mRNA was detected by RT-qPCR. (D) Effect of CYPA knockdown on binding of EBNA1 to oriP in the ChIP assay. CYPA was depleted from HEK293 with shRNA. Antibodies against Myc and IgG control were respectively used for the pulldown in ChIP assaysHEK293. The precipitation of oriP DNA was quantitated by RT-qPCR. CYPA and EBNA1 proteins were detected by WB. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗ P < 0.001.
FIGURE 5
FIGURE 5
The effect of CYPA-specific inhibitor CsA on EBNA1–mediated oriP transcription and EBNA1-oriP binding. (A) The concentration determination of CsA by Cell Counting Kit-8 (CCK-8) assay. The concentration of 40 μM at 48 h was determined as the working concentration for treatment. (B) Rescue experiment of EBNA1 protein level after CsA treatment in C2089 cells. (C) The protein expression of CYPA and EBNA1 detected by WB assay at 48 h post-treatment with CsA. The mRNA expression of EBNA1 measured by RT-qPCR. EBV-positive C2089 cell lines were used for the test. (D) C2089 cells were transfected with oriP-SV40-Luc reporter plasmid and CYPA expression plasmids. Overexpressed CYPA significantly increased EBNA1- oriP-dependent luciferase activity, but had no effect on SV40 promoter dependent luciferase activity (left). EBNA1 mRNA was measured by RT-qPCR (right). (E) OriP-SV40-Luc reporter plasmid was transfected into C2089 cells, following the CsA treatment. CsA treatment greatly reduced EBNA1- oriP luciferase activity compared with untreatment. (F) CsA and elevated CYPA on EBNA1-oriP-mediated transcription activity in the luciferase reporter assay in HEK293 cells. (G) ChIP- qPCR was used to determine EBNA1-oriP binding. CsA treatment reduced EBNA1-oriP binding while elevated CYPA increased binding. CYPA and EBNA1 proteins were analyzed by WB. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.
FIGURE 6
FIGURE 6
The effect of ectopic CYPA expression on USP7-EBNA1 and EBNA1-oriP binding. (A) Effect of CYPA overexpression on EBNA1-USP7 binding in HEK293-shCYPA and HEK293-shNC cells. These cells were transiently transfected with pCAGGS-Flag-CYPA alone or with pCAGGS-Myc-EBNA1, a co-IP assay was performed with the anti-Myc antibody for the pulldown. (B) The inhibition of CsA on CYPA-USP7 antagonism in the binding with EBNA1. pCAGGS-Myc-EBNA1 was transfected with pCAGGS-Flag-CYPA or alone, and treated with CsA. The anti-Myc antibody was used for the pulldown, and proteins were detected by WB. (C) Effect of CYPA overexpression and EBNA1Δ376-459 mutation on the EBNA1-oriP binding detected by ChIP assay. ∗P < 0.05, ∗∗∗P < 0.001.
FIGURE 7
FIGURE 7
Schematic for the mechanism of CYPA in supporting the replication function of EBNA1. EBV genomic DNA exists within the host genome in the form of extrachromosomal episomes. Cytoplasmic CYPA can be hijacked by EBNA1 into the nucleus. Overexpressed CYPA can overcome the suppression of USP7 in binding to EBNA1. Nuclear CYPA mediates EBNA1-oriP transcription, and thus contributing to the viral genome replication and maintenance.
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