Assembly of Epstein-Barr Virus Capsid in Promyelocytic Leukemia Nuclear Bodies

Abstract

The Epstein-Barr virus (EBV) capsid contains a major capsid protein, VCA; two minor capsid proteins, BDLF1 and BORF1; and a small capsid protein, BFRF3. During the lytic cycle, these capsid proteins are synthesized and imported into the host nucleus for capsid assembly. This study finds that EBV capsid proteins colocalize with promyelocytic leukemia (PML) nuclear bodies (NBs) in P3HR1 cells during the viral lytic cycle, appearing as nuclear speckles under a confocal laser scanning microscope. In a glutathione S-transferase pulldown study, we show that BORF1 interacts with PML-NBs in vitro. BORF1 also colocalizes with PML-NBs in EBV-negative Akata cells after transfection and is responsible for bringing VCA and the VCA-BFRF3 complex from the cytoplasm to PML-NBs in the nucleus. Furthermore, BDLF1 is dispersed throughout the cell when expressed alone but colocalizes with PML-NBs when BORF1 is also present in the cell. In addition, this study finds that knockdown of PML expression by short hairpin RNA does not influence the intracellular levels of capsid proteins but reduces the number of viral particles produced by P3HR1 cells. Together, these results demonstrate that BORF1 plays a critical role in bringing capsid proteins to PML-NBs, which may likely be the assembly sites of EBV capsids. The mechanisms elucidated in this study are critical to understanding the process of EBV capsid assembly. IMPORTANCE Capsid assembly is an important event during the Epstein-Barr virus (EBV) lytic cycle, as this process is required for the production of virions. In this study, confocal microscopy revealed that the EBV capsid protein BORF1 interacts with promyelocytic leukemia (PML) nuclear bodies (NBs) in the host nucleus and is responsible for transporting the other EBV capsid proteins, including VCA, BDLF1, and BFRF3, to these subnuclear locations prior to initiation of capsid assembly. This study also found that knockdown of PML expression by short hairpin RNA significantly reduces EBV capsid assembly capabilities. This enhanced understanding of capsid assembly offers potential for the development of novel antiviral strategies and therapies that can prevent the propagation and spread of EBV.

FIG 1

Nuclear entry of EBV capsid proteins. (A) P3HR1 cells were treated with TPA and sodium butyrate for 48 h and then incubated with monoclonal anti-VCA (a, e), rabbit anti-BDLF1 (b, f), rabbit anti-BORF1 (c, g), or rabbit anti-BFRF3 (d, h) antibodies. (B) EBV-negative Akata cells were transfected with pcDNA-VCA (a, e), pcDNA-BDLF1 (b, f), pEGFP-BORF1 (c, g), or pEGFP-BFRF3 (d, h). HA-tagged proteins were detected using rabbit anti-HA antibody and goat anti-rabbit IgG conjugated with Alexa Fluor 594. Cells were stained with DAPI to reveal the nucleus. Cells were examined using a confocal laser scanning microscope (LSM 510 META; Zeiss). Bar, 10 μm.

FIG 2

Involvement of BORF1 in the nuclear entry of BDLF1 and VCA. (A) EBV-negative Akata cells were cotransfected with pcDNA-VCA and pEGFP-BDLF1 (a to d), pcDNA-VCA and pEGFP-BORF1 (e to h), pcDNA-BDLF1 and pEGFP-BORF1 (i to l), pcDNA-VCA and pEGFP-BVRF2 (m to p), pcDNA-VCA and pEGFP-BdRF1 (q to t), or pcDNA-VCA and pEGFP-BVRF2(S116C) (GFP-BVS116C) (u to x). (B) EBV-negative Akata cells were transfected with pEGFP-mBORF1 (a to c) or cotransfected with either pEGFP-mBORF1 and pcDNA-VCA (d to g) or pEGFP-mBORF1 and pcDNA-BDLF1 (h to k). (C) EBV-negative Akata cells were cotransfected with pHcRed-BORF1 (b), pEGFP-BDLF1 (c), and pcDNA-VCA (d). (e) Merged image of the images in panels b, c, and d. HA-VCA and HA-ORF1 were detected using rabbit anti-HA antibody and goat anti-rabbit IgG conjugated with Alexa Fluor 594. Cells were examined using a confocal laser scanning microscope (LSM 510 META NLO; Zeiss). Bars, 10 μm.

FIG 3

Nuclear entry of BFRF3. EBV-negative Akata cells were cotransfected with pcDNA-VCA and pcDNA-myc-BFRF3 (a to d), pEGFP-BORF1 and pcDNA-myc-BFRF3 (e to h), or pcDNA-VCA, pEGFP-BORF1, and pcDNA-myc-BFRF3 (i to m). HA-VCA was detected using anti-HA antibody and goat anti-rabbit IgG antibody conjugated with Alexa Fluor 594. (i to m) Myc-BFRF3 was detected using mouse anti-Myc antibody and goat anti-mouse IgG antibody conjugated with Cy5. Bar, 10 μm.

FIG 4

Colocalization of EBV capsid proteins at PML-NBs. (A) P3HR1 cells were treated with TPA and sodium butyrate for 72 h. BORF1 and PML-NBs were stained using rabbit anti-BORF and mouse anti-PML antibodies, respectively. Goat anti-rabbit IgG antibody conjugated with Alexa Fluor 488 and goat anti-mouse IgG antibody conjugated with Alexa Fluor 594 were used as secondary antibodies. (B) EBV-negative Akata cells were transfected with pEGFP-BORF1 (a to d) or cotransfected with pcDNA-VCA and pEGFP-BORF1 (e to i), pcDNA-BDLF1 and pEGFP-BORF1 (j to n), or pcDNA-VCA, pTag-BORF1, and pcDNA-myc-BFRF3 (o to r). HA-VCA and HA-BDLF1 were detected using mouse anti-HA antibody and goat anti-mouse IgG antibody conjugated with Alexa Fluor 594. PML-NBs were detected using rabbit anti-PML antibody and goat anti-rabbit IgG antibody conjugated with Alexa Fluor-cy5 or Alexa Fluro-594. (C) 293 cells carrying bacmid 2089 (a to o) and 293 cells carrying bacmid M68 (p to ad) were treated with TPA and sodium butyrate for 3 days. VCA, BDLF1, and BORF1 were detected using rabbit anti-VCA, rabbit anti-BDLF1, and rabbit anti-BORF1 antibodies, respectively. Goat anti-rabbit IgG antibody conjugated with Alexa Fluor 594 was used as a secondary antibody. PML was detected using mouse anti-PML antibody and goat anti-mouse IgG antibody conjugated with Alexa Fluor 488. Cell nuclei were stained with DAPI. (D) 293T cells were cotransfected with pEGFP-PML and pHcRed-BORF1 for 24 h and then treated with 0.02% MMS. Images of a live cell were captured at 20-min intervals under a confocal laser scanning microscope, following MMS treatment (LSM 510 META NLO; Zeiss). Bars, 10 μm.

FIG 5

RNF4 and degradation of misfolded proteins. 293T cells were cotransfected with pcDNA-BORF1 and pEGFP-RNF4 (A) or pAtaxin3-79Q-HA and pCMV-RNF4 (B). Proteins in the lysates were detected by immunoblot analysis using anti-HA, anti-GFP, and anti-α-tubulin antibodies.

FIG 6

Interaction between PML and BORF1. (A) Bacterially expressed GST (lane 2), GST-VCA (lane 3), GST-BORF1 (lane 4), GST-BDLF1 (lane 5), and GST-BFRF3 (lane 6) were bound to glutathione-Sepharose beads and mixed with the lysates of 293T cells transfected with pEGFP-PML (lanes 2 to 6). Lane 1 was loaded with 1% of the lysate. Proteins pulled down by glutathione-Sepharose beads were analyzed via immunoblotting (IB) with anti-GFP antibody (lanes 1 to 6). Proteins on the beads were analyzed using anti-GST antibody (lanes 7 to 11). (B) The E. coli BL21(DE3)(pET32a-BORF1) lysate was mixed with glutathione-Sepharose beads labeled with GST (lane 2), GST-BDLF1 (lane 3), or GST-PML (lane 4). The lysate from 293T cells was also added to the reaction mixture for reactions involving GST-PML-glutathione-Sepharose beads (lane 5). Proteins on the beads were analyzed by immunoblotting, using rabbit anti-His antibody (lanes 2 to 5) and rabbit anti-GST antibody (lanes 6 to 9). Input lanes were loaded with 1% of the bacterial lysate.

FIG 7

PML and capsid assembly by EBV. (A) P3HR1 cells were untreated (Latent) or treated with TPA and sodium butyrate (T-S). After treatment, cells were transfected with pCMV-BDLF1 or pCMV-BFRF3 or cotransfected with these two plasmids to express BDLF1 and BFRF3. (B) Cells were infected with lentivirus expressing control shRNA (Ctrl-sh) or PML-specific shRNA (PML-sh) for 5 days and then treated with TPA and sodium butyrate or left untreated. Three days after lytic induction, the quantity of capsids produced by the cells was analyzed via ELISA. EBV capsids were captured by anti-BFRF3 antibody and detected using rabbit anti-BDLF1 antibody and goat HRP-conjugated anti-rabbit immunoglobulin antibodies. (C) P3HR1 cells (1 × 10⁵) were transfected with control shRNA and PML-specific shRNA. Cells were treated with TPA and sodium butyrate and cultured in 30 ml medium for 5 days. EBV particles were collected from the culture medium by centrifugation. The EBV genome in the viral particles was measured by real-time qPCR. (D) The PML, Rta, VCA, BORF1, BDLF1, and α-tubulin expressed by the cells were analyzed via immunoblotting 3 days after lytic induction.