EBV tegument protein BNRF1 disrupts DAXX-ATRX to activate viral early gene transcription

Abstract

Productive infection by herpesviruses involve the disabling of host-cell intrinsic defenses by viral encoded tegument proteins. Epstein-Barr Virus (EBV) typically establishes a non-productive, latent infection and it remains unclear how it confronts the host-cell intrinsic defenses that restrict viral gene expression. Here, we show that the EBV major tegument protein BNRF1 targets host-cell intrinsic defense proteins and promotes viral early gene activation. Specifically, we demonstrate that BNRF1 interacts with the host nuclear protein Daxx at PML nuclear bodies (PML-NBs) and disrupts the formation of the Daxx-ATRX chromatin remodeling complex. We mapped the Daxx interaction domain on BNRF1, and show that this domain is important for supporting EBV primary infection. Through reverse transcription PCR and infection assays, we show that BNRF1 supports viral gene expression upon early infection, and that this function is dependent on the Daxx-interaction domain. Lastly, we show that knockdown of Daxx and ATRX induces reactivation of EBV from latently infected lymphoblastoid cell lines (LCLs), suggesting that Daxx and ATRX play a role in the regulation of viral chromatin. Taken together, our data demonstrate an important role of BNRF1 in supporting EBV early infection by interacting with Daxx and ATRX; and suggest that tegument disruption of PML-NB-associated antiviral resistances is a universal requirement for herpesvirus infection in the nucleus.

Figure 1. BNRF1 binds the cellular protein Daxx.

(A) Colloidal blue stained SDS-PAGE of FLAG-immunoprecipitated BNRF1 and interacting partners. 293T cells were stably transfected with empty FLAG vector (V) or FLAG-tagged BNRF1 (B). Cell lysates were subject to Immunoprecipitation (IP) by anti-FLAG antibodies, then analyzed by SDS-PAGE. Bands unique to lane B were cut out and identified by LC/MS/MS. (B) IP confirmation of BNRF1/Daxx interaction. 293T cells were transiently transfected with empty vector (V) or wild-type BNRF1 (B). Cells harvested two days post-transfection were subject to IP with non-specific IgG, anti-FLAG or anti-Daxx antibodies, and analyzed by Western blot (WB) with anti-FLAG or anti-Daxx antibodies. (C) Summary of LC/MS/MS data from FLAG-BNRF1 purification. Genebank accession number (GI), percent of peptide coverage, number of peptides identified, and protein name are indicated.

Figure 2. Mapping the Daxx-interaction domain on BNRF1, and the effect of BNRF1 on the Daxx-ATRX complex.

(A) Diagram of wild-type BNRF1 (WT) and mutation constructs with 300 aa deletions (d1-d5). Dark gray block denotes the amino-terminal FLAG tag. Light gray blocks denote regions with sequence homology to the cellular enzymes Aminoimidazole ribonucleotide synthetase (AIR_S) and Type 1 glutamine amidotransferase (GATase1, an enzymatic domain of FGARAT), as identified by the National Center for Biotechnology Information (NCBI) conserved domain search. (B) IP pull down analysis of the BNRF1 deletion constructs. 293T cells were either transfected with empty FLAG vector (V), the BNRF1 constructs WT, or mutants d1-d5. Cell lysates of transfected cells were then subject to IP pull-downs with non-specific IgG, anti-FLAG, or anti-Daxx- antibodies, then Western blots were probed for Daxx (top panels), and FLAG-tagged proteins (lower panels). Input is shown for each mutant in the left most panels.

Figure 3. The Daxx-interaction domain on BNRF1 is located between sites 360-600 aa, and BNRF1 disrupts Daxx-ATRX binding.

(A) Diagram of BNRF1 mutation constructs with 60 aa deletions (d21–d25) within the 360–600 aa Daxx-interaction domain (DID) and the DID only. Blocks in the diagram drown as Fig. 2A. (B) IP analysis of BNRF1 60 aa deletion constructs. 293T cells were transfected with various BNRF1 expression vector constructs, and cell lysates were subject to IP pull downs with non-specific IgG and anti-FLAG antibodies, then Western blots were probed for Daxx (top panel), or FLAG-tagged proteins (lower panel). (C) IP analysis of 293T cells transfected with vector control (V), WT BNRF1 (WT), BNRF1 deletion mutants d22, d26, or DID. Input, IgG control IP, FLAG IP, or Daxx IP (panels left to right) as indicated above each panel. Western blots of IPs were probed with antibody to Daxx, ATRX, PML, FLAG, or Actin, as indicated to the left of each panel. (D) IP Western of 293T cells transfected with vector control (V), WT-BNRF1 (WT), or BNRF1 deletion mutants d1, d2, d3, d4, or d5. FLAG-IPs were analyzed by Western blot with antibodies to ATRX (top panel) Daxx (middle panel), or FLAG (lower panel).

Figure 4. BNRF1 co-localizes with Daxx at PML-NBs and disperses ATRX from PML-NBs.

Hep2 cells were transfected with either FLAG empty vector, WT BNRF1, or the deletion constructs d26 and DID. Cells were fixed 2 days post transfection and co-stained with anti-FLAG, and DAPI, and either anti-Daxx (A), anti-PML (B), or anti-ATRX (C) antibodies. Yellow regions in the merged panels denote co-localization of red and green signals. Remaining un-fixed, transfected Hep2 cells were subject to cell lysate Western blot analysis to confirm transfection efficiency and expression levels of the BNRF1 constructs (D). The number of nuclear bodies per cell nucleus was quantified by computational analysis of immunofluorescent microscopy images. A total of ten 40x magnification microscopy images of random fields were analyzed for each foci count. Foci counts of either Daxx (E), PML (F), or ATRX (G) were grouped into BNRF1 non-expressing and expressing sets. Bars on scatter plots denote the average foci per cell.

Figure 5. Stability and subcellular fractionation of BNRF1 proteins.

(A) FLAG vector (clone C) or FLAG-BNRF1 (clones 3 and 9) stably transfected 293T cells were lysed and analyzed by Western blot. Blots were probed with antibodies to PML, Daxx, ATRX, FLAG (BNRF1), or Actin, as indicated to the right. (B) 293T cells transfected with FLAG vector (V), WT-BNRF1 (WT), d26, or DID mutant for 48 hrs were prepared as total cell extracts (input), cytoplasmic or nuclear fractions. Transfected cells fractions were assayed by Western blot with antibody to FLAG (BNRF1), PARP1 (nuclear marker) or αTubulin (cytoplasmic marker).

Figure 6. △BNRF1 mutant virus infection can be rescued by ectopic complementation with wild type, but not Daxx-interaction-deficient BNRF1.

Primary B cells were infected with equal volumes of GFP expressing virus, either produced from wild type bacmids or △BNRF1 mutant virus complemented with empty FLAG vector, wild type, or the d26 Daxx-interaction-deficient mutant BNRF1. Infected cells would express the GFP carried in the virus. Infection rate as shown in fluorescent microscopy photos (A), or a manual count of the average number of GFP-positive cells per well (B). Aliquots of the virus used for infection were isolated for measurement of viral titers by real-time PCR analysis of the number of viral OriLyt DNA copy numbers (C). Viral particles reconstituted with FLAG vector, FLAG-tagged WT-BNRF1 or d26 mutant were concentrated and analyzed by Western blot with antibody to FLAG (D).

Figure 7. BNRF1 promotes viral gene expression.

(A) Primary B cells were infected with virus produced from △BNRF1 mutant bacmids either complemented with empty vector, wild type BNRF1 or the d26 Daxx-interaction-deficient mutant BNRF1. Virus infected cells were subject to reverse transcription PCR assay of viral gene expression for EBNA1, EBNA2, or BZLF1. (B) 293HEK cells were co-transfected with wild type EBV genomes and either empty vector, WT BNRF1 or the d26 Daxx-interaction-deficient mutant BNRF1. Transfected cells were subject to reverse transcription PCR assay of viral gene expression for EBNA1, EBNA2, EBNA3C, or BZLF1.

Figure 8. ATRX and Daxx depletion leads to reactivation of latent EBV.

Mutu I cells were transduced with lentivirus shRNA with non-targeting sequence (shNeg), or targeting for ATRX (shATRX), Daxx (shDaxx) or positive control ZEB1 (shZEB1.1). Transduced cells were selected for puromycin resistance for 9 days and then assayed by Western blot with antibodies to Zta, EA-D, Daxx, ATRX, ZEB1, or αTubulin, as indicated and visualized by HRP (A) and densitometric scanning of Western blot band intensities (B). Three replicates of Mutu I transduced as described above were assayed by flow cytometry with antibody to EBV viral capsid antigen VCA and quantified as % VCA positive fold change relative to shNeg. Error bars denote the standard error among the independent experiments (C).