Nucleosome assembly proteins bind to Epstein-Barr virus nuclear antigen 1 and affect its functions in DNA replication and transcriptional activation

Abstract

The EBNA1 protein of Epstein-Barr virus (EBV) plays several important roles in EBV latent infection, including activating DNA replication from the latent origin of replication (oriP) and activating the transcription of other latency genes within the EBV chromatin. These functions require EBNA1 binding to the DS and FR elements within oriP, respectively, although how these interactions activate these processes is not clear. We previously identified interactions of EBNA1 with the related nucleosome assembly proteins NAP1 and TAF-I, known to affect the replication and transcription of other chromatinized templates. We have further investigated these interactions, showing that EBNA1 binds directly to NAP1 and to the beta isoform of TAF-I (also called SET) and that these interactions greatly increase the solubility of EBNA1 in vitro. These interactions were confirmed in EBV-infected cells, and chromatin immunoprecipitation with these cells showed that NAP1 and TAF-I both localized with EBNA1 to the FR element, while only TAF-I was detected with EBNA1 at the DS element. In keeping with these observations, alteration of the NAP1 or TAF-Ibeta level by RNA interference and overexpression inhibited transcriptional activation by EBNA1 in FR reporter assays. In addition, EBNA1-mediated DNA replication was stimulated when TAF-I (but not NAP1) was downregulated and was inhibited by TAF-Ibeta overexpression. The results indicate that the interaction of EBNA1 with NAP1 and TAF-I is important for transcriptional activation and that EBNA1 recruits TAF-I to the DS element, where it negatively regulates DNA replication.

FIG. 1.

Coimmunoprecipitation of EBNA1 with nucleosome assembly proteins. Nucleosome assembly proteins were immunoprecipitated from Raji nuclear lysates by use of antibodies against NAP1, NAP2, TAF-I (α and β forms), or TAF-Iβ, and recovered proteins were Western blotted (WB) for EBNA1, NAP1, NAP2, or TAF-I. Immunoprecipitation was also performed with a nonspecific antibody (IgG) as a negative control. A 1/20 sample of the nuclear lysate used for immunoprecipitation is also shown (input).

FIG. 2.

Glycerol gradient sedimentation analysis of EBNA1-nucleosome assembly protein complexes. Equal molar ratios of EBNA1 and NAP1 (A) or EBNA1 and TAF-Iβ or TAF-Iα (B) were preincubated, analyzed on a glycerol gradient, and compared to the same proteins analyzed individually. Equal-volume fractions were collected from the top of each gradient and analyzed by SDS-PAGE and silver staining, and the pellet at the bottom of the tube (P) is also shown. Sedimentation positions are shown (at the top) for the molecular mass markers aldolase (158 kDa) and catalase (232 kDa), which were analyzed on an identical gradient. A sample of the protein(s) loaded on the gradients is also shown (input).

FIG. 3.

Effects of nucleosome assembly proteins on EBNA1 solubility. (A) EBNA1 and NAP1 were incubated alone or together (at an equal molar ratio) in buffer containing 50 to 250 mM NaCl. Soluble (S) and precipitated (P) proteins were then separated by centrifugation and analyzed by SDS-PAGE and Coomassie blue staining. A sample of the protein(s) prior to incubation is shown as “input.” The same experiment was also performed with an EBNA1 mutant lacking amino acids 325 to 376 (EBNA1Δ325-376). (B) Same experiment as in panel A, except that TAF-Iβ, TAF-Iα, or NAP2 was used in place of NAP1.

FIG. 4.

Effects of nucleosome assembly proteins on transcriptional activation by EBNA1. (A) Western blots of extracts of CNE2Z cells after transfection with siRNA against GFP (negative control), NAP1, NAP2, or TAF-I. Duplicate samples are shown. (B) After transfection with the indicated siRNA (or with siGFP in the second columns), cells were transfected with an EBNA1 expression plasmid or an empty plasmid (first column in each histogram), an FR-CAT reporter plasmid that is EBNA1 dependent, and a SEAP reporter plasmid that is independent of EBNA1. Effects on CAT expression (left) and SEAP expression (middle) were determined separately. CAT levels were also normalized to SEAP levels to account for any nonspecific transcriptional effects (right). **, P < 0.001 relative to the EBNA1 positive control. (C) CNE2Z cells were transfected with a plasmid overexpressing NAP1, NAP2, TAF-Iα, or TAF-Iβ or with empty plasmid (second columns) prior to transfection with EBNA1 expression, CAT reporter, and SEAP reporter plasmids and calculation of transcriptional activities as in panel B.

FIG. 5.

Effects of nucleosome assembly proteins on DNA replication activity of EBNA1. (A) CNE2Z cells were treated with siRNA against NAP1, NAP2, TAF-I, or GFP (negative control) and then transfected with an oriP plasmid expressing EBNA1 or lacking EBNA1 (first lane only). For TAF-I, two different siRNA sequences were used (oligo 1 and oligo 2). Three days later, oriP plasmids were harvested from the cells and linearized. One-tenth of the sample was saved as a recovery control (input), and then the remaining sample was digested with DpnI to digest any transfected plasmid that had not replicated in the human cells. Samples were analyzed by Southern blotting and probing for the oriP plasmid. The DpnI-resistant plasmid band is shown in the top panel. (B) Quantification of multiple experiments performed as in panel A. For TAF-I, the left panel is for oligo 1 and the right panel is for oligo 2. Values are shown relative to that for the sample with EBNA1 and siGFP within each experiment, which was set to 1.0. P values of <0.01 (**) and <0.05 (*) are indicated. (C) CNE2Z cells were transfected with plasmids overexpressing NAP1, NAP2, TAF-Iα, or TAF-Iβ or with empty plasmid (vector) along with the oriP plasmid expressing EBNA1 or lacking EBNA1. Replication of the oriP plasmids was then determined as in panel A. (D) Quantification of multiple experiments performed as in panel C. Values are shown relative to that for EBNA1 with an empty overexpression plasmid within each experiment, which was set to 1.0, and P values of <0.01 are indicated.

FIG. 6.

Localization of NAP1 and TAF-I to oriP elements by ChIP. (A) ChIP experiments were performed with Raji cells, using antibodies against EBNA1 (left), NAP1, TAF-I, or nonspecific rabbit IgG (right). Recovered DNA fragments were quantified by real-time PCR, using primer sets for the oriP DS and FR elements and the BZLF1 promoter region. The amplification signals were normalized to those from the same cell lysates prior to IP, using the same primer pairs. Signals from NAP1 and TAF-I antibody samples were expressed relative to that for the control IgG samples, which was set to 1. The results shown are from three independent experiments, with PCR quantification performed in triplicate for each experiment. (B and C) D98/Raji (B) and AGS-rEBV (C) cells were treated with siRNA against EBNA1 or GFP, and ChIP assays were performed for EBNA1 (left) and TAF-I (right) as in panel A.