Interferon regulatory factor 7 is negatively regulated by the Epstein-Barr virus immediate-early gene, BZLF-1

Abstract

Virus infection stimulates potent antiviral responses; specifically, Epstein-Barr virus (EBV) infection induces and activates interferon regulatory factor 7 (IRF-7), which is essential for production of alpha/beta interferons (IFN-alpha/beta) and upregulates expression of Tap-2. Here we present evidence that during cytolytic viral replication the immediate-early EBV protein BZLF-1 counteracts effects of IRF-7 that are central to host antiviral responses. We initiated these studies by examining IRF-7 protein expression in vivo in lesions of hairy leukoplakia (HLP) in which there is abundant EBV replication but the expected inflammatory infiltrate is absent. This absence might predict that factors involved in the antiviral response are absent or inactive. First, we detected significant levels of IRF-7 in the nucleus, as well as in the cytoplasm, of cells in HLP lesions. IRF-7 activity in cell lines during cytolytic viral replication was examined by assay of the IRF-7-responsive promoters, IFN-alpha4, IFN-beta, and Tap-2, as well as of an IFN-stimulated response element (ISRE)-containing reporter construct. These reporter constructs showed consistent reduction of activity during lytic replication. Both endogenous and transiently expressed IRF-7 and EBV BZLF-1 proteins physically associate in cell culture, although BZLF-1 had no effect on the nuclear localization of IRF-7. However, IRF-7-dependent activity of the IFN-alpha4, IFN-beta, and Tap-2 promoters, as well as an ISRE promoter construct, was inhibited by BZLF-1. This inhibition occurred in the absence of other EBV proteins and was independent of IFN signaling. Expression of BZLF-1 also inhibited activation of IRF-7 by double-stranded RNA, as well as the activity of a constitutively active mutant form of IRF-7. Negative regulation of IRF-7 by BZLF-1 required the activation domain but not the DNA-binding domain of BZLF-1. Thus, EBV may subvert cellular antiviral responses and immune detection by blocking the activation of IFN-alpha4, IFN-beta, and Tap-2 by IRF-7 through the medium of BZLF-1 as a negative regulator.

FIG. 1.

IRF-7 is expressed in lesions of oral hairy leukoplakia and can be detected in cellular nuclei. Frozen serial sections of HLP tissue were stained with antibodies to the indicated proteins and subjected to confocal microscopy. (A) IRF-7 (red) is detected in the nucleus, as well as the cytoplasm, of cells in the middle and upper stratum spinosum of an HLP lesion (arrows indicate nuclei). (B) BZLF-1 (green) is detected in the nucleus of cells in a corresponding region of a serial section of the same HLP lesion. (C) BZLF-1 is not detected in normal tongue (NT). (D) IRF-7 (red) expression in both the nucleus and the cytoplasm of cells in the middle stratum spinosum. (E) LMP-1 (green) is detected on the cell surface of cells in the middle stratum spinosum. (F) LMP-1 is not detected in normal tongue. (G) Preimmune rabbit serum. (H) IRF-2 is not detected in HLP. (I) IRF-7 is not detected in normal tongue. Magnification, ×400.

FIG. 2.

IRF-7 and BZLF-1 proteins physically associate. (A) 293 cells were transfected with the indicated expression plasmids, and IRF-7 (FL-IRF-7) and BZLF-1 were immunoprecipitated with Flag antibody or BZ-1 antibody, respectively. Immunocomplexes were subjected to SDS-PAGE and immunoblotted for IRF-7 (arrows, left panel) or BZLF-1 (arrows, right panel). (B) Endogenous IRF-7 and BZLF-1 proteins physically associate in Raji cells in which virus replication has been induced. Raji cells were treated with 5 μg of methotrexate/ml for 48 h, BZLF-1 was immunoprecipitated from the cell lysates, and the immunocomplexes were resolved by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted for IRF-7 and BZLF-1. Arrows indicate IRF-7 (right panels) and BZLF-1 (left panels).

FIG. 3.

Nuclear accumulation of GFP-IRF7 induced by LMP-1 is not inhibited by BZLF1. GFP fluorescence was detected by inverted light microscopy in 293 cells transfected with EGFP-IRF-7 (A); EGFP-IRF-7 and LMP-1 (B); EGFP-IRF-7 and BZLF-1 (C); or EGFP-IRF-7, LMP-1, and BZLF-1 (D). Subcellular localization of IRF-7 was examined 24 h after transfection. Fields of cells were scored to determine percentage of nuclear localization.

FIG. 4.

IFN-β, IFN-α4, and ISRE promoter activity is reduced in EBV-positive cells in which cytolytic viral reactivation has been induced. X50-7 or Raji cells were transfected with 10 μg of reporter plasmid and either BZLF-1 expression vector or control vector. At 24 h after transfection, cells were harvested for firefly luciferase assay and normalized to Renilla luciferase expression. (A) IFN-β promoter-reporter activity in induced X50-7 and Raji cells. (B) ISRE reporter construct activity in induced X50-7 cells. (C) IFN-α4 reporter-promoter activity in induced X50-7 cells.

FIG. 5.

TAP2 promoter activity is decreased in an ISRE-dependent manner in cells in which cytolytic replication has been induced. (A) X50-7 cells were nucleoporated (Amaxa) with either Tap-2 or mTap-2 promoter-reporter constructs, together with BZLF-1 or the vector control plasmids, and CD4 plasmid to a total of 5 μg of DNA. At 24 h after transfection, cells were harvested, a portion of which were used for CAT assays. The results were normalized with Renilla luciferase expression. Positions of point mutations are indicated at the top. (B) Endogenous TAP2 mRNA level is decreased during cytolytic viral reactivation. Remaining portions of the samples in panel A were pooled and enriched for transfected cells by CD4 selection. RNA was purified and used for real-time RT-PCR with Tap-2 mRNA specific probes. ΔΔC_T values represent the GAPDH-normalized ratio between the empty vector control and the BZLF-1-induced sample.

FIG. 6.

BZLF1 modulates the activation of IRF-7 by LMP1 in a dose dependent manner. 293 cells in 12-well plates were transfected with reporter plasmids (0.1 μg of IFN-β-luc, IFN-α4-luc, Tap-2 CAT, and ISRE-luc; 0.025 μg of pRL-TK) and expression plasmids encoding IRF-7 (0.1 μg), LMP-1 (0.1 μg), BZLF-1 (amounts as indicated), and BKRF-4 (amounts as indicated). Luciferase (A, B, and D) or CAT (C) assays were performed, and data were normalized to Renilla luciferase. Values are shown as the fold activation relative to IRF-7. (A) BZLF-1 inhibits LMP-1-induced activation of the IFN-β-luc promoter reporter by IRF-7. (B) BZLF-1 inhibits LMP-1-induced activation of the ISRE-luc reporter by IRF-7. (C) BZLF-1 inhibits LMP-1-induced activation of the Tap-2-CAT promoter reporter by IRF-7. (D) BZLF-1 inhibits LMP-1-induced activation of the IFN-α4-luc reporter by IRF7.

FIG. 7.

Deletion of the transactivation domain of BZLF1 inhibits its ability to deactivate IRF-7. 293 cells were transfected with reporter plasmids and expression plasmids encoding IRF-7, LMP-1, BZLF-1, BZLF-1 ΔTA, and BZLF-1 A185K as indicated. Luciferase assays were performed, and data were normalized to Renilla luciferase. Values are shown as the fold activation relative to IRF-7. Lower panel shows immunoblot for WT BZLF-1, BZLF-1 ΔTA, and BZLF-1 A185K.

FIG. 8.

Modulation of IRF7 by BZLF1 is downstream of signaling. (A) IRF-7 activation through the TLR-3/dsRNA pathway is inhibited by BZLF1. 293 cells and 293 cells stably expressing TLR-3 were transfected with reporter plasmids and expression plasmids for IRF-7, LMP-1, and BZLF-1 as indicated. Luciferase assays were performed, and data were normalized with Renilla luciferase. Values are shown as the fold activation relative to IRF-7. (B) Activation of the ISRE promoter by a constitutively active IRF7 mutant is blocked by BZLF-1. 293 cells were transfected with reporter plasmids and expression plasmids encoding IRF-7, IRF7 (D477/479), LMP-1, and BZLF-1 as indicated. Luciferase assays were performed as in panel A.