Viral induction of the zinc finger antiviral protein is IRF3-dependent but NF-kappaB-independent

Abstract

The zinc finger antiviral protein (ZAP) is an interferon-stimulated gene that restricts the replication of retroviruses, alphaviruses, and filoviruses. Relatively little is known, however, regarding the detailed mechanism of ZAP induction during viral infections. We show that, although being inducible by either interferon or virus, expression of ZAP is more efficiently activated by virus than are several other classical interferon-stimulated genes and that viral induction of ZAP occurs under the direct control of interferon regulatory factor 3 (IRF3) independent of interferon paracrine/autocrine signaling. ZAP was up-regulated in cells unresponsive to type I and III interferons upon engagement of TLR3, retinoic inducible gene I/melanoma differentiation-associated gene 5 pathways, or ectopic expression of a constitutively active IRF3 mutant. Conversely, induction of ZAP by virus or dsRNA was severely impaired in cells expressing a dominant-negative mutant IRF3 and completely abrogated in cells lacking IRF3. In contrast to IRF3, ZAP induction was independent of NF-kappaB activity. Mutational analysis of the human ZAP promoter revealed that multiple interferon-stimulated response elements far distal to the transcription start site serve redundantly to control IRF3-dependent induction of ZAP transcription. Chromatin immunoprecipitation assays demonstrated that IRF3 selectively binds the distal interferon-stimulated response elements in human ZAP promoter following viral infection. Collectively, these data suggest that ZAP is a direct target gene of IRF3 action in cellular antiviral responses.

FIGURE 1.

IRF3-dependent induction of ZAP and OASL mRNAs by virus or dsRNA in cells deficient in both type I and III IFN signaling. A, immunoblot analysis of ISG56 in Hec1B cells mock treated, infected with 100 HAU/ml of SeV, or treated with 1000 units/ml of IFNα for 16 h. The asterisk denotes a nonspecific band. B, immunoblot analysis of ISG15 in Hec1B cells stably expression the control vector (HecNeo) or DN IRF3 (HecF3DN), 293FT, and Huh7 cells treated with 10 ng/ml of recombinant human IL-29 (lanes 1–4) or 500 units/ml of recombinant human IFNα (lanes 5–8) for 18 h. C, HecNeo and HecF3DN cells were mock infected or infected with SeV and subsequently immunoblotted for IRF3, SeV, and actin. In the IRF3 panel, FL denotes full-length IRF3, whereas ΔN denotes DN IRF3. D, IFN production in culture supernatants of HecNeo and HecF3DN cells that were mock infected or infected with SeV for 24 h, determined by VSV plaque reduction assay. The dashed line indicates the detection limit of the assay (∼3 units/ml). E, Northern blot analysis of ZAP, ISG56, and OASL mRNAs in HecNeo (lanes 1–7) and HecF3DN (lanes 8–11) cells growing in 100-mm dishes following the indicated treatments: IL-29 (10 ng/ml); IFNα (1000 units/ml); poly I:C transfection (T-pIC, 6 μg); poly(I·C) added to culture medium (M-pIC, 50 μg/ml); SeV (300 HAU); and transfection of a vector expressing IRF3/5D (6 μg). All of the treatments were done for 8 h except IRF3/5D, which was transfected into cells for 20 h before cell lysis and RNA extraction.

FIGURE 2.

IRF3 deficiency abrogates viral induction of ZAP. A, left panel, HeLa and HeLa cells stably expressing BVDV Npro (HeLaNpro) were mock infected or infected with 100 HAU/ml of SeV for 16 h. The cell lysates were immunoblotted for Npro (using anti-HA monoclonal antibody), IRF3, actin, and ISG56. Right panel, HeLa and HeLaNpro cells were mock treated or treated with 100 nm of epoxomicin for 12 h prior to immunoblot analysis of Npro, IRF3, and actin. B, Q-PCR analysis of ZAP, IFN-β, and OAS1 mRNA levels in HeLa and HeLaNpro cells mock treated or stimulated with 500 units/ml IFNα, 100 HAU/ml of SeV or transfected with 2 μg of in vitro transcribed HCV RNA for 8 h. mRNA abundance was normalized to cellular 28 S ribosomal RNA. Fold changes were calculated by dividing normalized mRNA abundance following various treatments by that of the mock treated HeLa cells.

FIGURE 3.

Induction of ZAP and MxA by IFNα and virus in nonmalignant human cells. MRC-5 and PH5CH8 cells were mock treated or treated with the indicated stimuli for 8 h. Total cellular RNA were subjected to Q-PCR analysis of ZAP (left panel) and MxA (right panel) transcripts. mRNA abundance was normalized to cellular 28 S ribosomal RNA. Fold changes were calculated by dividing normalized mRNA abundance following various treatments by that of the mock treated MRC-5 cells. Note that PH5CH8 cells expressed 6.8- and 6.4-fold higher basal level of ZAP and MxA mRNAs, respectively, than did MRC-5 cells.

FIGURE 4.

Activation of hZAP and hOASL promoters by induction of the TLR3 and RIG-I/MDA5 signaling pathways. A and B, promoter activities of hZAP(−2881) and pGL3basic in HeLa and Hec1B (A) and PH5CH8 cells (B) following indicated treatments. M-pIC, poly(I·C) added to culture medium. C, activities of hZAP(−2881) and hOASL promoters in Hec1B cells transiently expressing an control vector or individual signaling components in the RIG-I/MDA5 or TLR3 pathways. N-RIG and N-MDA5 denote the constitutively active CARD domain of RIG-I and MDA5, respectively. D, activation of hOASL promoter in Hec1B cells by ectopic expression of various forms of IRFs or the constitutively active RelA. IRF7Δ is a constitutive IRF7 mutant which has a internal deletion (deletion of amino acids 238–408). E, activation of wild type (WT) or the indicated mutant hOASL promoters or human OAS2 p69 promoter in Hec1B cells by IRF3/5D. mISRE_p and mISRE_d denote mutation in the proximal (−291 to −271) and distal (−335 to −315) ISRE, respectively (see supplemental Fig. S2A for further details). F, activation of various hZAP promoters by IRF3/5D in Hec1B cells. The wild type hZAP(−2881) promoter was also tested for its activation by other indicated IRFs or RelA.

FIGURE 5.

The five ISREs distal to transcription start site are essential and function in redundancy for viral induction of hZAP promoter, whereas they are dispensable for activation the promoter by IFN. A, schematic representation of the hZAP promoter reporter plasmids with various length of 5′-flanking sequence. The putative ISRE/IRF-E and STAT binding sites were indicated as filled circles and hatched bars, respectively. B and C, activation of various hZAP promoters by SeV in Hec1B cells (B) and by IFNα in Huh7 cells (C). D and E, activities of various hZAP promoter deletion mutants in Hec1B cells (D) and Huh7 cells (E) following mock treatment, SeV infection, or IFNα stimulation for 17 h. Note in B and C that the hZAP(−1438) always had a lower basal activity than other mutants, indicating the presence of a negatively regulatory element between positions −1438 and −800.

FIGURE 6.

The proximal STAT site (STAT V) is important for both virus- and IFN-induced activation of hZAP promoter. A, schematic representation of the hZAP promoter reporter plasmids bearing mutations at the STAT IV (mST4) or STAT V (mST5) sites or both (mST4 + 5). B, activities of various hZAP promoters in A549 (upper panel) and HeLa (lower panel) cells following IFNα stimulation (400 units/ml) or SeV infection (200 HAU/ml) for 8 h.

FIGURE 7.

Differential binding of IRF3 and STAT transcription factors to the hZAP promoter following virus infection or IFN stimulation. A, immunoblot analysis of ISG56 expression and phosphorylation status of STAT1 and STAT2 in HeLa cells following stimulation of IFNα (500 units/ml) for 1 and 8 h or infection with SeV (100 HAU/ml) for 16 h. Actin blot was shown as a loading control. B, ChIP analyses of IRF3 and STAT binding to the ISRE1–2, ISRE3–5, and STAT V sites within hZAP promoter in HeLa cells mock treated, stimulated with IFNα (400 units/ml) for 1 h, or infected with SeV (200 HAU/ml) for 8 h. The ChIP-enriched DNA levels analyzed by Q-PCR were normalized to input DNA, followed by subtraction of nonspecific binding determined with control IgG.

FIGURE 8.

NF-κB activation is neither sufficient nor required for induction of hZAP promoter by virus or dsDNA. A, promoter activities of hZAP(−2881), hOASL(−795), and PRDII in Hec1B cells mock treated or treated with TNFα (10 ng/ml), SeV (50 HAU), or IFNα (1000 units/ml) for 17 h. B and C, promoter activities of hZAP(−2486) (B) and hIL-8 (−162/+44) and PRDII (C) in HeLa Tet-Off cells conditionally expressing the NF-κB super-suppressor, IκBαMut (bearing S32A and S36A double mutations) that were manipulated to induce or repress IκBαMut expression. Where indicated, the cells were treated with TNFα, infected with SeV, or transfected with poly(dA·dT) for 17 h. Note in B that although the basal activity of hZAP promoter was lower in cells expressing IκBαMut, induction of the promoter by SeV or poly(dA·dT) was actually more than cells without IκBαMut expression (16.9- versus 9.1-fold and 9.9- versus 6.0-fold by SeV and poly(dA·dT), respectively).