Human cytomegalovirus induces the interferon response via the DNA sensor ZBP1 - PubMed (original) (raw)

Human cytomegalovirus induces the interferon response via the DNA sensor ZBP1

Victor R DeFilippis et al. J Virol. 2010 Jan.

Abstract

Human cytomegalovirus (HCMV) is a member of the betaherpesvirus family that, unlike other herpesviruses, triggers a strong innate immune response in infected cells that includes transcription of the beta interferon gene via activation of interferon regulatory factor 3 (IRF3). IRF3 activation requires signaling from pattern recognition receptors that is initiated by their interaction with specific pathogen-associated molecules. However, while IRF3-activating pathways are increasingly well characterized, the cellular molecules involved in HCMV-mediated IRF3-dependent beta interferon transcription are virtually unknown. We undertook a systematic examination of new and established IRF3-terminal pathway components to identify those that are essential to HCMV-triggered IRF3 activation. We show here that IRF3 activation induced by HCMV infection involves the newly identified protein STING but, in contrast to infections with other herpesviruses, occurs independently of the adaptor molecule IPS-1. We also show that the protein DDX3 contributes to HCMV-triggered expression of beta interferon. Moreover, we identify Z-DNA binding protein 1 (ZBP1) as being essential for IRF3 activation and interferon beta expression triggered by HCMV, as well as being sufficient to enhance HCMV-stimulated beta interferon transcription and secretion. ZBP1 transcription was also found to be induced following exposure to HCMV in a JAK/STAT-dependent manner, thus perhaps also contributing to a positive feedback signal. Finally, we show that constitutive overexpression of ZBP1 inhibits HCMV replication. ZBP1 was recently identified as a cytosolic pattern recognition receptor of double-stranded DNA, and thus, we propose a model for HCMV-mediated IRF3 activation that involves HCMV-associated DNA as the principal innate immune-activating pathogen-associated molecular pattern.

PubMed Disclaimer

Figures

FIG. 1.

FIG. 1.

IPS-1 does not contribute to UV-HCMV-mediated IRF3-dependent transcription, IFN-β transcription, or IFN-β secretion. (A) THF-P55C1B cells were treated in duplicate for 6 h with UV-HCMV at the indicated MOIs. The values presented are average levels of detected luciferase plus standard errors of the mean (SEM) from cell lysates; (B) THF-ISRE cells were treated in duplicate for 6 h with IFN-β at the indicated concentrations. The values presented are average levels of detected luciferase (plus SEM) from cell lysates. (C) Immunoblot (IB) showing diminishment of IPS-1 and IRF3 proteins from THF-ISRE cells treated with the indicated siRNAs. (D) IFN-β-dependent luciferase expression (plus SEM) from THF-ISRE cells following treatment with the indicated siRNAs and subsequent exposure to either SeV or UV-HCMV (MOI = 1). (E) IRF3-dependent luciferase expression (plus SEM) from THF-P55C1B cells following treatment with the indicated siRNAs and subsequent exposure to UV-HCMV (MOI = 1). (F) IFN-β gene transcription change relative to untreated cells following exposure of siRNA-treated THFs to UV-HCMV. The values are the changes of UV-HCMV-treated cells relative to untreated cells normalized to the changes obtained from THFs treated with NS siRNA (set to 1).

FIG. 2.

FIG. 2.

IRF3 activation and STAT1-dependent signaling contribute to UV-HCMV-induced upregulation of ZBP1 mRNA. (A) Change as determined by qPCR of ZBP1 mRNA in THFs exposed to UV-HCMV relative to that in unexposed cells following transfection of NS or IRF3-directed siRNA. (B) Change of ZBP1 mRNA relative to untreated cells in THFs and THF-STAT1-DN cells treated with IFN-β (100 U/ml) or UV-HCMV in the presence and absence of CHX (200 μg/ml; untreated control cells were also treated with CHX). The values are the changes in treated relative to untreated cells normalized to the change observed in IFN-β-treated THFs (set to 1).

FIG. 3.

FIG. 3.

ZBP1 contributes to UV-HCMV-triggered IRF3-dependent transcription and IFN-β secretion. (A) qPCR showing UV-HCMV-triggered induction of ZBP1 mRNA relative to untreated THFs in the presence of ZBP1-targeted or NS siRNA. The values represent ZBP1 mRNA change relative to that of unexposed cells normalized to the change observed in cells treated with NS siRNA (set to 1). (B) Change in IFN-β gene transcription relative to that in unexposed cells following exposure of siRNA-treated THFs to UV-HCMV. The values (plus standard errors of the mean) are the changes in UV-HCMV-treated cells relative to untreated cells normalized to the changes obtained from THFs treated with NS siRNA (set to 1). (C) Expression of IRF3-dependent luciferase following treatment of THF-P55C1B cells with UV-HCMV in the presence of NS or ZBP1-specific siRNAs. The values presented (plus standard errors of the mean) are normalized to NS siRNA-treated cells (set to 1). (D) Expression of IFN-β-dependent luciferase following treatment of THF-ISRE reporter cells with UV-HCMV in the presence of NS or ZBP1-specific siRNA. The values displayed (plus standard errors of the mean) are normalized to UV-HCMV-induced luciferase expression in NS siRNA-treated cells (set to 1). (E) IFN-β gene transcription change relative to unexposed cells following exposure of siRNA-treated THFs to HCMV and CHX (200 μg/ml). The values (plus standard errors of the mean) are the changes in HCMV- and CHX-treated cells relative to those in non-HCMV-treated, CHX-treated cells that were normalized to HCMV-induced changes obtained from THFs treated with NS siRNA and CHX (set to 1).

FIG. 4.

FIG. 4.

Ectopic expression of ZBP1 increases UV-HCMV-induced IFN-β transcription independently of IPS-1. (A) Immunoblot showing overexpression of ZBP1 in two stable THF lines. (B) Induction of IFN-β mRNA as measured by qPCR in UV-HCMV-exposed relative to unexposed cells; the values shown are normalized to the IFN-β induction observed in THF parental cells (set to 1). (C) Immunoblot showing siRNA-mediated knockdown of stably expressed ZBP1 from THF-ZBP1 cells using two different ZBP1-targeted siRNA sequences. (D) Induction of IFN-β mRNA as measured by qPCR in UV-HCMV-exposed relative to unexposed THF-ZBP1 cells following treatment with the indicated siRNAs; the values shown are normalized to the IFN-β induction observed in THF-ZBP1 cells treated with NS siRNA (set to 1). (E) IFN-β secretion as measured by luciferase expression from THF-ISRE cells following exposure to media collected from THF or THF-ZBP1 cells that were either untreated or treated with UV-HCMV. The values (plus standard errors of the mean) are normalized to untreated THFs that were not exposed to UV-HCMV (set to 1). *, P < 0.05.

FIG. 5.

FIG. 5.

Overexpression of ZBP1 inhibits replication of HCMV. In duplicate, THF or THF-ZBP1 no. 1 cells were infected with HCMV as described in the text. Virus from infected cell media was then quantified using endpoint dilution assays of primary human fibroblasts. The values presented are PFU/ml plus the standard error of the mean for each cell type.

FIG. 6.

FIG. 6.

STING contributes to UV-HCMV-induced IRF3-dependent transcription, IFN-β transcription, and IFN-β secretion. (A) Immunoblot (IB) showing siRNA-mediated depletion of STING protein from UV-HCMV-treated THFs treated with STING-specific, but not NS, siRNA. (B) Change of STING gene transcription in cells treated with UV-HCMV in the presence of either NS or STING-directed siRNA relative to that in cells treated with NS siRNA only. (C) Change of IFN-β gene transcription in UV-HCMV-exposed relative to unexposed THFs following treatment with the indicated siRNAs. The values displayed (plus standard errors of the mean) represent UV-HCMV-induced change with siRNA treatment normalized to the changes obtained from THFs treated with NS siRNA (set to 1). (D) Expression of IRF3-dependent luciferase following treatment of THF-P55C1B reporter cells with UV-HCMV in the presence of NS or STING-specific siRNA. The values displayed (plus standard errors of the mean) are normalized to UV-HCMV-induced luciferase expression in NS siRNA-treated cells (set to 1). (E) Expression of IFN-β-dependent luciferase following treatment of THF-ISRE reporter cells with UV-HCMV in the presence of NS or STING-specific siRNA. The values displayed (plus standard errors of the mean) are normalized to UV-HCMV-induced luciferase expression in NS siRNA-treated cells (set to 1).

FIG. 7.

FIG. 7.

DDX3 is necessary for HCMV-induced IRF3-dependent transcription, IFN-β transcription, and IFN-β secretion. (A) Immunoblot (IB) showing siRNA-mediated depletion of DDX3 protein from THFs treated with DDX3-specific, but not NS, siRNA. (B) Change of IFN-β gene transcription in UV-HCMV-exposed relative to unexposed THFs following treatment with the indicated siRNAs. The values displayed (plus standard errors of the mean) represent the UV-HCMV-induced change with siRNA treatment normalized to the changes obtained from THFs treated with NS siRNA (set to 1). (C) Expression of IRF3-dependent luciferase following treatment of THF-P55C1B reporter cells with UV-HCMV in the presence of NS or DDX3-specific siRNA. The values displayed (plus standard errors of the mean) are normalized to UV-HCMV-induced luciferase expression in NS siRNA-treated cells (set to 1). (D) Expression of IFN-β-dependent luciferase following treatment of THF-ISRE reporter cells with UV-HCMV in the presence of NS or DDX3-specific siRNA. The values displayed (plus standard errors of the mean) are normalized to UV-HCMV-induced luciferase expression in NS siRNA-treated cells (set to 1).

FIG. 8.

FIG. 8.

STING, DDX3, and ZBP1, but not IPS-1, are required for UV-HCMV-stimulated IRF3 dimerization. Native immunoblots show the IRF3 dimerization status in THFs treated with the indicated siRNAs following mock (−) or actual (+) exposure to UV-HCMV.

FIG. 9.

FIG. 9.

Proposed model of HCMV-triggered, IRF3-dependent transcriptional induction and ZBP1 expression. The schematic shows HCMV-triggered IRF3 activation and HCMV-mediated induction of ZBP1 expression in human fibroblasts. The dashed arrows indicate de novo transcription or translation, and the solid arrows indicate molecular interaction or signal transduction (e.g., phosphorylation).

Similar articles

Cited by

References

    1. Abate, D., S. Watanabe, and E. Mocarski. 2004. Major human cytomegalovirus structural protein pp65 (ppUL83) prevents interferon response factor 3 activation in the interferon response. J. Virol. 78:10995-11006. - PMC - PubMed
    1. Ablasser, A., F. Bauernfeind, G. Hartmann, E. Latz, K. A. Fitzgerald, and V. Hornung. 2009. RIG-I-dependent sensing of poly(dA:dT) through the induction of an RNA polymerase III-transcribed RNA intermediate. Nat. Immunol. 10:1065-1072. - PMC - PubMed
    1. Alford, C. A., S. Stagno, R. F. Pass, and W. J. Britt. 1990. Congenital and perinatal cytomegalovirus infection. Rev. Infect. Dis. 12:745-753. - PubMed
    1. Boehme, K. W., J. Singh, S. T. Perry, and T. Compton. 2004. Human cytomegalovirus elicits a coordinated cellular antiviral response via envelope glycoprotein B. J. Virol. 78:1202-1211. - PMC - PubMed
    1. Boyle, K. A., R. L. Pietropaolo, and T. Compton. 1999. Engagement of the cellular receptor for glycoprotein B of human cytomegalovirus activates the interferon-responsive pathway. Mol. Cell. Biol. 19:3607-3613. - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources