学位論文内容の要旨
博士の専攻分野の名称

博士（医 学）氏 名
白鳥 聡一
学 位 論 文 題 名
Identification of ZAPS as a positive regulator of RIG-I-mediated antiviral response(パターン認識受容体 RIG-I を介した抗ウイルス応答を増強する新規調節因子 ZAPS の同定)
【Background and purpose】 Pathogen invasion is sensed by pattern recognition receptors(PRRs) of the innate immune system via its recognition of pathogen-associated molecular patterns(PAMPs). Viral RNAs can serve as a PAMP, and the retinoic acid-inducible gene I (RIG-I) is a key
PRR for the detection of positive- and negative-stranded RNA viruses in the cytoplasm of cells.
RNA carrying 5'-triphosphate modification (3pRNA) is shown to be an essential determinant forviral RNA recognition by RIG-I, which results in the activation of the downstream both interferon(IFN)-regulatory factor (IRF) 3 and nuclear factor-B (NF-B) pathways for the production of type
I IFNs and inflammatory cytokines. Accumulating evidence has been shown that RIG-I-mediatedpathways are important for the activation of innate immune responses to viral infection.
Poly(ADP-ribose) polymerases (PARPs) are known to regulate not only cell survival and celldeath, but also other diverse biological processes and pathogeneses of diseases. It has been reported
that some PARPs, including PARP-1 and PARP-13 (also termed ZAP; zinc-finger CCCH-type
antiviral protein 1), interact with viral molecules and are likely to show a direct regulatory effect ofcertain viral subsets. However, no interaction of the PARPs with host innate immune responses has
been defined. In this study, we clarify the role of PARPs in innate immunity, particularly in termsof its possible involvement in pattern recognition receptor-mediated signaling.
【Methods and Results】 To investigate the role of the PARPs in nucleic acid-induced innate
immune responses, we exogenously expressed PARP-1, PARP-2, PARP-7, PARP-9, PARP-12 andPARP-13, all of which were reported to be involved in microbial infection, inflammation andimmunity. We performed quantitative RT-PCR (qRT-PCR) analysis, and found that PARP-13/ZAP
uniquely showed a marked enhancing effect on the expression of IFN- mRNA upon transfection
with 3pRNA in HEK293T human embryonic kidney cells. Next, we examined whether there is any
difference between the two isoforms of PARP-13/ZAP: qRT-PCR analysis also revealed that theshorter isoform (we hereafter call this isoform ZAPS (zinc-finger CCCH-type antiviral protein 1,short form)) rather than the full-form ZAP, was selectively induced by IFN- or 3pRNA and had astronger effect on IFN- mRNA induction by stimulation with 3pRNA.
By conducting qRT-PCR experiments, we found that exogenous expression of ZAPS inHEK293T cells remarkably enhanced the induction of type I IFN genes with 3pRNA stimulation.
Consistent with this result, luciferase reporter analyses showed that 3pRNA stimulation resulted in
activation of the IFNB promoter in a manner dependent on the levels of ZAPS expression. Inaddition, we conducted RNA interference-based knockdown experiments with small interfering
RNA (siRNA) targeting ZAPS mRNA (siZAPS). qRT-PCR analyses showed that the induction ofIFN- mRNA was severely abrogated in siZAPS-treated CD14+ monocytes as well as similarly
treated other cell lines (HEK293T cells and A549 human lung epithelial adenocarcinoma cells).
The production of IFN- protein was also verified by enzyme-linked immunosorbent assay(ELISA). We also found that ZAPS overexpression in HEK293T cells strongly enhanced the3pRNA-induced activation of NF-B-driven luciferase reporter gene. Consistently, qRT-PCR
analyses showed that the induction of genes encoding other cytokines, such as tumor necrosis
factor (TNF), interleukin 6 (IL-6) and CXCL10, in response to 3pRNA stimulation, was diminishedin A549 cells, in which ZAPS mRNA was knocked down. These data indicated that ZAPS stronglypotentiates the activation of not only type I IFN but also NF-B in the RIG-I-mediated pathway.
Next, we investigated how ZAPS functions as a potentiator of RIG-I pathway. The IFN-
mRNA expression induced by the exogenous expression of ZAPS in HEK293T cells wasabolished by RNAi-mediated downregulation of not only several molecules situateddownstream of RIG-I but also RIG-I itself. Therefore, we speculated that ZAPS plays apossible role as a novel regulator in the proximal process of RIG-I-mediated signaling.
Confocal analysis and immunoprecipitation assay revealed that ZAPS is in association with RIG-I
after 3pRNA stimulation. Moreover, native page and immunoprecipitation assay showed that theformation of RIG-I oligomers induced by Newcastle disease virus (NDV) infection or stimulation
with 3pRNA was suppressed in siZAPS-treated HEK293T cells. These findings suggest that ZAPS
associates with RIG-I to promote the RIG-I oligomerization.
To evaluate the function of ZAPS in RIG-I-mediated antiviral responses, we infected A549 cellswith influenza virus, which are known to activate RIG-I-mediated signaling. Knockdown of ZAPS
expression by siRNA impaired influenza virus-induced mRNA expression of type I IFN, IL-6, TNF
and CXCL10 and the production of IFN- protein, which were assessed by qRT-PCR and ELISA,respectively. By qRT-PCR analyses, we also detected higher expression of a viral nucleoprotein
gene in siZAPS-treated A549 cells than control siRNA-treated A549 cells following influenza virus
infection. Moreover, qRT-PCR analysis showed that exogenous expression of ZAPS in HEK293T
cells resulted in much more induction of IFN- mRNA after influenza virus infection, andconsistent with this result, a notable suppression of viral replication was observed byplaque-forming assay. A similar observation was also obtained for NDV infection. These suggestthat ZAPS exerts its antiviral activity through the RIG-I-mediated pathways.
【Discussion】 In this study, we demonstrated that PARP-13 is a regulator of RIG-I-mediatedantiviral signaling in human cells. We also found that ZAPS, the shorter isoform of PARP-13, isselectively upregulated by stimulation with 3pRNA, possibly through type I IFN signaling. It has
been speculated that ZAPS acts as a positive feedback regulator for RIG-I-mediated type I IFN
pathway. In this regard, it is the next issue to clarify how each isoform is differently regulated.
Activation of RIG-I after ligand binding is thought to be a multistep process that includes theactivation of its ATPase activity, conformational changes and oligomerization. The present data
have demonstrated that ZAPS interacts with RIG-I after 3pRNA stimulation, and oligomerizationof RIG-I, possibly by stabilizing the RNA-RIG-I complex, although the underlying mechanism
needs to be investigated further. The results also indicated that ZAPS expression alone slightly
enhances the expression of IFN- mRNA and the activation of the IFNB promoter independently of
any ligands. Consistent with these data, we detected the ZAPS-RIG-I interaction before stimulation,albeit very weakly. This disconnection of ligand dependence may suggest that this interaction issufficient to promote small amounts of signaling but that the overall signaling program is mostefficiently triggered by the binding of ligand to RIG-I in addition to its activation by ZAPS.
The result showing that ZAPS is critically involved in antiviral innate immune responses toinfluenza virus and NDV raises the possibility that ZAPS have an important role against otherviruses which activate RIG-I signaling. It is also needed to investigate the role of ZAPS in other
PRR-activated innate signaling. Previous reports show that ZAP has a role in the decay of mRNAs,
which are derived from certain viral subsets. Together with this observation, our present data
indicate that ZAPS might exert a "dual-mode" defense activity against viral infection. How theseactivities are regulated remains to be clarified.
【Conclusion】 This study indicates that ZAPS has a key role as a positive regulator ofRIG-I signaling during innate antiviral immune responses, suggesting that ZAPS may be an
important target for therapeutic intervention in the control of viral diseases.