A novel role of Zebrafish TMEM33 in negative regulation of interferon production by two distinct mechanisms.

Long-Feng Lu,Fang Zhou,Zhuo-Cong Li,Jing-Yu Jiang,Yong-An Zhang,Can Zhang,Dan-Dan Chen,Shun Li,Xiao-Yu Zhou,Feng Xiong

doi:10.1371/journal.ppat.1009317

Abstract

The transmembrane protein 33 (TMEM33) was originally identified as an endoplasmic reticulum (ER) protein that influences the tubular structure of the ER and modulates intracellular calcium homeostasis. However, the role of TMEM33 in antiviral immunity in vertebrates has not been elucidated. In this article, we demonstrate that zebrafish TMEM33 is a negative regulator of virus-triggered interferon (IFN) induction via two mechanisms: mitochondrial antiviral signaling protein (MAVS) ubiquitination and a decrease in the kinase activity of TANK binding kinase 1 (TBK1). Upon stimulation with viral components, tmem33 was remarkably upregulated in the zebrafish liver cell line. The IFNφ1 promoter (IFNφ1pro) activity and mRNA level induced by retinoic acid-inducible gene (RIG)-I-like receptors (RLRs) were significantly inhibited by TMEM33. Knockdown of TMEM33 increased host ifn transcription. Subsequently, we found that TMEM33 was colocalized in the ER and interacted with the RLR cascades, whereas MAVS was degraded by TMEM33 during the K48-linked ubiquitination. On the other hand, TMEM33 reduced the phosphorylation of mediator of IFN regulatory factor 3 (IRF3) activation (MITA)/IRF3 by acting as a decoy substrate of TBK1, which was also phosphorylated. A functional domain assay revealed that the N-terminal transmembrane domain 1 (TM1) and TM2 regions of TMEM33 were necessary for IFN suppression. Finally, TMEM33 significantly attenuated the host cellular antiviral capacity by blocking the IFN response. Taken together, our findings provide insight into the different mechanisms employed by TMEM33 in cellular IFN-mediated antiviral process.

Highlights

The innate immune system constitutes the first line of host cell defense against invading pathogens [1]
We identify that zebrafish transmembrane protein 33 (TMEM33) negatively regulates IFN production by two distinct mechanisms: TMEM33 interacted with and degraded mitochondrial antiviral signaling protein (MAVS) through K48-linked ubiquitination; TMEM33 acted as a decoy substrate for cellular TANK-binding kinase 1 (TBK1) and reduced the phosphorylation of MITA/IFN regulatory factor 3 (IRF3)
The ORF of zebrafish TMEM33 was amplified from a cDNA library of Zebrafish liver (ZFL) cells stimulated with poly I:C to assess whether TMEM33 is involved in the antivirus process

Summary

Introduction

The innate immune system constitutes the first line of host cell defense against invading pathogens [1]. The RLR family comprises three members: RIG-I, melanoma differentiation-associated factor 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2) [3, 4] Upon activation, both RIG-I and MDA5 interact with downstream mitochondrial antiviral signaling protein (MAVS, known as VISA, IPS-1, and Cardif) [5,6,7,8] and mitochondrial- and endoplasmic reticulum (ER)-localized adapter protein mediator of IFN regulatory factor 3 (IRF3) activation (MITA) ( called STING, MPYS, and ERIS) [9,10,11,12]. These observations suggest that the RLR pathway is highly conserved from teleost fish to mammals [28]

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Results

Discussion

Conclusion