Assembly of the WHIP-TRIM14-PPP6C Mitochondrial Complex Promotes RIG-I-Mediated Antiviral Signaling
Authors: Changsheng Xing
Xin Cao
Peng Tan
Meng Lin
Xiao Yu
Qingyuan Zhu
Lian He
Yinyin Li
Jun Cui
Helen Y Wang
Chen Qian
Rong-Fu Wang
Journal: Molecular Cell (2017)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: ATPase domain; K63 ubiquitination; RIG-I-like receptor signaling; dephosphorylation; mitochondrial signalosome
Affiliations: Institute of Biosciences and Technology, College of Medicine, Texas A&M University, Houston, TX 77030, USA; Center for Inflammation and Epigenetics, Houston Methodist Research Institute, Houston, TX 77030, USA., Institute of Biosciences and Technology, College of Medicine, Texas A&M University, Houston, TX 77030, USA., Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, College of Life Sciences, Collaborative Innovation Center of Cancer Medicine, Sun Yat-sen University, Guangzhou 510275, China., Center for Inflammation and Epigenetics, Houston Methodist Research Institute, Houston, TX 77030, USA., Center for Inflammation and Epigenetics, Houston Methodist Research Institute, Houston, TX 77030, USA. Electronic address: ., Institute of Biosciences and Technology, College of Medicine, Texas A&M University, Houston, TX 77030, USA; Center for Inflammation and Epigenetics, Houston Methodist Research Institute, Houston, TX 77030, USA; Department of Microbiology and Immunology, Weill Cornell Medical College, Cornell University, New York, NY 10065, USA. Electronic address: .

Abstract:  Mitochondrial antiviral signaling platform protein (MAVS) acts as a central hub for RIG-I receptor proximal signal propagation. However, key components in the assembly of the MAVS mitochondrial platform that promote RIG-I mitochondrial localization and optimal activation are still largely undefined. Employing pooled RNAi and yeast two-hybrid screenings, we report that the mitochondrial adaptor protein tripartite motif (TRIM)14 provides a docking platform for the assembly of the mitochondrial signaling complex required for maximal activation of RIG-I-mediated signaling, consisting of WHIP and protein phosphatase PPP6C. Following viral infection, the ubiquitin-binding domain in WHIP bridges RIG-I with MAVS by binding to polyUb chains of RIG-I at lysine 164. The ATPase domain in WHIP contributes to stabilization of the RIG-I-dsRNA interaction. Moreover, phosphatase PPP6C is responsible for RIG-I dephosphorylation. Together, our findings define the WHIP-TRIM14-PPP6C mitochondrial signalosome required for RIG-I-mediated innate antiviral immunity. 

Retracted: No

PubPeer comments:
 As TRIM14 is an ISG, so polyI:C or Sev infection should increase the expression of TRIM14, however in 1B, nothing is increased, even worsely in 1C, Sev infection decrease its expression.  , 
 As TRIM14 is an ISG, so RIG-I KO lane should express less exdogenous Trim14.  , 
 In lane 4, p-IRF3 did not increase, but they claim that it increase.  , 
 In this experiment, there were two sets of experiments: one is whole cell lysates with western blot; and the second one is IP followed by IB experiments. In WCL part, VSV stimulation increased p-IRF3 level (lane 3, 4 vs 1, 2). That is exactly right result, as expected. In IP plus IB part, we described that  Further experiments using a WHIP antibody showed that pull-down of ubiquitinated RIG-I after VSV infection contained RIG-I with a higher molecular weight, suggesting that WHIP binds to ubiquitinated RIG-I through polyUb chains (Figure 4H). . , 
 #2 That s the overexpressed TRIM14 not endogenous. Please also refer to the response to comment #1. , 
 For comment#1 and 2, It was reported that TRIM14 is an ISG upon DNA virus stimulation (Chen et al. Mol Cell, 2016) or HBV infection in hepatic cells (Tan et al. Front Immunol, 2018) or Sev-stimulated A549 cells (Fig1C and 4C; Zhou et al. PNAS, 2014). However, it is not increased in cell line such as in THP-1 cells (Fig6C, WCE; Zhou et al. PNAS, 2014). In our paper, we reported that  human TRIM14 or mouse Trim14 mRNA had no change or little change after vesicular stomatitis virus (VSV) or encephalomyocarditis virus (EMCV) infection in both human monocyte THP-1 or RAW264.7 cells (Figures S1C and S1D). Furthermore, we did not observe much changes in protein level of TRIM14 after VSV or synthetic dsRNA stimulations in 293T, THP-1 or RAW264.7 cells. Therefore, our data suggest that TRIM14 is not an ISG in 293T, THP-1 or RAW264.7 cells (at least in our hand). In Fig1C, we did not use SeV but VSV stimulation. In Supplemental Figure S1 I, we showed that ectopic expression of Myc-TRIM14 in RIG-I WT and KO cells markedly increased p-IRF3 level in WT cells but not in RIG-I KO cells. These results suggest that TRIM14 regulates IRF3 activation through RIG-I. For comment #2, That s the overexpressed TRIM14 not endogenous. Please also refer to the response to comment #1. For comment #3, In this experiment, there were two sets of experiments: one is whole cell lysates with western blot; and the second one is IP followed by IB experiments. In WCL part, VSV stimulation increased p-IRF3 level (lane 3, 4 vs 1, 2). That is exactly right result, as expected. In IP plus IB part, we described that  Further experiments using a WHIP antibody showed that pull-down of ubiquitinated RIG-I after VSV infection contained RIG-I with a higher molecular weight, suggesting that WHIP binds to ubiquitinated RIG-I through polyUb chains (Figure 4H). . ,