Hepatitis C Virus NS4B Can Suppress STING Accumulation To Evade Innate Immune Responses
Authors: Chang Shu
Kouacou V Konan
Pingwei Li
C Cheng Kao
Guanghui Yi
Yahong Wen
Qingxia Han
Journal: Journal of virology (2016)
Discipline: MICROBIOLOGY
Keywords: cyclic dinucleotide; cGAMP; STING; HCV; genotype 1b
Affiliations: Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, USA ., Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, USA., Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA., Center for Immunology and Microbial Disease, Albany Medical College, Albany, New York, USA.

Abstract:  The cyclic dinucleotide 2',3'-cGAMP can bind the adaptor protein STING (stimulator of interferon [IFN] genes) to activate the production of type I IFNs and proinflammatory cytokines. We found that cGAMP added to the culture medium could suppress the replication of the hepatitis C virus (HCV) genotype 1b strain Con1 subgenomic replicon in human hepatoma cells. Knockdown of STING expression diminished the inhibitory effect on replicon replication, while overexpression of STING enhanced the inhibitory effects of cGAMP. The addition of cGAMP into 1b/Con1 replicon cells significantly increased the expression of type I IFNs and antiviral interferon-stimulated genes. Unexpectedly, replication of the genotype 2a JFH1 replicon and infectious JFH1 virus was less sensitive to the inhibitory effect of cGAMP than was that of 1b/Con1 replicon. Using chimeric replicons, 2a NS4B was identified to confer resistance to cGAMP. Transient expression of 2a NS4B resulted in a pronounced inhibitory effect on STING-mediated beta IFN (IFN-β) reporter activation compared to that of 1b NS4B. 2a NS4B was found to suppress STING accumulation in a dose-dependent manner. The predicted transmembrane domain of 2a NS4B was required to inhibit STING accumulation. These results demonstrate a novel genotype-specific inhibition of the STING-mediated host antiviral immune response. Importance: The cyclic dinucleotide cGAMP was found to potently inhibit the replication of HCV genotype 1b Con1 replicon but was less effective for the 2a/JFH1 replicon and infectious JFH1 virus. The predicted transmembrane domain in 2a NS4B was shown to be responsible for the decreased sensitivity to cGAMP. The N terminus of NS4B has been reported to suppress STING-mediated signaling by disrupting the interaction of STING and TBK1 and/or MAVS. We show that 2a/JFH1 NS4B has an additional mechanism to evade STING signaling through suppressing STING accumulation. 

Retracted: No

PubPeer comments:
 January 2018 correction for Figure 1D,  due to a labeling error  . ![file] Here is the original Figure 1D, with red boxes marking a possible duplication. Note that this is not a simple duplication, but rather a duplication that involved repositioning and adjustment of the brightness in the green channel. ![file] , 
 #1 **Below please find the original images taken by SP5 confocal microscopy using Leica LAS X software. ** The scale bars in the left bottom of images were automatically generated by software (default setting). Scale bars were cropped out in Fig. 1D because the images were flipped horizontally when transferring data from PC (software only works on PC) to Mac, and they were too small for publication. ![file] Both images shown in Fig1D (mock and 25 ug ml) were taken from mock-treated samples. I took about 10-20 images for each cGAMP (concentration from 0, 25, 50, 100, to 120 ug ml)-treated sample from microscope glass cover slide. Please also see the images record computer record taken at 5-8-2014. the number of images were also generated by software. Some images taken from the same glass slide may have some overlapping area (such as shown in red box). **The week overlapping signal in red box was due to photobleaching (also known as fading) in fluorescence microscopy (a common issue caused by laser of fluorescence microscopy), but NOT by adjusting the contrast brightness of images**.![file] ![file] The middle panel was mislabeled as 25 ug ml cGAMP-treated sample during preparation of images. Because it only serves as a control, similar to mock, in immuno-fluorescence assay, I didnot identify this mistake from submission to publication. All the original images including the original 25 ug ml treated images were sent to Journal of Virology ASM publisher for correction. ,