Grass Carp Laboratory of Genetics and Physiology 2 Serves As a Negative Regulator in Retinoic Acid-Inducible Gene I- and Melanoma Differentiation-Associated Gene 5-Mediated Antiviral Signaling in Resting State and Early Stage of Grass Carp Reovirus Infection

Abstract

Laboratory of genetics and physiology 2 (LGP2) is a key component of RIG-I-like receptors (RLRs). However, the lack of the caspase recruitment domains (CARDs) results in its controversial functional performance as a negative or positive regulator in antiviral responses. Especially, no sufficient evidence uncovers the functional mechanisms of LGP2 in RLR signaling pathways in teleost. Here, negative regulation mechanism of LGP2 in certain situations in retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5)-mediated antiviral responses was identified in Ctenopharyngodon idella kidney cells. LGP2 overexpression inhibits synthesis and phosphorylation of interferon regulatory factor 3/7 (IRF3/7), and mRNA levels and promoter activities of IFNs and NF-κBs in resting state and early phase of grass carp reovirus (GCRV) infection. Knockdown of LGP2 obtains opposite effects. Luciferase report assay indicates that LGP2 works at the upstream of RIG-I and MDA5. LGP2 binds to RIG-I and MDA5 with diverse domain preference and which is independent of GCRV infection. Furthermore, LGP2 restrains K63-linked ubiquitination of RIG-I and MDA5 in various degrees. These differences result in disparate repressive mechanisms of LGP2 to RIG-I- and MDA5-mediated signal activations of IFN-β promoter stimulator 1 and mediator of IRF3 activation. Interestingly, LGP2 also inhibits K48-linked RIG-I and MDA5 ubiquitination to suppress proteins degradation, which guarantees the basal protein levels for subsequently rapid signal activation. All these results reveal a mechanism that LGP2 functions as a suppressor in RLR signaling pathways to maintain cellular homeostasis in resting state and early phase during GCRV infection.

ROS-induced HSP70 promotes cytoplasmic translocation of high-mobility group box 1b and stimulates antiviral autophagy in grass carp kidney cells

“…For viral infection, CIK cells were plated for 24 h in advance and then infected with GCRV-GZ1208 at a multiplicity of infection (m.o.i.) of 0.1 as described previously (46).…”

“…Cytosol and nuclear proteins were extracted using cytosol/ nuclear protein isolation kits (Beyotime). WB and co-IP experiments were conducted as in our previous study (46). IP of GFP fusion proteins was performed using GFP trap beads (Chromotek) that consist of a single-domain anti-GFP Ab conjugated to an agarose bead matrix.…”

“…qRT-PCR amplification was carried out in a total volume of 15 l containing 7.5 l of BioEasy Master Mix (SYBR Green) (Hangzhou Bioer Technology Co., Ltd.), 5.1 l of nuclease-free water, 2 l of diluted cDNA (200 ng), and 0.2 l of each genespecific primer (10 M) (Table S2). The data were analyzed as described previously (22,46).…”

Grass Carp Reovirus Major Outer Capsid Protein VP4 Interacts with RNA Sensor RIG-I to Suppress Interferon Response

“…GCRV-II strain GCRV-097 was conserved in our lab. For viral infection, CIK cells were plated for 24 h in advance and then infected with GCRV-097 at a multiplicity of infection (MOI) of 1 as previously described [21].…”

“…In mammals, binding to the K63 ubiquitin chain in the CARDs domain is essential for activation of RIG-I [20]. The ability to bind the K63 polyubiquitin chain of grass carp RIG-I CARDs is similar to that in mammals [21].…”