Abstract
The antiviral cGMP-AMP (cGAMP)-stimulator of interferon genes (STING) pathway is well characterized in mammalian cells. However, whether this pathway also plays a role in insect antiviral immunity is unknown. In this study, we found that cGAMP is produced in silkworm (Bombyx mori) cells infected with nucleopolyhedrovirus (NPV). In searches for STING-related sequences, we identified BmSTING, a potential cGAMP sensor in B. mori We observed that BmSTING overexpression effectively inhibits NPV replication in silkworm larvae, whereas dsRNA-mediated BmSTING knockdown resulted in higher viral load. Cleavage and nuclear translocation of BmRelish, a NF-κB-related transcription factor, was also observed when BmSTING was overexpressed and was enhanced by cGAMP stimulation or viral infection of B. mori larvae. Moreover, we identified a caspase-8-like protein (BmCasp8L) as a BmSTING-interacting molecule and as a suppressor of BmSTING-mediated BmRelish activation. Interestingly, cGAMP stimulation decreased BmCasp8L binding to BmSTING and increased BmRelish activity. Of note, an interaction between death-related ced-3/Nedd2-like caspase (BmDredd) and BmSTING promoted BmRelish cleavage for efficient antiviral signaling and protection of insect cells from viral infection. Our findings have uncovered BmSTING as a critical mediator of antiviral immunity in the model insect B. mori and have identified several BmSTING-interacting proteins that control antiviral defenses.
Highlights
Over recent decades, the innate immune responses elicited by nucleic acids derived from viruses, bacteria, or protozoan
Based on the calibration curves of chemical-synthesized cGMP– AMP (cGAMP), we found that the concentrations of cytosolic cGAMP increased upon viral infection and reached a maximal level at ϳ6 h postinfection
stimulator of interferon genes (STING) is a critical signaling molecule required for the immune response to cytosolic nucleic acids, dsDNAs derived from pathogens and viruses as well as endogenous second messengers, such as c-di-GMP, cyclic-di-AMP [10], and cGAMP [22], in mammalian cells
Summary
CGAMP, was reported to be induced by DNA and DNA viruses [21, 22]. To determine whether DNA viral infection leads to the production of an activator in insect cells that triggers the innate immunity, we infected BmE cells with BmNPV-GFP and performed a LC coupled with tandem MS (LC-MS/MS) analysis of the cytosolic extract. The concentrations of cGAMP decreased and sustained from 24 to 72 hpi (Fig. 1, A and B) These results demonstrate the production of cGAMP in BmE cells after BmNPV infection. To determine whether cGAMP activates innate immunity in insects, we examined the mRNA levels of the antibacterial peptides BmCecA and BmCecB. Quantitative RT-PCR analysis showed that chemically synthesized cGAMP induced BmCecA and BmCecB in BmE cells (Fig. 1, C and D) as well as in the fat body of silkworm larvae (Fig. 1E)
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