Functional characterization of cytosolic ddx41 sensor in mediating antiviral immune response in black tiger shrimp penaeus monodon

Abstract

DEAD (Asp-Glu-Ala-Asp) box polypeptide 41 (DDX41), a receptor belonging to the DExD family, was recently identified as an intracellular DNA sensor in vertebrates. Here, we investigated the presence of cytosolic DNA sensing, DDX41 in shrimp Penaeus monodon. By searching for gene involved in DNA sensing cascade within the shrimp P. monodon EST database, 3 cDNA fragments exhibited similarity to DDX41 in various species were identified. Sequences assembly resulted in a complete ORF of Penaeus monodon DDX41 (PmDDX41) which has 1868-bp encoding a putative protein of 620 amino acid residues. The multiple sequence alignment of deduced amino acid sequence of PmDDX41 with other species revealed that there are three conserved domains in the protein: DEADc domain, HELICc domain and Zinc finger domain. PmDDX41 shares 83% similarity with DDX41 in bee and crab. The transcript of PmDDX41 was detected in all tested tissues. Gene expression profile of PmDDX41 in hemocyte of pathogen challenged was analyzed by quantitative RT-PCR. The results showed that the PmDDX41 transcript of White Spot Syndrome Virus (WSSV) challenged shrimp was up-regulated at 6 and 48 hpi when compared to the control shrimp. On the contrary, after challenge with Vibrio harveyi, PmDDX41 mRNA expression was not significantly changed at all time points when compared with the control PBS-injected shrimp. Knockdown of PmDDX41 by dsRNA-mediated gene silencing resulted in a decrease of the mRNA expression level of PmIKKβ, PmIKKɛ, PmPEN3 and PmPEN5 genes. Moreover, PmDDX41-knockdown shrimp exhibited increase in the cumulative mortality after WSSV infection. In addition, PmDDX41 was localized in the cytoplasm of shrimp hemocytes and upon WSSV infection or stimulation the nucleic acid mimics, poly(dA:dT) and poly(I:C), it was found in both the cytoplasm and nucleus of hemocytes. Similar results were observed when PmDDX41 was transfected into human embryonic kidney 293T (HEK293T) cells. Immunoprecipitation further demonstrated that PmDDX41 bound to biotin-labeled poly(dA:dT) but not poly(I:C). The overexpression of shrimp PmDDX41 and mouse stimulator of interferon gene (MmSTING) in HEK293T cells synergistically promoted IFN-β and NF-κB promoter activity via the DEADc domain. Co-immunoprecipitation (Co-IP) also confirmed that there was an interaction between PmDDX41 and STING after stimulation with poly(dA:dT) but not poly(I:C). Our study is the first to demonstrate that PmDDX41 acts as a cytosolic DNA sensor that interacts with STING via its DEADc domain to trigger the IFN-β and NF-κB signaling pathways, thus activating antiviral innate immune responses.