Identification of a Shrimp E3 Ubiquitin Ligase TRIM50-Like Involved in Restricting White Spot Syndrome Virus Proliferation by Its Mediated Autophagy and Ubiquitination.

Chao Zhao,Lihua Qiu,Pengfei Wang,Chao Peng,Lulu Yan,Sigang Fan

doi:10.3389/fimmu.2021.682562

Abstract

Most tripartite motif (TRIM) family proteins are critical components of the autophagy machinery and play important roles in host defense against viral pathogens in mammals. However, the roles of TRIM proteins in autophagy and viral infection have not been studied in lower invertebrates, especially crustaceans. In this study, we first identified a TRIM50-like gene from Penaeus monodon (designated PmTRIM50-like), which, after a white spot syndrome virus (WSSV) challenge, was significantly upregulated at the mRNA and protein levels in the intestine and hemocytes. Knockdown of PmTRIM50-like led to an increase in the WSSV quantity in shrimp, while its overexpression led to a decrease compared with the controls. Autophagy can be induced by WSSV or rapamycin challenge and has been shown to play a positive role in restricting WSSV replication in P. monodon. The mRNA and protein expression levels of PmTRIM50-like significantly increased with the enhancement of rapamycin-induced autophagy. The autophagy activity induced by WSSV or rapamycin challenge could be inhibited by silencing PmTRIM50-like in shrimp. Further studies showed that rapamycin failed to induce autophagy or inhibit WSSV replication after knockdown of PmTRIM50-like. Moreover, pull-down and in vitro ubiquitination assays demonstrated that PmTRIM50-like could interact with WSSV envelope proteins and target them for ubiquitination in vitro. Collectively, this study demonstrated that PmTRIM50-like is required for autophagy and is involved in restricting the proliferation of WSSV through its ubiquitination. This is the first study to report the role of a TRIM family protein in virus infection and host autophagy in crustaceans.

Highlights

Protein post-translational modifications, such as phosphorylation, acetylation, and ubiquitination, are important biological processes that regulate many intracellular signaling pathways [1]
The replication of white spot syndrome virus (WSSV) in the dsPmTRIM50-like+Rap+WSSV group increased significantly compared with the dsGFP+Rap+ WSSV group, as a higher VP28 expression (Figures 5A, B, upper panel), higher WSSV copies (Figure 5C), and lower survival rate (Figure 5D) were detected in the dsPmTRIM50-like+ Rap+WSSV group. These results further showed that PmTRIM50-like is indispensable for autophagy induction and a possible mechanism for PmTRIM50-like-mediated inhibition of WSSV replication may be via mediating autophagy in shrimp
tripartite motif (TRIM) proteins are widely recognized as important antiviral restriction factors or modulators of signaling pathways that lead to the induction of antiviral or proinflammatory cytokines

Summary

Introduction

Protein post-translational modifications, such as phosphorylation, acetylation, and ubiquitination, are important biological processes that regulate many intracellular signaling pathways [1]. Ubiquitination is a post-translational modification pathway that regulates many host cellular processes, including DNA repair, differentiation, and regulation of immune responses [2]. E3 ubiquitin ligases are mainly responsible for determining the substrate specificity [5]. These enzymes operate in a concerted manner to polyubiquitinate a substrate protein and define its subsequent fate [6]. The chain of ubiquitin molecules regulates molecular signaling depending on the lysine linkage type of the poly-ubiquitin moiety, its length, and additional posttranslational modifications. The type of lysine linkage generally determines the fate of the substrate protein. K48-linked chain-directed ubiquitination of substrate protein leads to its proteasomal degradation, whereas a substrate protein conjugated with K63-linked chain is targeted for kinase-mediated cell signaling [7]

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Conclusion