SpCrus2 Glycine-Rich Region Contributes Largely to the Antiviral Activity of the Whole-Protein Molecule by Interacting with VP26, a WSSV Structural Protein.

Yue Wang,Wen-Hong Fang,Chao Zhang,Hong-Yu Ma,Xin-Cang Li

doi:10.3390/md19100544

Abstract

Crustins are cysteine-rich cationic antimicrobial peptides with diverse biological functions including antimicrobial and proteinase inhibitory activities in crustaceans. Although a few crustins reportedly respond to white spot syndrome virus (WSSV) infection, the detailed antiviral mechanisms of crustins remain largely unknown. Our previous research has shown that SpCrus2, from mud crab Scylla paramamosain, is a type II crustin containing a glycine-rich region (GRR) and a cysteine-rich region (CRR). In the present study, we found that SpCrus2 was upregulated in gills after WSSV challenge. Knockdown of SpCrus2 by injecting double-stranded RNA (dsSpCrus2) resulted in remarkably increased virus copies in mud crabs after infection with WSSV. These results suggested that SpCrus2 played a critical role in the antiviral immunity of mud crab. A GST pull-down assay showed that recombinant SpCrus2 interacted specifically with WSSV structural protein VP26, and this result was further confirmed by a co-immunoprecipitation assay with Drosophila S2 cells. As the signature sequence of type II crustin, SpCrus2 GRR is a glycine-rich cationic polypeptide with amphipathic properties. Our study demonstrated that the GRR and CRR of SpCrus2 exhibited binding activities to VP26, with the former displaying more potent binding ability than the latter. Interestingly, pre-incubating WSSV particles with recombinant SpCrus2 (rSpCrus2), rGRR, or rCRR inhibited virus proliferation in vivo; moreover, rSpCrus2 and rGRR possessed similar antiviral abilities, which were much stronger than those of rCRR. These findings indicated that SpCrus2 GRR contributed largely to the antiviral ability of SpCrus2, and that the stronger antiviral ability of GRR might result from its stronger binding activity to the viral structural protein. Overall, this study provided new insights into the antiviral mechanism of SpCrus2 and the development of new antiviral drugs.

Highlights

Antimicrobial peptides (AMPs) are evolutionarily conserved immune-defense components found in all living organisms, ranging from bacteria to mammals [1,2]
Our previous studies have demonstrated that SpCrus2 protein comprises three regions [17]: an signal peptide (SP) (1–17 AA), a glycine-rich region (GRR) (18–81 AA), and a cysteinerich region (CRR) (82–164 AA) (Figure 1a)
We noticed that the glycine content of SpCrus2 GRR was about 40%, and that most glycine residues were in the form of short glycine-rich repeats, such as GGF, GGH, GGG, GGGF, and GGGH, which are the common polypeptide repetitions in other GRRs

Summary

Introduction

Antimicrobial peptides (AMPs) are evolutionarily conserved immune-defense components found in all living organisms, ranging from bacteria to mammals [1,2]. AMPs are multifunctional molecules exhibiting broad-spectrum activity against bacteria, fungi, protozoa, and viruses. The notable characteristic of AMPs is that they are small, amphipathic proteins with hydrophobic and cationic properties [2,3]. The special amphipathic structure of AMPs enables the killing of target organisms by disrupting their cell membrane. The antibacterial mechanism of AMPs relatively differs from that of antibiotics, which lead to the development of drug-resistant bacteria through long-term abuse. AMPs are considered ideal therapeutic reagents of the future

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Conclusion