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

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.

A New Crustin Gene Homolog SpCrus8 Identified in Scylla paramamosain Exerting In Vivo Protection Through Opsonization and Immunomodulation

“…The tissues with high expression levels of crustacean crustins are mostly hemocytes, gills, intestines and stomachs. Of the crustins in S. paramamosain, except for CruSp, which was most expressed in the hemocytes, SpCrus2-6 were highly expressed in the gills (8,17,46). Additionally, it is noted that most crustins in shrimp were identified from hemocytes, such as SWDPm1 (47), Pc-crustin 4 (8), Pc-DWD (35), Fi-crustin (22), and LvSWD3 (48).…”

Characterisation of a novel crustin isoform from mud crab, Scylla serrata (Forsskål, 1775) and its functional analysis in silico

“…Instability index greater than 40, slightly negative GRAVY score and abundance of aliphatic residues like I, V and L (total 15% for MC-crustin) are properties associated to be observed with antiviral peptides (Chang and Yang 2013 ). Crustins are known to show antiviral properties (Du et al 2019 ; Wang et al 2021a , b ). iAMP pred supported the anti-viral potential for MC-crustin in the present study.…”

“…Since then, several crustin like sequences have been reported from various crustaceans such as Penaeus subtilis (Rosa et al 2007 ), Hyas araneus and Paralithodes camtschaticus (Sperstad et al 2009 ), Fenneropenaeus indicus (Antony et al 2010 ; Sruthy et al 2017 ), P. monodon (Antony et al 2011 ), Scylla serrata (Afsal et al 2011 ), Macrobrachium rosenbergii (Huang et al 2016 ) and Rimicaris sp. (Wang et al 2021a , b ). Crustins are 7–22 kDa antibacterial proteins in the crustacean hemolymph with the WAP domain containing protein superfamily (Smith et al 2008 ; Wang et al 2021a , b ).…”

“…(Wang et al 2021a , b ). Crustins are 7–22 kDa antibacterial proteins in the crustacean hemolymph with the WAP domain containing protein superfamily (Smith et al 2008 ; Wang et al 2021a , b ). They have a distinctive signature of 12 cysteine residues out of which four form the cysteine-rich region, while the remaining eight participate in four disulphide bonds, recognized as the 4-disulphide core domain/whey acidic protein domain (4DSC/WAP).…”

SpgC1qR interacts with WSSV VP28 exhibiting antiviral activity

“…GST pull-down assay was performed according to a previously reported method with slight modifications [42] . A total of 150 μL of glutathione–Sepharose 4B resin (50% beads slurry) after washing with PBS was incubated with a mixture of GST-fused Sp gC1qR (15 μg) and the recombinant viral protein VP28 (rVP28; 15 μg) for 2 h at room temperature.…”