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

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.

Involvement of a newly identified atypical type II crustin (SpCrus5) in the antibacterial immunity of mud crab Scylla paramamosain.

Crustins, the main AMP family in Crustacea, are generated as isoforms in many species and implicated in innate immune responses, but their detailed molecular mechanisms on susceptible bacteria remain largely unclear. Type II and type I crustins are distinguished by glycine-rich region (GRR), which is a major marker motif, and some type II crustins exhibit stronger antibacterial activities than their GRR deletion mutants. In the present study, a novel crustin, namely, SpCrus5, was functionally characterized from a commercially valuable crab Scylla paramamosain. SpCrus5 contained a typical cysteine-rich domain at the N-terminus, a conserved WAP domain in the center, and a special GRR at the C-terminus, which is located in a site that differs from that of GRRs in typical type II crustins found between signal peptides and cysteine-rich domains. SpCrus5 shared high similarities with most type II crustins, and it was more closely related to type II crustins than to other retrieved crustins. SpCrus5 was predominantly expressed in gills and remarkably upregulated after the crabs were challenged with Vibrio parahemolyticus or Staphylococcus aureus, suggesting that SpCrus5 might participate in antibacterial immune responses. To further elucidate how this C-terminal GRR affects the function of SpCrus5, we harvested a GRR deletion mutant (SpCrus5-ΔGRR) by deleting the GRR. Liquid growth inhibition assays demonstrated that the antimicrobial activity of SpCrus5 was stronger than that of SpCrus5-ΔGRR, and the antibacterial spectrum of the former toward Gram-negative bacteria was broader than that of the latter. Binding assays revealed that the microorganism-binding ability and polysaccharide-binding activity of SpCrus5 were stronger than those of SpCrus5-ΔGRR. SpCrus5 or SpCrus5-ΔGRR agglutinated all tested Gram-positive bacteria. Therefore, the antibacterial activities of SpCrus5 were stronger and broader than those of SpCrus5-ΔGRR, and the binding ability and agglutination activity might contribute to the antimicrobial activity of SpCrus5. These results revealed that the C-terminal GRR was necessary to produce an efficient antibacterial activity of SpCrus5. SpCrus5 was highly identical with most type II crustins and it functioned as many type II crustins did, indicating that SpCrus5 was more likely an atypical type II crustin than a type I crustin. This study revealed that SpCrus5 participated as an essential antimicrobial effector in immune responses and provided new insights into the underlying mechanisms of the sequence and function diversity of crustins.

Peptide matching between Epstein-Barr virus and human proteins.

Epstein-Barr virus proteins were examined for amino acid sequence matching to human proteins at the decapeptide level. We report that numerous EBV peptides of different length (from 10- to 13-mer) are present in 28 human proteins. The viral vs. human peptide overlap mainly involves the glycine-rich region allocated in the NH2 terminus of Epstein-Barr nuclear antigen 1 protein and host cellular components that play crucial roles in basic biochemical pathways, such as chromatin remodeling, RNA splicing, transmission across chemical/electrical synapses, and neurogenesis, and that, when altered, may characterize various pathologies such as immunodeficiency, systemic lupus erythematosus, myelination, and speech disorders. The present results might contribute to understand and define the (physio) pathological relationships and interactions occurring between EBV and the human host.