RESUMEN
Secreted protein acidic and rich in cysteine (SPARC) is an extracellular and non-structural glycoprotein. In shrimp, a significant function of SPARC in WSSV infection remains unclear. In this study, the full-length cDNA sequence of a secreted protein acidic and rich in cysteine -like was cloned from shrimp Litopenaeus vannamei (named as LvSPARC-L). LvSPARC-L contained an open reading frame of 1002 bp, encoding 333 amino acids. Bioinformatics analysis showed that LvSPARC-L contained a SPARC Ca2+-binding region in the C-terminus, a Kazal-type serine protease inhibitor domain and a BUD22 domain. Tissue distribution assay indicated that LvSPARC-L generally expressed in all tissues selected with a higher expression in hemocyte, stomach and pleoplod. In hepatopancreas and intestine, the relative expression of LvSPARC-L was significantly up-regulated following the WSSV challenge. Besides, the relative expression of viral immediately early gene IE1 and a late gene VP28 was significantly increased in the LvSPARCL-silenced shrimp. Furthermore, the relative expression of LvP53 and LvCaspase3 was extremely decreased in the stomach of dsLvSPARC-L treated shrimp, while that of LvP38 was not affected significantly. All data together suggest that LvSPARC-L might play an antiviral role by regulating apoptosis.
Asunto(s)
Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Osteonectina/genética , Osteonectina/inmunología , Penaeidae/genética , Penaeidae/inmunología , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos/química , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/inmunología , Secuencia de Bases , Perfilación de la Expresión Génica , Osteonectina/química , Filogenia , Alineación de SecuenciaRESUMEN
As an extremely virulent pathogen, white spot syndrome virus (WSSV) greatly threatens shrimp aquaculture worldwide. The interaction between virus and host is important for viral infection. In the present study, a yeast two-hybrid (Y2H) library was constructed to clarify the functions of wsv006, and the interaction between wsv006 and shrimp Litopenaeus vannamei (L. vannamei) was analyzed. Furthermore, we explored the role of the wsv006-interacting molecule L. vannamei COP9 constitutive photomorphogenic-like protein subunit 5 (LvCSN5) in WSSV infection. Y2H assay showed that wsv006 interacted with LvCSN5, and co-immunoprecipitation (Co-IP) assay confirmed such interaction. Multiple alignments of amino acid sequences with other species revealed that the LvCSN5 had high identity with Penaeusmonodon CSN5 (PmCSN5). LvCSN5 was mainly expressed in intestine, eye and hepatopancreas. In addition, the relative expression of LvCSN5 was significantly up-regulated both in intestine and hepatopancreas following the WSSV challenge. Besides, the relative expressions of IE1 and VP28, as well as the viral copy numbers were significantly increased in the LvCSN5-silenced shrimp. Our findings suggested that LvCSN5 was involved in WSSV infection by interacting with wsv006.
Asunto(s)
Proteínas de Artrópodos , Complejo del Señalosoma COP9 , Infecciones por Virus ADN , Hepatopáncreas , Intestinos , Penaeidae , Proteínas Virales , Virus del Síndrome de la Mancha Blanca 1 , Animales , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/metabolismo , Complejo del Señalosoma COP9/genética , Complejo del Señalosoma COP9/metabolismo , Infecciones por Virus ADN/inmunología , Infecciones por Virus ADN/metabolismo , Hepatopáncreas/metabolismo , Interacciones Huésped-Patógeno , Proteínas Inmediatas-Precoces/metabolismo , Inmunidad Innata , Intestinos/metabolismo , Penaeidae/inmunología , Unión Proteica , ARN Interferente Pequeño/genética , Técnicas del Sistema de Dos Híbridos , Regulación hacia Arriba , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral , Virus del Síndrome de la Mancha Blanca 1/fisiologíaRESUMEN
Cystatins B is an endogenous cysteine cathepsin inhibitor. In shrimp, cystatins B-like (CSTB-L) has not been characterized and its role in WSSV infection is largely unknown. In this study, a full-length 699 bp CSTB-L sequence with 291 bp open reading frame encoding a 96 amino acid from L.vannamei (Lv) was first cloned. The tissue distribution assay indicated that LvCSTB-L presented ubiquitous expression in most examined tissues, with the most predominant expression in the hepatopancreas and the weakest expression in the muscles. LvCSTB-L transcripts could be induced in the intestine and hepatopancreas by WSSV challenge. The relative expression level of IE1 and VP28 in the LvCSTB-L knockdown shrimp were increased significantly. In addition, the shrimp cumulative mortality was remarkably (p < 0.01) increased after LvCSTB-L knockdown. Moreover, following the LvCSTB-L silencing, significant decreases in the mRNA levels of p53, p38, caspase3, STAT and ERK were also observed. The results suggested that LvCSTB-L could play positively roles in antiviral immune response by JAK-STAT, MAPK and apoptotic pathway. These findings would further our understanding of shrimp antiviral response, and therefore help for virus control and prevention.