Characterization of a novel protein containing TBC1 domain in the black tiger shrimp Penaeus monodon and its role in reponse to white spot syndrome virus infection

Abstract

TBC domain (TRE2/BUB2/CDC16) is a 200-amino-acid protein containing a Rab-GTPase-activating protein (GAP) homology domain required for regulating the activity of Rab proteins which are center of endocytic membrane trafficking in cells. From cDNA microarray analysis, a novel PmTBC1D20 gene with deduced amino acid sequences that showed significant similarity with TBC1D20 protein in many organisms was up-regulated in response to White Spot Syndrome Virus (WSSV) in the black tiger shrimp (Penaeus monodon). In this study, a full-length sequence of PmTBC1D20 gene was determined by Rapid Amplification of cDNA Ends technique. The full-length sequence contained 2,004 bp with an open reading frame encoding a 480 amino acid protein. The PmTBC1D20 mRNA was expressed in many tissues including hemocytes and up-regulated about 2.3 and 2.0 fold at 24 and 48 h after WSSV infection, respectively. The effect of PmTBC1D20 suppression upon WSSV infection in primary hemocyte cell culture using RNA interference technique was investigated. The result revealed that the transcription level of VP28 gene, an envelope protein of WSSV, was decreased by about 50%. Moreover, the suppression of PmTBC1D20 transcript in WSSV-infected shrimp reduced the expression of three WSSV genes; VP28, ie1, and WSV477 by 90% compared to the control shrimp. When observed under the fluorescence microscope using coimmunofluorescense technique, the VP28 transcription level in PmTBC1D20 suppressed WSSV-infected hemocyte cells detected with anti-VP28 antibody was also decreased. Localization of PmTBC1D20 protein using anti-TBC antibody in shrimp hemocyte revealed that PmTBC1D20 protein localized both in cytoplasm and nuclear membrane in all types of hemocyte cells and the PmTBC1D20 protein signal in hemocytes of 48 h-WSSV-infected shrimp was higher than in the hemocytes of uninfected shrimp.These results suggested that PmTBC1D20 is involved in WSSV infection.