ABSTRACT
TBC (TRE2/BUB2/CDC16) domain proteins contain an ≈ 200-amino-acid motif and function as Rab GTPase-activating proteins that are required for regulating the activity of Rab proteins, and so, in turn, endocytic membrane trafficking in cells. TBC domain family member 20 (TBC1D20) has recently been reported to mediate Hepatitis C virus replication. Herein, PmTBC1D20 identified from the black tiger shrimp, Penaeus monodon, was characterized and evaluated for its role in white spot syndrome virus (WSSV) infection. The full-length cDNA sequence of PmTBC1D20 contains 2003 bp with a predicted 1443 bp open reading frame encoding a deduced 480 amino acid protein. Its transcript levels were significantly up-regulated at 24 and 48 h by ≈ 2.3- and 2.1-fold, respectively, after systemic infection with WSSV. In addition, depletion of PmTBC1D20 transcript in shrimps by double stranded RNA interference led to a decrease in the level of transcripts of three WSSV genes (VP28, ie1 and wsv477). This suggests the importance of PmTBC1D20 in WSSV infection. This is the first report of TBC1D20 in a crustacean and reveals the possible mechanism used by WSSV to modulate the activity of the host protein, PmTBC1D20, for its benefit in viral trafficking and replication.
Subject(s)
Penaeidae/immunology , Penaeidae/virology , White spot syndrome virus 1/immunology , rab1 GTP-Binding Proteins/immunology , Amino Acid Sequence , Animals , Base Sequence , Molecular Sequence Data , RNA Interference , RNA, Small Interfering , Sequence Alignment , Sequence Analysis, DNA , Transcription, Genetic , Up-Regulation , rab1 GTP-Binding Proteins/geneticsABSTRACT
A unique isoform of crustin, crustinPm5, was identified from a gill-epipodite cDNA library of the tiger shrimp, Penaeus monodon. The crustinPm5 cDNA contains an open reading frame (ORF) of 510 bp encoding a 169 amino acid protein. The deduced amino acid sequence of crustinPm5 showed 38% and 37% overall sequence identity with those of crustinPm1 and crustin-likePm, respectively, two crustin isoforms previously reported. The crustinPm5 gene contained four exons interrupted by three introns whilst the upstream sequence contains a putative promoter with two potential binding sites for NF-kappaB, one complete heat-shock regulatory element (HSE) and five putative GATA factor binding sites. The transcripts of crustinPm5 were primarily observed in the epipodite and eyestalk and not in hemocytes. Expression analysis revealed that the transcript levels of crustinPm5, crustinPm1 and crustin-likePm in epipodite were up-regulated upon heat treatment and hyperosmotic salinity stress. The recombinant crustinPm5 exhibited antimicrobial activity against some Gram-positive bacteria in vitro, but did not inhibit the growth of any Gram-negative bacteria tested. These results, together with the transcript expression pattern, indicate a diverse function of the proteins in the crustin family particularly crustinPm5 that might function as a stress mediator in addition to its antibacterial action.
