Characterization of viral responsive protein 15 and its possible role in nuclear import/export of virus in black tiger shrimp Penaeus monodon

Abstract

A viral responsive protein 15 (PmVRP15) has been identified from suppression subtractive hybridization library at the early (24 h) and late phase (48/72h). PmVRP15 was highly up-regulated in the hemocyte of white spot syndrome virus-infected Penaeus monodon. A protein domain prediction indicated that PmVRP15 consisted of a transmembrane helix. In primary hemocyte cultures, PmVRP15 mRNA expression was up-regulated about 1.1, 2.6, 3.6, 6.7 and 4.1 fold at 6, 12, 24, 48 and 72 h post-WSSV infection, respectively. After PmVRP15 gene silencing by RNA interference (RNAi), VP28 transcript was reduced by 4.2 fold at 24 h post WSSV-infection, compared to normal WSSV-infected P. monodon hemocytes. These results suggested that PmVRP15 is important to WSSV propagation. Confocal laser scanning microscopy study, revealed that PmVRP15 localized at the nuclear membrane. In this study, subcellular fractionation of WSSV-infected hemocytes showed that PmVRP15 was probed in soluble nuclear and chromatin-bound fractions. Taken together, PmVRP15 may function in nucleus as a part of membrane protein or related with membrane protein. It is possible that PmVRP15 involves in nuclear import/export of WSSV. To test this hypothesis, ration of WSSV DNA in nuclear and cytoplasmic fractions of normal and PmVRP15-dilenced hemocytes were compared. Knockdown of PmVRP15 resulted in a lower ratio of WSSV copy number in nuclear to cytoplasmic fractions by 9.3 fold, compared to that of control, indicating that PmVRP15 may involve in nuclear entry of WSSV. For crystallization purpose, rPmVRP15 was expressed in Saccharomyces cerevisiae and Escherichia coli expression system. Full-length rPmVRP15 was expressed at low level in S. cerevisiae (FGY217) but overexpressed in E. coli C.43 (DE3) at 1-3 h after IPTG-induction The rPmVRP15 was purified by Ni-NTA and DEAE columns, respectively. MALDI-TOF MS revealed that the molecular weight of PmVRP15 is 15.899 kDa. Circular dichroism spectroscopy has been used to examine secondary structure of PmVRP15. PmVRP15 contains 48.45 % of alpha-helix and 13.57 % of beta-sheet. In addition, screening of PmVRP15 crystallization conditions was performed in order to obtain a crystal for structure determination by X-ray crystallography. Crystals produced in Index™ D12 gave close spots in diffraction, suggesting the presence of protein. This condition will be optimized for further X-ray crystallographic study.