Guangxi Key Laboratory of Aquatic Genetic Breeding and Healthy Aquaculture, Guangxi Academy of Fisheries Sciences, Nanning, China.
Guangxi Shrimp Breeding Engineering Technology Research Center, Guangxi Academy of Fisheries Sciences, Nanning, China.
Fish Shellfish Immunol. 2019 May;88:198-206. doi: 10.1016/j.fsi.2019.02.057. Epub 2019 Mar 1.
Viral capsid proteins play an important role in the viral infection process. To identify the cellular proteins in shrimp that interact with the Penaeus stylirostris densovirus capsid protein (PstDNV-CP), we constructed a yeast two-hybrid (Y2H) cDNA library of the muscle tissue of Litopenaeus vannamei, and hybridized the bait vector pGBKT7-CP with this library. Cloning and sequencing showed that the shrimp protein interacting with PstDNV-CP was a homolog of BRCA2 and CDKN1A(p21)-interacting protein (BCCIP). We named this protein L. vannamei BCCIP (LvBCCIP). Further analysis showed that LvBCCIP interacted with L. vannamei calmodulin (LvCaM). We validated the interactions between PstDNV-CP and LvBCCIP, and between LvBCCIP and LvCaM, with GST pulldown assays. The gene expression of LvBCCIP increased significantly after PstDNV challenge. In addition, the PstDNV titer of PstDNV-challenged shrimp was significantly reduced after LvBCCIP expression was inhibited using double-stranded RNA (dsRNA) interference. These results indicated that LvBCCIP is critical to PstDNV pathogenesis in L. vannamei. Interestingly, the growth rate of L. vannamei was significantly reduced when LvBCCIP gene expression was silenced, indicating that LvBCCIP may also be associated with growth regulation in L. vannamei. Thus, the interaction between PstDNV-CP and LvBCCIP might explain why PstDNV infection leads to runt-deformity syndrome in shrimp.
病毒衣壳蛋白在病毒感染过程中发挥重要作用。为了鉴定与凡纳滨对虾浓核症病毒衣壳蛋白(PstDNV-CP)相互作用的虾细胞蛋白,我们构建了凡纳滨对虾肌肉组织的酵母双杂交(Y2H)cDNA 文库,并将诱饵载体 pGBKT7-CP 与该文库杂交。克隆和测序表明,与 PstDNV-CP 相互作用的虾蛋白是 BRCA2 和 CDKN1A(p21)-相互作用蛋白(BCCIP)的同源物。我们将这种蛋白命名为凡纳滨对虾 BCCIP(LvBCCIP)。进一步分析表明,LvBCCIP 与凡纳滨对虾钙调蛋白(LvCaM)相互作用。我们使用 GST 下拉实验验证了 PstDNV-CP 与 LvBCCIP 之间以及 LvBCCIP 与 LvCaM 之间的相互作用。LvBCCIP 的基因表达在 PstDNV 挑战后显著增加。此外,在用双链 RNA(dsRNA)干扰抑制 LvBCCIP 表达后,PstDNV 感染虾的 PstDNV 滴度显著降低。这些结果表明 LvBCCIP 对凡纳滨对虾 PstDNV 发病机制至关重要。有趣的是,当沉默 LvBCCIP 基因表达时,凡纳滨对虾的生长速度显著降低,这表明 LvBCCIP 可能也与凡纳滨对虾的生长调节有关。因此,PstDNV-CP 和 LvBCCIP 之间的相互作用可能解释了为什么 PstDNV 感染会导致虾的畸形综合征。