State Key Laboratory of Marine Environmental Science, State-Province Joint Engineering Laboratory of Marine Bioproducts and Technology, Xiamen University, Xiamen, 361102, Fujian, China.
State Key Laboratory of Marine Environmental Science, State-Province Joint Engineering Laboratory of Marine Bioproducts and Technology, Xiamen University, Xiamen, 361102, Fujian, China; Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology (Qingdao), China.
Fish Shellfish Immunol. 2019 Nov;94:934-943. doi: 10.1016/j.fsi.2019.10.012. Epub 2019 Oct 7.
Really Interesting New Gene (RING) finger proteins are highly conserved molecules that participate in a variety of biological processes such as regulation of development, apoptosis and antiviral immunity in vertebrates. However, the functions of RING finger proteins are still poorly understood in crustaceans. Previously, we found that the transcript of a homolog of RING finger protein 152 (CqRNF152-like) was up-regulated in a differentially expressed transcriptome library of the haematopietic tissue (Hpt) cells from red claw crayfish Cherax quadricarinatus upon white spot syndrome virus (WSSV) infection, which is one of the most devastating viral diseases for crustaceans like shrimp and crayfish. The full-length cDNA sequence of CqRNF152-like was then identified with 975 bp, including an ORF of 685 bp that encoded a 195 amino acids protein, a 5'- UTR of 180 bp, and a 3'-UTR with a poly (A) tail of 207 bp. The conserved domain prediction showed that CqRNF152-like contained a conserved RING-finger domain. Gene expression analysis showed that CqRNF152-like was distributed in all tissues examined and the transcript is significantly up-regulated after WSSV challenge both in vivo in Hpt tissue and in vitro in cultured Hpt cells. Furthermore, the transcripts of both an immediate early gene ie1 and a late envelope protein gene vp28 of WSSV were clearly increased in the Hpt tissues, hemocytes and cultured Hpt cells after gene silencing of CqRNF152-like, which were further proved to be significantly decreased after overloading of recombinant CqRNF152-like protein in Hpt cell cultures. Meanwhile, CqRNF152-like was found to bind with WSSV envelope protein VP28 by proteins pull-down assay. Similar to most of RNF proteins, CqRNF152-like protein sequence contained a conserved RING-finger domain and showed self-ubiquitination activity in a RING finger domain dependent manner. Taken together, CqRNF152-like is likely to function as an antiviral molecular against WSSV infection through interaction with the envelope protein VP28 in a crustacean C. quadricarinatus. This is the first report that a RING finger protein with directly antiviral functions via interaction with viral protein and self-ubiquitination activity in crustacean, which sheds new light on the molecular mechanism of WSSV infection and the control of white spot disease.
真核生物 RING 指蛋白是高度保守的分子，参与脊椎动物的多种生物学过程，如发育调控、细胞凋亡和抗病毒免疫。然而，甲壳动物中 RING 指蛋白的功能仍知之甚少。之前，我们发现，螯虾血细胞（Hpt）组织差异表达转录组文库中，真核生物 RING 指蛋白 152（CqRNF152-like）的同源物的转录本在白斑综合征病毒（WSSV）感染后上调，这是对虾和螯虾等甲壳类动物最具破坏性的病毒性疾病之一。然后用 975bp 鉴定出 CqRNF152-like 的全长 cDNA 序列，包括一个 685bp 的 ORF，编码一个 195 个氨基酸的蛋白质，一个 5'-UTR 为 180bp，和一个 3'-UTR 带有 207bp 的多聚（A）尾。保守结构域预测表明，CqRNF152-like 含有一个保守的 RING 指结构域。基因表达分析表明，CqRNF152-like 分布在所有检测的组织中，在体内 Hpt 组织和体外培养的 Hpt 细胞中，WSSV 攻击后，转录本明显上调。此外，在 CqRNF152-like 基因沉默后，WSSV 的即刻早期基因 ie1 和晚期包膜蛋白基因 vp28 的转录本在 Hpt 组织、血细胞和培养的 Hpt 细胞中均明显增加，而过载重组 CqRNF152-like 蛋白后进一步证实其显著降低。同时，通过蛋白质下拉实验发现 CqRNF152-like 与 WSSV 包膜蛋白 VP28 结合。与大多数 RNF 蛋白一样，CqRNF152-like 蛋白序列含有一个保守的 RING 指结构域，并表现出自依赖 RING 指结构域的自我泛素化活性。总之，CqRNF152-like 可能通过与甲壳动物 C. quadricarinatus 中的包膜蛋白 VP28 相互作用，作为一种抗病毒分子发挥作用，抵抗 WSSV 感染。这是第一个报道具有直接抗病毒功能的 RING 指蛋白通过与病毒蛋白相互作用和自我泛素化活性在甲壳动物中，这为 WSSV 感染和白斑病的控制提供了新的视角。
