Characterization of the interaction between outer-fiber protein VP55 of genotype III grass carp reovirus and Fibulin-4 of grass carp
Characterization of the interaction between outer-fiber protein VP55 of genotype III grass carp reovirus and Fibulin-4 of grass carp
复制标题
基因Ⅲ型草鱼呼肠孤病毒外纤维蛋白VP55与草鱼Fibulin-4相互作用的表征
DOI：
10.1016/j.fsi.2018.11.067
复制
发表时间：
2019
影响因子：
4.7
通讯作者：
Lu Liqun
中科院分区：
文献类型：
--
作者：
Wang Longlong;Yu Fei;Sun Hao;Lu Liqun
基因型III草鱼呼肠孤病毒（GCRV;代表株，GCRV-104）属于Spinareovirinae亚科并且编码外纤维蛋白VP 55，其负责介导病毒对靶组织的感染并协助病毒进入细胞。Fibulin-4/EFEMP 2蛋白先前在酵母双杂交筛选中被鉴定为VP 55的推定结合伴侣。在这里，我们通过使用蛋白质相互作用测定进一步表征了Fibulin-4和VP 55之间的关联。细胞内共定位实验表明，RFP-Fibulin-4与GFP-VP 55在草鱼卵巢（GCO）细胞内共定位。细菌表达的GST-标记的Fibulin-4显示与杆状病毒表达的His-标记的VP 55在斑点印迹重叠测定中缔合;此外，杆状病毒表达的His-标记的VP 55能够拉下GCO细胞中表达的GFP-Fibulin-4。我们进行了实时PCR和免疫印迹分析，并表明，内源性Fibulin-4，虽然抑制到一个较低的水平，在晚期感染阶段，是存在于整个感染过程中的GCRV-104在CIK细胞。总之，我们的研究结果表明，草鱼Fibulin-4与VP 55相互作用。在GCRV-104感染草鱼细胞的过程中，Fibulin-4（一种已知的分泌蛋白）的存在暗示了其在病毒通过与VP 55相互作用而逃逸过程中的潜在作用。
Genotype III grass carp reovirus (GCRV; representative strain, GCRV-104) belongs to the subfamily Spinareovirinae and encodes an outer-fiber protein, VP55, responsible for mediating the infection of target tissues by the virus and assisting the virus into cells. Fibulin-4/EFEMP2 protein was previously identified as a putative binding partner for VP55 in a yeast two-hybrid screening. Here, we have further characterized the association between Fibulin-4 and VP55 by using protein interaction assays. An intracellular co-localization assay showed that RFP-Fibulin-4 co-localized with GFP-VP55 in grass carp ovary (GCO) cells. Bacterially expressed GST-tagged Fibulin-4 was shown to associate with baculovirus-expressed His-tagged VP55 in a dot-blot overlay assay; moreover, baculovirus-expressed His-tagged VP55 was able to pull down GFP-Fibulin-4 expressed in the GCO cells. We performed real-time PCR and immunoblotting analysis and showed that endogenous Fibulin-4, although suppressed to a lower level in the late infection phase, is present throughout the infection course of GCRV-104 in CIK cells. In conclusion, our results indicate that grass carp Fibulin-4 interacts with VP55. The presence of Fibulin-4, a well-known secreted protein, during the infection course of GCRV-104 in grass carp cells implies its potential role during viral egression through interaction with VP55.