C-terminal region of apoptin affects chicken anemia virus replication and virulence.
C-terminal region of apoptin affects chicken anemia virus replication and virulence.
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DOI：
10.1186/s12985-017-0713-9
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发表时间：
2017-02-21
期刊：
影响因子：
4.8
通讯作者：
Chen H
中科院分区：
文献类型：
--
作者：
Wang Y;Song X;Gao H;Wang X;Hu Y;Gao Y;Qi X;Qin L;Lin H;Gao L;Yao S;Han C;Wang X;Chen H
鸡贫血病毒（Chicken Anemia Virus，CAV）引起的贫血和免疫抑制是养禽业的重要疾病。CAV VP 3也被称为“抗肿瘤素”，已被证明可以选择性地杀死肿瘤细胞，这为它作为抗癌疗法的应用带来了很大的希望。Eschertin诱导细胞凋亡的能力与其核定位密切相关。C-terminin的C-末端区域包含一个二分核定位信号（NLS），核输出信号（内斯）位于NLS的臂之间。以往的研究大多是在体外表达不同长度的reflectin，以了解其定位和诱导细胞凋亡之间的关系。在这项研究中，我们研究了复制的CAV和诱导细胞凋亡在体外和体内与VP 3截短的感染性病毒。采用定量PCR检测MDCC-MSB 1细胞中病毒的复制情况，并通过共聚焦显微镜观察病毒的定位。流式细胞仪检测病毒诱导MDCC-MSB 1细胞凋亡的情况。此外，将拯救病毒感染鸡与亲本病毒rM 9905进行比较，以评价病毒在体内的复制和毒力。在此基础上，我们成功地拯救了两株在Ekettin C端缺失NES-NLS 2（rCAV-VP 3 N88）和NLS 1-NES-NLS 2（rCAV-VP 3 N80）的病毒。rCAV-VP 3 N88的病毒载量在60和108 hpi之间显著降低，并且总是比亲本病毒rM 9905的病毒载量低10-100倍。rCAV-VP 3 N80的水平也比rM 9905低10-100倍，并且在三个时间点显著下降。rCAV-VP 3 N88和rCAV-VP 3 N80的病毒载量几乎没有差异。另外，rM 9905在96 hpi时诱导85.39 ± 2.18%的细胞凋亡，而rCAV-VP 3 N88和rCAV-VP 3 N80分别诱导63.08 ± 4.78%和62.56 ± 7.35%的细胞凋亡，这两种病毒诱导的细胞凋亡显著低于亲本病毒诱导的细胞凋亡（约20%）。拯救的病毒改变了MDCC-MSB 1细胞中的核定位。此外，cavitin的C-末端区域的缺失损害了病毒在体内的复制，并降低了CAV在鸡中的毒力。总之，我们已经证明，在感染性CAV中的C-末端缺失的reflectin影响病毒的复制。apoptin C末端区域的缺失不仅显着降低了病毒在体外的复制，而且还降低了其对细胞凋亡的诱导，这与其核定位的丧失相关。C-terminin基因的缺失也削弱了CAV在鸡体内的复制能力，降低了其毒力。
Chicken anemia virus (CAV) causes anemia and immune suppression, which are important diseases in the poultry industry. CAV VP3, also referred as ‘apoptin’, has been shown to selectively kill tumor cells, raising great hopes for its utilization as an anticancer therapy. The ability of apoptin to induce apoptosis is closely related to its nuclear localization. The C-terminal region of apoptin contains a bipartite nuclear localization signals (NLS), and a nuclear export signal (NES) is located between the arms of the NLS. Most previous studies have expressed apoptin of different lengths in vitro to understand the relationship between its localization and its induction of apoptosis. In this study, we investigated the replication of CAV and its induction of apoptosis in vitro and in vivo with VP3-truncated infectious virus. Quantitative PCR was used to detect viral replication in MDCC-MSB1 cells, and the viral localization was observed by confocal microscopy. Flow cytometry was uesed to analyze virus-induced apoptosis in MDCC-MSB1 cells. Additionally, chickens infected with the rescued viruses compared with the parental virus rM9905 to evaluate the viral replication in vivo and virulence. Based on the infectious clone, we rescued two viruses in which were deleted NES–NLS2 (rCAV-VP3N88) or NLS1–NES–NLS2 (rCAV-VP3N80) in the C-terminal region of apoptin. The viral load of rCAV-VP3N88 decreased significantly between 60 and 108 hpi, and was always 10–100-fold lower than that of the parental virus rM9905. The levels of rCAV-VP3N80 were also 10–100-fold lower than that of rM9905 and declined significantly at three time points. There was almost no difference in the viral loads of rCAV-VP3N88 and rCAV-VP3N80. Additionally, rM9905 induced 85.39 ± 2.18% apoptosis at 96 hpi, whereas rCAV-VP3N88 and rCAV-VP3N80 induced 63.08 ± 4.78% and 62.56 ± 7.35% apoptosis, respectively, which were significantly (about 20%) lower than that induced by the parental virus. The rescued viruses altered the nuclear localization in MDCC-MSB1 cells. Moreover, deletion of C-terminal region of apoptin impaired viral replication in vivo and reduced the virulence of CAV in chickens. In summary, we have demonstrated that the C-terminal deletion of apoptin in infectious CAV affected the replication of the virus. The deletion of the C-terminal region of apoptin not only significantly reduced viral replication in vitro but also reduced its induction of apoptosis, which correlated with the loss of its nuclear localization. The deletion of the C-terminal region of apoptin also impaired the replication of CAV and attenuated its virulence in chickens.