Functional analysis of the putative antiapoptotic genes, p49 and iap4, of Spodoptera litura nucleopolyhedrovirus with RNAi.
Functional analysis of the putative antiapoptotic genes, p49 and iap4, of Spodoptera litura nucleopolyhedrovirus with RNAi.
复制标题
DOI：
10.1099/vir.0.2008/001008-0
复制
发表时间：
2008-08
期刊：
影响因子：
--
通讯作者：
Qian Yu;Tiehao Lin;Guozhong Feng;Kai Yang;Y. Pang
中科院分区：
文献类型：
--
作者：
Qian Yu;Tiehao Lin;Guozhong Feng;Kai Yang;Y. Pang
通过对公共数据库的同源性检索发现，斜纹夜蛾核多角体病毒（SpltNPV）具有两个推测的抗凋亡基因p49和凋亡抑制剂4 (iap4)，但它们在其原生宿主细胞中的功能尚未被研究。在本研究中，我们使用RNA干扰（RNAi）来单独或共同沉默Splt-iap4和Splt-p49的表达，以确定它们在SpltNPV生命周期中的作用。RT-PCR分析和Western blot分析显示，Splt-p49或Splt-iap4双链RNA （dsRNA）分别处理spltnpv感染的SpLi-221细胞后，靶基因表达被敲除，证实这两个基因被有效沉默。在Splt-p49 dsRNA处理的spltnpv感染细胞中，凋亡开始于14 h，几乎所有细胞在48 h内都发生了凋亡。相比之下，Splt-iap4 dsRNA处理的感染细胞中，出芽病毒的产生和多面体的形成正常进行。细胞活力分析显示Splt-IAP4对SpLi-221细胞受SpltNPV感染后的凋亡抑制无协同作用。有趣的是，在Splt-iap4 dsRNA处理后，细胞在早期感染阶段不像SpltNPV感染的细胞那样聚集，这意味着杆状病毒iap4的作用未知。我们的研究结果表明Splt-p49是防止细胞凋亡所必需的；然而，Splt-iap4在SpltNPV感染过程中没有抗凋亡功能。
A homology search of a public database revealed that Spodoptera litura nucleopolyhedrovirus (SpltNPV) possesses two putative, antiapoptotic genes, p49 and inhibitor of apoptosis 4 (iap4), but their function has not been investigated in its native host cells. In the present study, we used RNA interference (RNAi) to silence the expression of Splt-iap4 and Splt-p49, independently or together, to determine their roles during the SpltNPV life cycle. RT-PCR analysis and Western blot analysis showed the target gene expression had been knocked out in the SpltNPV-infected SpLi-221 cells after treatment with Splt-p49 or Splt-iap4 double-stranded RNA (dsRNA), respectively, confirming that the two genes were effectively silenced. In SpltNPV-infected cells treated with Splt-p49 dsRNA, apoptosis was observed beginning at 14 h, and almost all cells had undergone apoptosis by 48 h. In contrast, budded virus production and polyhedra formation progressed normally in infected cells treated with Splt-iap4 dsRNA. Cell viability analysis showed that Splt-IAP4 had no synergistic effect on the inhibition of apoptosis of SpLi-221 cells induced by SpltNPV infection. Interestingly, after Splt-iap4 dsRNA treatment, cells did not congregate like those infected with SpltNPV in the early infection phase, implying an unknown role of baculovirus iap4. Our results determine that Splt-p49 is necessary to prevent apoptosis; however, Splt-iap4 has no antiapoptotic function during SpltNPV infection.