Autographa californica multiple nucleopolyhedrovirus nucleocapsid protein BV/ODV-C42 mediates the nuclear entry of P78/83
Autographa californica multiple nucleopolyhedrovirus nucleocapsid protein BV/ODV-C42 mediates the nuclear entry of P78/83
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苜蓿银纹夜蛾多核多角体病毒核衣壳蛋白BV/ODV-C42介导P78/83入核
DOI：
10.1128/jvi.02510-07
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发表时间：
2008-05-01
影响因子：
5.4
通讯作者：
Chen, Xinwen
中科院分区：
文献类型：
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作者：
Wang, Yun;Wang, Qian;Chen, Xinwen
链球绦虫多核多角体病毒BV/ODV-C42(orf101；C42)是杆状病毒科成员中高度保守的基因，编码41.5 kDa的病毒核衣壳蛋白，C末端有核定位信号(NLS)基序。C42被证明是AcMNPV繁殖所必需的，并能与核衣壳蛋白P78/83结合，核衣壳蛋白P78/83是肌动蛋白相关蛋白2/3(Arp2/3)复合体的病毒活化剂，启动核肌动蛋白聚合，这对AcMNPV感染过程中病毒核衣壳的形态形成是必不可少的。在这里，我们报告了C42在核衣壳组装中起作用的一条新途径的鉴定。将C42和P78/83共转染Sf9细胞，结果表明C42能够将P78/83募集到未感染细胞的胞核，C42的NLS基序在这一过程中是必不可少的。为了验证这一核定位模式，我们制备了C42破坏的BAc(C42ko-GFP)和带有野生型C42(vAcc42(res-GFP))或带有NLS编码序列突变的C42(vAcc42(NLS-GFP))的挽救的BAc。免疫组织化学染色发现P78/83定位于VAc(c421ko-GFP)和VAc(c42nls-GFP)细胞的胞浆内，而P78/83定位于VAC(c42res-GFP)细胞的胞核。此外，F-肌动蛋白特异性染色证实，无论是vAc(c42ko-gfp)还是vav(c42nls-gfp)转基因细胞的细胞核中都没有肌动蛋白聚合活性，这可能是由于核内没有启动肌动蛋白聚合的Arp2/3复合体的激活剂P78/83。因此，我们假设在AcMNPV感染过程中，C42与细胞质中的P78/83结合形成蛋白质复合体，并在C42的NLS基序的指导下共转运到细胞核。
Autographa californica multiple nucleopolyhedrovirus (AcMNPV) BV/ODV-c42 (orf101; c42), which encodes a 41.5-kDa viral nucleocapsid protein with a putative nuclear localization signal (NLS) motif at the C terminus, is a highly conserved gene among members of the Baculoviridae family. C42 is demonstrated to be essential for AcMNPV propagation and can bind to nucleocapsid protein P78/83, a viral activator for the actin-related protein 2/3 (ARP2/3) complex to initiate nuclear actin polymerization, which is essential for viral nucleocapsid morphogenesis during AcMNPV infection. Here, we report the identification of a novel pathway through which c42 functions in nucleocapsid assembly. Cotransfection of Sf9 cells with c42 and p78/83 plasmids demonstrated that C42 was capable of recruiting P78/83 to the nuclei of uninfected cells and that the NLS motif of C42 was essential for this process. To validate this nuclear relocation mode in bacmid-transfected cells, a c42-disrupted bacmid (vAc(c42ko-gfp)) and rescued bacmids with wild-type c42 (vAcc42(res-gfp)) or with NLS coding sequencemutated c42 (vAcc42(nls-gfp)) were prepared. By immuno-staining, P78/83 was found to be localized in the cytoplasm of either vAc(c421ko-gfp) - or vAc(c42nls-gfp)-transfected cells, whereas P78/83 was relocated to the nuclei of VAC(c42res-gfP)-transfected cells. Furthermore, F-actin-specific staining confirmed that there was no actin polymerization activity in the nuclei of either vAc(c42ko-gfp) - or vAv(c42nls-gfP)-transfected cells, which might be attributed to the absence of nuclear P78/83, an activator of the ARP2/3 complex to initiate nuclear actin polymerization. We therefore hypothesize a mode of action where C42 binds to P78/83 in the cytoplasm to form a protein complex and cotransports to the nucleus under the direction of the NLS motif in C42 during AcMNPV infection.