Mangor J T, Monsma S A, Johnson M C, Blissard G W
Boyce Thompson Institute at Cornell University, Ithaca, New York 14853, USA.
J Virol. 2001 Mar;75(6):2544-56. doi: 10.1128/JVI.75.6.2544-2556.2001.
The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) GP64 protein is an essential virion protein that is involved in both receptor binding and membrane fusion during viral entry. Genetic studies have shown that GP64-null viruses are unable to move from cell to cell and this results from a defect in the assembly and production of budded virions (BV). To further examine requirements for virion budding, we asked whether a GP64-null baculovirus, vAc(64-), could be pseudotyped by introducing a heterologous viral envelope protein (vesicular stomatitis virus G protein [VSV-G]) into its membrane and whether the resulting virus was infectious. To address this question, we generated a stably transfected insect Sf9 cell line (Sf9(VSV-G)) that inducibly expresses the VSV-G protein upon infection with AcMNPV Sf9(VSV-G) and Sf9 cells were infected with vAc(64-), and cells were monitored for infection and for movement of infection from cell to cell. vAc(64-) formed plaques on Sf9(VSV-G) cells but not on Sf9 cells, and plaques formed on Sf9(VSV-G) cells were observed only after prolonged intervals. Passage and amplification of vAc(64-) on Sf9(VSV-G) cells resulted in pseudotyped virus particles that contained the VSV-G protein. Cell-to-cell propagation of vAc(64-) in the G-expressing cells was delayed in comparison to wild-type (wt) AcMNPV, and growth curves showed that pseudotyped vAc(64-) was generated at titers of approximately 10(6) to 10(7) infectious units (IU)/ml, compared with titers of approximately 10(8) IU/ml for wt AcMNPV. Propagation and amplification of pseudotyped vAc(64-) virions in Sf9(VSV-G) cells suggests that the VSV-G protein may either possess the signals necessary for baculovirus BV assembly and budding at the cell surface or may otherwise facilitate production of infectious baculovirus virions. The functional complementation of GP64-null viruses by VSV-G protein was further demonstrated by identification of a vAc(64-)-derived virus that had acquired the G gene through recombination with Sf9(VSV-G) cellular DNA. GP64-null viruses expressing the VSV-G gene were capable of productive infection, replication, and propagation in Sf9 cells.
苜蓿银纹夜蛾多核型多角体病毒（AcMNPV）的GP64蛋白是一种必需的病毒粒子蛋白，在病毒进入细胞过程中参与受体结合和膜融合。遗传学研究表明，缺失GP64的病毒无法在细胞间移动，这是由于出芽病毒粒子（BV）的组装和产生存在缺陷所致。为了进一步研究病毒粒子出芽的条件，我们探讨了缺失GP64的杆状病毒vAc(64-)能否通过在其膜中引入异源病毒包膜蛋白（水泡性口炎病毒G蛋白[VSV-G]）进行假型化，以及产生的病毒是否具有感染性。为了解决这个问题，我们构建了一个稳定转染的昆虫Sf9细胞系（Sf9(VSV-G)），该细胞系在感染AcMNPV时可诱导表达VSV-G蛋白。用vAc(64-)感染Sf9(VSV-G)和Sf9细胞，并监测细胞的感染情况以及感染在细胞间的传播。vAc(64-)在Sf9(VSV-G)细胞上形成噬斑，但在Sf9细胞上不形成，且在Sf9(VSV-G)细胞上形成噬斑仅在延长的时间间隔后才观察到。vAc(64-)在Sf9(VSV-G)细胞上传代和扩增产生了含有VSV-G蛋白的假型化病毒颗粒。与野生型（wt）AcMNPV相比，vAc(64-)在表达G蛋白的细胞中的细胞间传播延迟，生长曲线显示假型化的vAc(64-)产生的滴度约为10^6至10^7感染单位（IU）/毫升，而wt AcMNPV的滴度约为10^8 IU/毫升。假型化的vAc(64-)病毒粒子在Sf9(VSV-G)细胞中的传播和扩增表明，VSV-G蛋白可能具有杆状病毒BV在细胞表面组装和出芽所需的信号，或者可能以其他方式促进感染性杆状病毒粒子的产生。通过鉴定一种通过与Sf9(VSV-G)细胞DNA重组而获得G基因的源自vAc(64-)的病毒，进一步证明了VSV-G蛋白对缺失GP64病毒的功能互补作用。表达VSV-G基因的缺失GP64的病毒能够在Sf9细胞中进行有效感染、复制和传播。
