Autographa californica Multicapsid Nucleopolyhedrovirus Efficiently Infects Sf9 Cells and Transduces Mammalian Cells via Direct Fusion with the Plasma Membrane at Low pH

The budded virus (BV) of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) infects insect cells and transduces mammalian cells mainly through the endocytosis pathway. However, this study revealed that the treatment of the virus bound to Sf9 cells at low pH could efficiently rescue the infectivity of AcMNPV in the presence of endocytosis pathway inhibitors. A colocalization assay of the major capsid protein VP39 with the early endosome marker EEA1 showed that at low pH, AcMNPV entered Sf9 cells via an endosome-independent pathway. Using a fluorescent probe (R18), we showed that at low pH, the viral nucleocapsid entered Sf9 cells via direct fusion at the cell surface. By using the myosin-specific inhibitor 2,3-butanedione monoxime (BDM) and the microtubule inhibitor nocodazole, the low pH-triggered direct fusion was demonstrated to be dependent on myosin-like proteins and independent of microtubules. The reverse transcription-PCR of the IE1 gene as a marker for viral entry showed that the kinetics of AcMNPV in cells triggered by low pH was similar to that of the normal entry via endocytosis. The low pH-mediated infection assay and VP39 and EEA1 colocalization assay also demonstrated that AcMNPV could efficiently transduce mammalian cells via direct membrane fusion at the cell surface. More importantly, we found that a low-pH trigger could significantly improve the transduction efficiency of AcMNPV in mammalian cells, leading to the potential application of this method when using baculovirus as a vector for heterologous gene expression and for gene therapy.

苜蓿银纹夜蛾多粒包埋核型多角体病毒（AcMNPV）的出芽型病毒（BV）主要通过内吞途径感染昆虫细胞并转导哺乳动物细胞。然而，本研究表明，在低pH值条件下处理与Sf9细胞结合的病毒，在内吞途径抑制剂存在的情况下，能够有效恢复AcMNPV的感染性。对主要衣壳蛋白VP39与早期内体标记物EEA1进行的共定位分析显示，在低pH值下，AcMNPV通过一种不依赖内体的途径进入Sf9细胞。利用一种荧光探针（R18），我们发现，在低pH值下，病毒核衣壳通过在细胞表面直接融合的方式进入Sf9细胞。通过使用肌球蛋白特异性抑制剂2,3 - 丁二酮单肟（BDM）和微管抑制剂诺考达唑，证明低pH值触发的直接融合依赖于类肌球蛋白，但不依赖于微管。以IE1基因作为病毒进入的标记进行的逆转录 - PCR显示，低pH值触发的AcMNPV在细胞内的动力学与通过内吞作用正常进入的情况相似。低pH值介导的感染实验以及VP39和EEA1的共定位实验还表明，AcMNPV能够通过在细胞表面直接膜融合的方式有效地转导哺乳动物细胞。更重要的是，我们发现低pH值触发能够显著提高AcMNPV在哺乳动物细胞中的转导效率，这使得在将杆状病毒用作异源基因表达和基因治疗的载体时，这种方法具有潜在的应用价值。