State Key Laboratory of Silkworm Genome Biology, Southwest Universitygrid.263906.8, Chongqing, China.
Chongqing Key Laboratory of Microsporidia Infection and Prevention, Southwest Universitygrid.263906.8, Chongqing, China.
Microbiol Spectr. 2022 Aug 31;10(4):e0091722. doi: 10.1128/spectrum.00917-22. Epub 2022 Jul 5.
Baculovirus is a powerful tool for biological control in agriculture and foreign gene expression and delivery in insect and mammalian cells. Baculovirus enters host cells by multiple endocytic pathways; however, the current understanding of the Bombyx mori nucleopolyhedrovirus (BmNPV) entry mechanism remains limited. Previous studies have identified NPC1 and NPC2 as important host factors for viral infection in insect cells, although their exact role in viral infection has not yet been determined. In this study, we demonstrate that the BmNPC1 protein is an important intracellular factor for BmNPV escape from the endosomal compartment, and the expression of BmNPC1 in Sf9 cells confers the virus the ability to enter into the nucleus of Sf9 cells. Additionally, the second luminal domain of BmNPC1 (BmNPC1-C) binds to the viral glycoprotein gp64, and preincubation of BmNPV with purified BmNPC1-C inhibits virus infection. Furthermore, knockout of the BmNPC2 protein results in reduced efficiency of viral fusion with the endosomal membrane, and BmNPC2 protein interacts directly with both viral envelope glycoprotein gp64 and the host BmNPC1 protein. BmNPC2 was found to be incorporated into progeny viral particles. Taken together, our results suggest that NPC2 protein incorporated into viral particles may facilitate viral infection through promoting the interaction of BmNPV and NPC1 in the endosome, thus enhancing viral fusion and escape from endosomes. These results, combined with those from previous studies, support that BmNPV hijacks two important cholesterol receptor members (NPC1 and NCP2) in the cholesterol intracellular transport pathway for viral entry into host cells. Baculovirus is an important biological factor for controlling insect populations and represents a powerful biological tool for gene delivery and expression. However, the host receptor of baculovirus is still unknown. In this study, we demonstrate that BmNPC1 protein is an important intracellular factor for BmNPV escape from the endosomal compartment, and the expression of BmNPC1 confers the ability of virus to enter into the host cell nucleus in nonpermissive Sf9 cells. BmNPC2 can bind to the virus and promote progeny virion infection through the NPC1-NPC2 endosome cholesterol transport pathway. We believe that our study on the BmNPV entry mechanism will further facilitate the application of baculovirus systems in eukaryotic gene delivery. Not only can the cholesterol transport pathway NPC1 protein be used by a variety of enveloped viruses, but the NPC2 protein can also be used by viruses to infect host cells. This will provide new insights into the study of enveloped virus infection mechanisms.
杆状病毒是农业生物防治和昆虫及哺乳动物细胞中外源基因表达和传递的有力工具。杆状病毒通过多种内吞途径进入宿主细胞；然而，目前对家蚕核型多角体病毒（BmNPV）进入机制的理解仍然有限。以前的研究已经确定 NPC1 和 NPC2 是病毒感染昆虫细胞的重要宿主因素，尽管它们在病毒感染中的具体作用尚未确定。在这项研究中，我们证明 BmNPC1 蛋白是 BmNPV 从内体区室逃逸的重要细胞内因子，并且在 Sf9 细胞中表达 BmNPC1 赋予病毒进入 Sf9 细胞核的能力。此外，BmNPC1 的第二个内腔结构域（BmNPC1-C）与病毒糖蛋白 gp64 结合，并且用纯化的 BmNPC1-C 预孵育 BmNPV 可抑制病毒感染。此外，BmNPC2 蛋白的敲除导致病毒与内体膜融合的效率降低，并且 BmNPC2 蛋白直接与病毒包膜糖蛋白 gp64 和宿主 BmNPC1 蛋白相互作用。发现 BmNPC2 蛋白被掺入到子代病毒颗粒中。总之，我们的结果表明，整合到病毒颗粒中的 NPC2 蛋白可能通过促进 BmNPV 和 NPC1 在内涵体中的相互作用来促进病毒感染，从而增强病毒融合和从内涵体逃逸。这些结果与以前的研究结果相结合，支持 BmNPV 劫持胆固醇细胞内运输途径中的两个重要胆固醇受体成员（NPC1 和 NPC2）以进入宿主细胞。杆状病毒是控制昆虫种群的重要生物因素，是基因传递和表达的有力生物工具。然而，杆状病毒的宿主受体仍然未知。在这项研究中，我们证明 BmNPC1 蛋白是 BmNPV 从内体区室逃逸的重要细胞内因子，并且在非允许性 Sf9 细胞中表达 BmNPC1 赋予病毒进入宿主细胞核的能力。BmNPC2 可以与病毒结合，并通过 NPC1-NPC2 内体胆固醇运输途径促进子代病毒粒子感染。我们相信，我们对 BmNPV 进入机制的研究将进一步促进杆状病毒系统在真核基因传递中的应用。不仅 NPC1 蛋白的胆固醇运输途径可以被多种包膜病毒利用，而且 NPC2 蛋白也可以被病毒用来感染宿主细胞。这将为包膜病毒感染机制的研究提供新的见解。
