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Abstract：
The baculovirus expression vector system (BEVS) has become an important platform for the expression of recombinant proteins and is especially useful for the production of large protein complexes such as virus-like particles (VLPs). An important application for VLPs is their use as vehicles for targeted delivery of drugs or toxins which requires the development of methods for efficient loading with the intended cargo. Our research intends to employ the BEVS for the production of VLPs for the delivery of insecticidal dsRNA molecules to targeted insect pests (as \"dsRNA-VLPs\"). A convenient strategy would be the co-expression of long dsRNAs with viral capsid proteins and their simultaneous encapsulation during VLP assembly but the capacity of the BEVS for the production of long dsRNA has not been assessed so far. In this study, the efficiency of production of long RNA hairpins targeting the luciferase gene (\"dsLuc\") by the polyhedrin promoter during baculovirus infection was evaluated. However, RNAi reporter assays could not detect significant amounts of dsLuc in Hi5 cells infected with recombinant baculovirus, even in the presence of co-expressed dsRNA-binding protein B2-GFP or the employment of the MS2-MCP system. Nevertheless, dot blot analyses using anti-dsRNA antibody revealed that baculovirus-mediated expression of B2-GFP resulted in significant increases in dsRNA levels in infected cells that may correspond to hybridized complementary viral transcripts. Using B2-GFP as a genetically encoded sensor, dsRNA foci were detected in the nuclei that partially co-localized with DAPI staining, consistent with their localization at the virogenic stroma. Co-localization experiments with the baculovirus proteins vp39, Ac93, ODV-E25 and gp64 indicated limited overlap between B2-GFP and the ring zone compartment where assembly of nucleocapsids and virions occurs. Stability experiments showed that exogenous dsRNA is resistant to degradation in extracts of non-infected and infected Hi5 cells and it is proposed that strong unwinding activity at the virogenic stroma in the infected nuclei may neutralize the annealing of complementary RNA strands and block the production of long dsRNAs. Because the strong stability of exogenous dsRNA, transfection can be explored as an alternative method for delivery of cargo for dsRNA-VLPs during their assembly in baculovirus-infected Hi5 cells.
摘要：
杆状病毒表达载体系统（BEVS）已成为表达重组蛋白的重要平台，尤其可用于生产大型蛋白质复合物，例如病毒样颗粒（VLP）。VLP的重要应用是它们用作靶向递送药物或毒素的载体,这需要开发有效装载预期货物的方法。我们的研究旨在使用BEVS生产VLP，将杀虫dsRNA分子递送至目标害虫（如“dsRNA-VLP”）。一种方便的策略是长dsRNA与病毒衣壳蛋白的共表达及其在VLP组装过程中的同时包封,但迄今为止尚未评估BEVS产生长dsRNA的能力。在这项研究中,评估了杆状病毒感染过程中多角体蛋白启动子靶向荧光素酶基因（\“dsLuc\”）的长RNA发夹的产生效率。然而,RNAi报告基因检测不能在感染重组杆状病毒的Hi5细胞中检测到显著量的dsLuc,即使在共表达的dsRNA结合蛋白B2-GFP的存在下或使用MS2-MCP系统。然而，使用抗dsRNA抗体的斑点印迹分析显示,杆状病毒介导的B2-GFP表达导致感染细胞中dsRNA水平的显著增加,其可能对应于杂交的互补病毒转录物。使用B2-GFP作为基因编码的传感器，在与DAPI染色部分共定位的细胞核中检测到dsRNA病灶，与它们在病毒发生基质中的定位一致。杆状病毒蛋白vp39，Ac93，ODV-E25和gp64的共定位实验表明，B2-GFP与发生核衣壳和病毒体组装的环区室之间的重叠有限。稳定性实验表明，外源dsRNA对未感染和感染的Hi5细胞的提取物中的降解具有抗性，并且提出在感染的细胞核中的病毒发生基质处的强解链活性可以中和互补RNA链的退火并阻断长dsRNA的产生。因为外源dsRNA的稳定性强，转染可以作为在杆状病毒感染的Hi5细胞中组装期间递送dsRNA-VLP的货物的替代方法进行探索。
