A New theraphosid Spider Toxin Causes Early Insect Cell Death by Necrosis When Expressed <i>In Vitro</i> during Recombinant Baculovirus Infection

Baculoviruses are the most studied insect viruses in the world and are used for biological control of agricultural and forest insect pests. They are also used as versatile vectors for expression of heterologous proteins. One of the major problems of their use as biopesticides is their slow speed to kill insects. Thus, to address this shortcoming, insect-specific neurotoxins from arachnids have been introduced into the baculovirus genome solely aiming to improve its virulence. In this work, an insecticide-like toxin gene was obtained from a cDNA derived from the venom glands of the theraphosid spider Brachypelma albiceps. The mature form of the peptide toxin (called Ba3) has a high content of basic amino acid residues, potential for three possible disulfide bonds, and a predicted three-stranded β-sheetDifferent constructions of the gene were engineered for recombinant baculovirus Autographa californica multiple nuclepolyhedrovirus (AcMNPV) expression. Five different forms of Ba3 were assessed; (1) the full-length sequence, (2) the pro-peptide and mature region, (3) only the mature region, and the mature region fused to an (4) insect or a (5) virus-derived signal peptide were inserted separately into the genome of the baculovirus. All the recombinant viruses induced cell death by necrosis earlier in infection relative to a control virus lacking the toxin gene. However, the recombinant virus containing the mature portion of the toxin gene induced a faster cell death than the other recombinants. We found that the toxin construct with the signal peptide and/or pro-peptide regions delayed the necrosis phenotype. When infected cells were subjected to ultrastructural analysis, the cells showed loss of plasma membrane integrity and structural changes in mitochondria before death. Our results suggest this use of baculovirus is a potential tool to help understand or to identify the effect of insect-specific toxic peptides when produced during infection of insect cells.杆状病毒（Baculoviruses）是全球研究最为深入的昆虫病毒类群，既可用于农林害虫的生物防治，也可作为通用载体实现异源蛋白的表达。作为生物农药应用时，其核心短板之一为杀虫速度缓慢。为解决这一缺陷，研究人员将蛛形纲动物来源的昆虫特异性神经毒素引入杆状病毒基因组，以针对性提升其毒力。本研究从捕鸟蛛科（Theraphosidae）蜘蛛Brachypelma albiceps的毒腺互补DNA（cDNA）中扩增得到类杀虫剂毒素基因。该肽毒素的成熟形式（命名为Ba3）富含碱性氨基酸残基，具备形成3个二硫键的结构潜力，且预测拥有三股β折叠片（β-sheet）结构。针对该基因的多种构建体被用于重组苜蓿银纹夜蛾核型多角体病毒（Autographa californica multiple nuclepolyhedrovirus, AcMNPV）的表达实验。本次研究共评估了5种不同形式的Ba3：(1) 全长序列；(2) 前肽区与成熟区片段；(3) 仅成熟区片段；以及成熟区分别融合(4) 昆虫来源信号肽或(5) 病毒来源信号肽，将上述构建体单独插入杆状病毒基因组中。相较于未携带毒素基因的对照病毒，所有重组病毒均可在感染早期诱导坏死性细胞死亡。其中，仅携带毒素基因成熟区段的重组病毒诱导细胞死亡的速度快于其余重组体。研究发现，融合了信号肽和/或前肽区的毒素构建体，会延缓坏死表型的出现进程。对感染细胞开展超微结构分析后可见，细胞在死亡前出现质膜完整性丧失及线粒体结构异常改变。本研究结果表明，此种改造后的杆状病毒有望成为解析昆虫细胞感染过程中表达的昆虫特异性毒性多肽功能的潜在研究工具。