Slack Jeffrey M, Lawrence Susan D, Krell Peter J, Arif Basil M
Great Lakes Forestry Centre, Sault Ste Marie, ON P6A 2E5, Canada.
US Department of Agriculture, Beltsville, MD 20705-2350, USA.
J Gen Virol. 2008 Oct;89(Pt 10):2388-2397. doi: 10.1099/vir.0.2008/002543-0.
Baculovirus occlusion-derived virions (ODVs) contain a number of infectivity factors essential for the initiation of infection in larval midgut cells. Deletion of any of these factors neutralizes infectivity by the per os route. We have observed that P74 of the group I alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is N-terminally cleaved when a soluble form of the protein was incubated with insect midgut tissues under alkaline conditions and that cleavage was prevented by soybean trypsin inhibitor (SBTI). Presently, biological assays were carried out that suggest SBTI inhibits and trypsin enhances baculovirus per os infectivity. We developed a method to rescue per os infectivity of a P74 null virus involving co-transfection of viral DNA with a plasmid that transiently expresses p74. We used this plasmid rescue method to functionally characterize P74. A series of site-directed mutants were generated at the N terminus to evaluate if trypsin cleavage sites were necessary for function. Mutagenesis of R195, R196 and R199 compromised per os infectivity and rendered P74 resistant to midgut trypsin.
杆状病毒包涵体衍生病毒粒子（ODV）包含许多对幼虫中肠细胞感染起始至关重要的感染性因子。缺失这些因子中的任何一个都会通过口服途径使感染性丧失。我们观察到，当将I组α杆状病毒苜蓿银纹夜蛾多角体病毒（AcMNPV）的P74蛋白的可溶形式在碱性条件下与昆虫中肠组织一起孵育时，其N端会被切割，并且大豆胰蛋白酶抑制剂（SBTI）可阻止这种切割。目前，进行的生物学试验表明，SBTI会抑制杆状病毒的口服感染性，而胰蛋白酶则会增强其口服感染性。我们开发了一种拯救P74缺失病毒口服感染性的方法，该方法涉及将病毒DNA与瞬时表达p74的质粒共转染。我们使用这种质粒拯救方法对P74进行功能表征。在N端产生了一系列定点突变体，以评估胰蛋白酶切割位点对功能是否必要。R195、R196和R199的诱变损害了口服感染性，并使P74对中肠胰蛋白酶具有抗性。
