Inactivation of the budded virus of Autographa californica M nucleopolyhedrovirus by gloverin.
Inactivation of the budded virus of Autographa californica M nucleopolyhedrovirus by gloverin.
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DOI：
10.1016/j.jip.2012.02.007
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发表时间：
2012-05
影响因子：
3.4
通讯作者：
Haas-Stapleton EJ
中科院分区：
文献类型：
--
作者：
Moreno-Habel DA;Biglang-awa IM;Dulce A;Luu DD;Garcia P;Weers PM;Haas-Stapleton EJ
抗微生物肽是在接触病原体的昆虫体内产生的，用于对抗感染。Gloverin是一种小的阳离子抗菌蛋白，在接触细菌的毛癣虫幼虫的血细胞和脂肪体细胞中诱导表达。本研究的目的是确定gloverin在杆状病毒感染中的作用。我们发现，在感染加州自状杆状病毒M型核多角体病毒（AcMNPV）的豚鼠体内，gloverin的表达被诱导。利用系统感染AcMNPV芽化病毒（BV）的T. ni幼虫血细胞中分离的RNA克隆了两个gloverin基因，并结合c -末端6x-His和V5表位标签进行gloverin的分离、检测和功能研究。用两种gloverin表达质粒和亲和纯化的gloverin蛋白稳定转染Sf9细胞的上清液，通过未转染Sf9细胞的斑块测定，可减少感染性AcMNPV BV的数量。纳米摩尔浓度亲和柱纯化的gloverin蛋白可使钙黄蛋白从磷脂酰甘油组成的单层囊泡中迅速释放，但不能从磷脂酰胆碱组成的囊泡中释放，这表明gloverin与膜的相互作用是迅速的，并受膜电荷的影响。gloverin的BV灭活活性和钙黄蛋白释放活性均随gloverin浓度的升高而升高。这些结果表明，gloverin是一种抗病毒蛋白，与囊泡膜相互作用，导致内容物释放。
Antimicrobial peptides are generated in insects exposed to pathogens for combating infection. Gloverin is a small cationic antibacterial protein whose expression is induced in the hemocytes and fat body cells of Trichoplusia ni larvae exposed to bacteria. The purpose of this study was to determine the role of gloverin during baculovirus infection. We found that gloverin expression is induced in T. ni systemically infected with the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV). Two gloverin genes were cloned using RNA isolated from the hemocytes of T. ni larvae that were systemically infected AcMNPV budded virus (BV) and C-terminal 6x-His and V5 epitope tags were incorporated to facilitate gloverin isolation, detection and functional studies. The supernatants of Sf9 cells stably transfected with the two gloverin expression plasmids and affinity purified gloverin proteins reduced the quantity of infectious AcMNPV BV as measured in vitro by plaque assay with untransfected Sf9 cells. Nanomolar concentrations of affinity column purified gloverin protein caused calcein to be rapidly released from unilamellar vesicles comprised of phosphatidylglycerol, but not from vesicles made up of phosphatidylcholine, suggesting that gloverin interaction with membranes is rapid and affected by membrane charge. Both the BV inactivation and calcein release activities of gloverin increased with higher concentrations of gloverin. These results demonstrate that gloverin is an antiviral protein that interacts with vesicle membranes to cause the contents to be released.