Identification and functional analysis of inter-subunit disulfide bonds of the F protein of Helicoverpa armigera nucleopolyhedrovirus.
Identification and functional analysis of inter-subunit disulfide bonds of the F protein of Helicoverpa armigera nucleopolyhedrovirus.
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DOI：
10.1099/vir.0.068122-0
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发表时间：
2014-12
期刊：
影响因子：
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通讯作者：
F. Yin;Manli Wang;Ying Tan;F. Dèng;J. Vlak;Zhìhóng Hú;Huálín Wáng
中科院分区：
文献类型：
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作者：
F. Yin;Manli Wang;Ying Tan;F. Dèng;J. Vlak;Zhìhóng Hú;Huálín Wáng
杆状病毒芽生病毒的主要囊膜融合蛋白F由两个二硫键连接的亚基组成：N端的F2亚基和C端的膜锚定F1亚基。F2中有1个半胱氨酸，F1中有15个半胱氨酸，但它们在二硫键连接中的作用很大程度上是未知的。本研究利用定点突变技术对棉铃虫单核衣壳核型多角体病毒F蛋白的亚基间和亚基内二硫键进行了分析。结果表明，在功能性F蛋白中，氨基酸C108(F2)和C241(F1)之间存在亚基间二硫键。当C241发生突变时，C108和C232之间形成了一个替代的二硫键，导致F不起作用。在F1的一个双C232/C241突变体中没有观察到亚基间桥。C403不参与亚基间二硫键的形成，但该氨基酸的突变显著降低了病毒的感染力，提示它可能参与亚基内二硫键的形成。测定了还原剂[三(2-羧乙基)膦(TCEP)]和游离硫醇抑制剂[4-乙酰氨基-4‘-马来酰亚胺基二苯乙烯(AMS)和5，5’-二硫双(2-硝基苯甲酸)(DTNB)]对心脏NPV感染性的影响。结果表明，TCEP能显著降低HzAm1细胞感染心脏NPV的能力。相反，AMS和DTNB对病毒感染性没有抑制作用。这些数据表明，游离硫醇/二硫键异构化不太可能在病毒进入和传染性方面发挥作用。
The major envelope fusion protein F of the budded virus of baculoviruses consists of two disulfide-linked subunits: an N-terminal F2 subunit and a C-terminal, membrane-anchored F1 subunit. There is one cysteine in F2 and there are 15 cysteines in F1, but their role in disulfide linking is largely unknown. In this study, the inter- and intra-subunit disulfide bonds of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) F protein were analysed by site-directed mutagenesis. Results indicated that in a functional F protein, an inter-subunit disulfide bond exists between amino acids C108 (F2) and C241 (F1). When C241 was mutated, an alternative disulfide bond was formed between C108 and C232, rendering F non-functional. No inter-subunit bridge was observed in a double C232/C241 mutant of F1. C403 was not involved in the formation of inter-subunit disulfide bonding, but mutation of this amino acid decreased viral infectivity significantly, suggesting that it might be involved in intra-subunit disulfide bonds. The influence of reductant [tris(2-carboxyethyl) phosphine (TCEP)] and free-thiol inhibitors [4-acetamido-4'-maleimidylstilbene 2,2'-disulfonic acid (AMS) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)] on the infectivity of HearNPV was tested. The results indicated that TCEP greatly decreased the infection of HzAm1 cells by HearNPV. In contrast, AMS and DTNB had no inhibitory effect on viral infectivity. The data suggested that free thiol/disulfide isomerization was not likely to play a role in viral entry and infectivity.