title: Identification and functional analysis of inter-subunit disulfide bonds of the F protein of Helicoverpa armigera nucleopolyhedrovirus.
abstract: 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.
authors.affiliation: School of Tropical and Laboratory Medicine, Hainan Medical University, Haikou 571101, PR China.|State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan 430071, PR China.
authors.forename: Feifei
authors.initials: F
authors.lastname: Yin
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan 430071, PR China.
authors.forename: Manli
authors.initials: M
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan 430071, PR China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Tan
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan 430071, PR China.
authors.forename: Fei
authors.initials: F
authors.lastname: Deng
authors.affiliation: Laboratory of Virology, Wageningen University, 6708 PB Wageningen, The Netherlands.
authors.forename: Just M
authors.initials: JM
authors.lastname: Vlak
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan 430071, PR China.
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Hu
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan 430071, PR China.
authors.forename: Hualin
authors.initials: H
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D009036:Moths / Q000166:cytology; D009154:Mutation; D017924:Nucleopolyhedroviruses / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits; D014760:Viral Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053586:Virus Internalization
chemical_list: D021122:Protein Subunits; D014760:Viral Fusion Proteins