Identification and functional analysis of inter-subunit disulfide bonds of F protein of HearNPV.

　　The major envelope fusion protein F of budded virus (BV) 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 (C) in F2 and there are fifteen 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 was analyzed by site-directed mutagenesis. Results indicate 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, but 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 (TCEP) and free-thiol inhibitors (AMS and 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. These data suggest that free thiol/disulfide isomerization was not likely to play roles in viral entry and infectivity.

http://vir.sgmjournals.org/content/early/2014/08/10/vir.0.068122-0.short