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.