Isomerization of the intersubunit disulphide-bond in Env controls retrovirus fusion

The membrane fusion activity of murine leukaemia virus Env is carried by the transmembrane (TM) and controlled by the peripheral (SU) subunit. We show here that all Env subunits of the virus form disulphide-linked SU - TM complexes that can be disrupted by treatment with NP-40, heat or urea, or by Ca2+ depletion. Thiol mapping indicated that these conditions induced isomerization of the disulphide-bond by activating a thiol group in a Cys-X-X-Cys (CXXC) motif in SU. This resulted in dissociation of SU from the virus. The active thiol was hidden in uninduced virus but became accessible for alkylation by either Ca2+ depletion or receptor binding. The alkylation inhibited isomerization, virus fusion and infection. DTT treatment of alkylated Env resulted in cleavage of the SU - TM disulphide-bond and rescue of virus fusion. Further studies showed that virus fusion was specifically inhibited by high and enhanced by low concentrations of Ca2+. These results suggest that Env is stabilized by Ca2+ and that receptor binding triggers a cascade of reactions involving Ca2+ removal, CXXC-thiol exposure, SU - TM disulphide-bond isomerization and SU dissociation, which lead to fusion activation.