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

ABSTRACT: 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 Ca(2+) 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 Ca(2+) 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 Ca(2+). These results suggest that Env is stabilized by Ca(2+) and that receptor binding triggers a cascade of reactions involving Ca(2+) removal, CXXC-thiol exposure, SU-TM disulphide-bond isomerization and SU dissociation, which lead to fusion activation.

SUBMITTER: Wallin M

PROVIDER: S-EPMC1271652 | biostudies-literature | 2004 Jan

REPOSITORIES: biostudies-literature

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

Wallin Michael M Ekström Maria M Garoff Henrik H

The EMBO journal 20031211 1

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 Ca(2+) 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 ...[more]

PMID: 14685283

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Project description:The protein disulphide-bond isomerization activity of highly active homogeneous protein disulphide-isomerase (measured by re-activation of 'scrambled' ribonuclease) is enhanced by EDTA and by phosphate buffers. As shown for previous less-active preparations, the enzyme has a narrow pH optimum around pH 7.8 and requires the presence of either a dithiol or a thiol. The dithiol dithiothreitol is effective at concentrations 100-fold lower than the monothiols reduced glutathione and cysteamine. The enzyme follows Michaelis-Menten kinetics with respect to these substrates; Km values are 4,620 and 380 microM respectively. The enzyme shows apparent inhibition by high concentrations of thiol or dithiol compounds (greater than 10 X Km), but the effect is mainly on the extent of reaction, not the initial rate. This is interpreted as indicating the formation of significant amounts of reduced ribonuclease in these more reducing conditions. The purified enzyme will also catalyse net reduction of insulin disulphide bonds by reduced glutathione (i.e. it has thiol:protein-disulphide oxidoreductase or glutathione:insulin transhydrogenase activity), but this requires considerably higher concentrations of enzyme and reduced glutathione than does the disulphide-isomerization activity. The Km for reduced glutathione in this reaction is an order of magnitude greater than that for the disulphide-isomerization activity, and the turnover number is considerably lower than that of other enzymes that can catalyse thiol-disulphide oxidoreduction. Conventional two-substrate steady-state analysis of the thiol:protein-disulphide oxidoreductase activity indicates that it follows a ternary-complex mechanism. The protein disulphide-isomerase and thiol:protein-disulphide oxidoreductase activities co-purify quantitatively through the final stages of purification, implying that a single protein species is responsible for both activities. It is concluded that previous preparations, from various sources, that have been referred to as protein disulphide-isomerase, disulphide-interchange enzyme, thiol:protein-disulphide oxidoreductase or glutathione:insulin transhydrogenase are identical or homologous proteins. The assay, nomenclature and physiological role of this enzyme are discussed.

Dataset Information

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

Publications

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

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