Michael Wallin

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.

Intersubunit Disulfide Isomerization Controls Membrane Fusion of Human T-Cell Leukemia Virus Env

ABSTRACT Human T-cell leukemia virus (HTLV-1) Env carries a typical disulfide isomerization motif, C225XXC, in the C-terminal domain SU. Here we have tested whether this motif is used for isomerization of the intersubunit disulfide of Env and whether this rearrangement is required for membrane fusion. We introduced the C225A and C228A mutations into Env and found that the former but not the latter mutant matured into covalently linked SU-TM complexes in transfected cells. Next, we constructed a secreted Env ectodomain and showed that it underwent incubation-dependent intersubunit disulfide isomerization on target cells. However, the rearrangement was blocked by the C225A mutation, suggesting that C225 carried the isomerization-active thiol. Still, it was possible to reduce the intersubunit disulfide of the native C225A ectodomain mutant with dithiothreitol (DTT). The importance of the CXXC-mediated disulfide isomerization for infection was studied using murine leukemia virus vectors pseudotyped with wild-type or C225A HTLV-1 Env. We found that the mutant Env blocked infection, but this could be rescued with DTT. The fusion activity was tested in a fusion-from-within assay using a coculture of rat XC target and transfected BHK-21 effector cells. We found that the mutation blocked polykaryon formation, but this could be reversed with DTT. Similar DTT-reversible inhibition of infection and fusion was observed when a membrane-impermeable alkylator was present during the infection/fusion incubation. We conclude that the fusion activity of HTLV-1 Env is controlled by an SU CXXC-mediated isomerization of the intersubunit disulfide. Thus, this extends the applicability of the isomerization model from gammaretroviruses to deltaretroviruses.

The Fusion-Controlling Disulfide Bond Isomerase in Retrovirus Env Is Triggered by Protein Destabilization

ABSTRACT The membrane fusion function of murine leukemia virus (MLV) is carried by the Env protein. This protein is composed of three SU-TM subunit complexes. The fusion activity is loaded into the transmembrane TM subunit and controlled by the peripheral, receptor-binding SU subunit. It is assumed that TM adopts a metastable conformation in the native Env and that fusion activation involves the folding of TM into a stable form. Activation is suppressed by the associated SU and triggered by its dissociation, which follows receptor binding. Recently we showed that the two subunits are disulfide linked and that SU dissociation and triggering of the fusion function are caused by a switch of the intersubunit disulfide into an intrasubunit disulfide isomer using an isomerization-active CWLC motif in SU (M. Wallin, M. Ekstrom, and H. Garoff, EMBO J. 23:54-65, 2004). In the present work we address how the SU disulfide isomerase is activated. Using Moloney MLV, we show that isomerization of the SU-TM disulfide bond can be triggered by heat, urea, or guanidinium hydrochloride. Such protein perturbation treatments also significantly increase the kinetics and efficiency of viral fusion. The threshold conditions for the effects on isomerization and fusion are virtually the same. This finding indicates that destabilization of interactions in the SU oligomer induces the disulfide bond isomerase and the subsequent activation of the fusion function in TM.

Kinetic Analyses of the Surface-Transmembrane Disulfide Bond Isomerization-Controlled Fusion Activation Pathway in Moloney Murine Leukemia Virus

ABSTRACT The surface (SU) and transmembrane (TM) subunits of Moloney murine leukemia virus (Mo-MLV) Env are disulfide linked. The linking cysteine in SU is part of a conserved CXXC motif in which the other cysteine carries a free thiol. Recently, we showed that receptor binding activates its free thiol to isomerize the intersubunit disulfide bond into a disulfide within the motif instead (M. Wallin, M. Ekström and H. Garoff, EMBO J. 23:54-65, 2004). This facilitated SU dissociation and activation of TM for membrane fusion. The evidence was mainly based on the finding that alkylation of the CXXC-thiol prevented isomerization. This arrested membrane fusion, but the activity could be rescued by cleaving the intersubunit disulfide bond with dithiothreitol (DTT). Here, we demonstrate directly that receptor binding causes SU-TM disulfide bond isomerization in a subfraction of the viral Envs. The kinetics of the isomerization followed that of virus-cell membrane fusion. Arresting the fusion with lysophosphatidylcholine did not arrest isomerization, suggesting that isomerization precedes the hemifusion stage of fusion. Our earlier finding that native Env was not possible to alkylate but required isomerization induction by receptor binding intimated that alkylation trapped an intermediate form of Env. To further clarify this possibility, we analyzed the kinetics by which the alkylation-sensitive Env was generated during fusion. We found that it followed the fusion kinetics. In contrast, the release of fusion from alkylated, isomerization-blocked virus by DTT reduction of the SU-TM disulfide bond was much faster. These results suggest that the alkylation-sensitive form of Env is a true intermediate in the fusion activation pathway of Env.

Furin Cleavage Potentiates the Membrane Fusion-Controlling Intersubunit Disulfide Bond Isomerization Activity of Leukemia Virus Env

ABSTRACT The membrane fusion protein of murine leukemia virus is a trimer of a disulfide-linked peripheral-transmembrane (SU-TM) subunit complex. The intersubunit disulfide bond is in SU linked to a disulfide bond isomerization motif, CXXC, with which the virus controls its fusion reaction (M. Wallin, M. Ekström, and H. Garoff, EMBO J. 23:54-65, 2004). Upon receptor binding the isomerase rearranges the intersubunit disulfide bond into a disulfide bond isomer within the motif. This facilitates SU dissociation and fusion activation in the TM subunit. In the present study we have asked whether furin cleavage of the Env precursor potentiates the isomerase to be triggered. To this end we accumulated the late form of the precursor, gp90, in the cell by incubation in the presence of a furin-inhibiting peptide. The isomerization was done by NP-40 incubation or by a heat pulse under alkylation-free conditions. The cells were lysed in the presence of alkylator, and the precursor was immunoprecipitated, gel isolated, deglycosylated, and subjected to complete trypsin digestion. Disulfide-linked peptide complexes were separated by sodium dodecyl sulfate-tricine-polyacrylamide gel electrophoresis under nonreducing conditions. This assay revealed the size of the characteristic major disulfide-linked peptide complex that differentiates the two isomers of the disulfide bond between Cys336 (or Cys339) and Cys563, i.e., the bond corresponding to the intersubunit disulfide bond. The analyses showed that the isomerase was five- to eightfold more resistant to triggering in the precursor than in the mature, cleaved form. This suggests that the isomerase becomes potentiated for triggering by a structural change in Env that is induced by furin cleavage in the cell.

R-Peptide Cleavage Potentiates Fusion-Controlling Isomerization of the Intersubunit Disulfide in Moloney Murine Leukemia Virus Env

ABSTRACT Fusion of the membrane of the Moloney murine leukemia virus (Mo-MLV) Env protein is facilitated by cleavage of the R peptide from the cytoplasmic tail of its TM subunit, but the mechanism for this effect has remained obscure. The fusion is also controlled by the isomerization of the intersubunit disulfide of the Env SU-TM complex. In the present study, we used several R-peptide-cleavage-inhibited virus mutants to show that the R peptide suppresses the isomerization reaction in both in vitro and in vivo assays. Thus, the R peptide affects early steps in the activation pathway of murine leukemia virus Env.

Receptor-Triggered but Alkylation-Arrested Env of Murine Leukemia Virus Reveals the Transmembrane Subunit in a Prehairpin Conformation

ABSTRACT A central feature of the prevailing model for retrovirus fusion is conversion of the transmembrane (TM) subunit from a prehairpin to a hairpin-like structure. The fusion inhibition of many retroviruses, except murine leukemia virus (MLV), with peptides corresponding to interacting regions in the hairpin supports the model. MLV fusion is controlled by isomerization of the intersubunit disulfide in Env. We show here that TM peptides bind to MLV Env that has been arrested at an intermediate stage of activation by alkylation of the isomerization-active thiol in the surface subunit. This inhibits fusion rescue by dithiothreitol-mediated reduction of the surface protein-TM disulfide.

Turning of the receptor‐binding domains opens up the murine leukaemia virus Env for membrane fusion

The activity of the membrane fusion protein Env of Moloney mouse leukaemia virus is controlled by isomerization of the disulphide that couples its transmembrane (TM) and surface (SU) subunits. We have arrested Env activation at a stage prior to isomerization by alkylating the active thiol in SU and compared the structure of isomerization‐arrested Env with that of native Env. Env trimers of respective form were isolated from solubilized particles by sedimentation and their structures were reconstructed from electron microscopic images of both vitrified and negatively stained samples. We found that the protomeric unit of both trimers formed three protrusions, a top, middle and a lower one. The atomic structure of the receptor‐binding domain of SU fitted into the upper protrusion. This was formed similar to a bent finger. Significantly, in native Env the tips of the fingers were directed against each other enclosing a cavity below, whereas they had turned outward in isomerization‐arrested Env transforming the cavity into an open well. This might subsequently guide the fusion peptides in extended TM subunits into the target membrane.