Disulphide-linked SU and TM in Mo-MLV. [³⁵S]Cys-labelled virus was purified by floatation centrifugation. Fractions were subjected to IP with anti-MLV pAb (A,B), anti-SU pAb (C,D) or anti-Env mAb 500 (E) and analysed by reducing (A,C) or nonreducing (B–E) SDS–PAGE (12%). The disulphide-linked SU–TM complexes, the nonlinked SU and TM (Pr15E and p15E) subunits, the internal viral proteins capsid (CA), matrix (MA) and nucleocapsid (NC), and contaminating host protein (hp) are indicated. Internal viral protein p12 lacks Cys and is not detected. The asterisk in (D) indicates a weak TM band. Lane 9 in all panels shows unfractionated medium (s). Note that three times less of loading zone (54%) has been analysed as compared to other fractions.

Induction of the SU–TM disulphide-bond isomerization in Mo-MLV. (A) Suppressed isomerization in DMEM. Virus, isolated by floatation centrifugation, was incubated for 0–36 h at 37°C in DMEM. NEM was added and the samples were subjected to IP with anti-MLV pAb and SU–TM disulphide-bond analyses by nonreducing SDS–PAGE (lanes 1–4). Samples that received NEM before the incubations were used as controls (lanes 5–8). (B) Heat induces isomerization. Virus in DMEM was incubated at elevated temperatures for 5–60 min before disulphide-bond analysis as in (A). (C) Urea induces isomerization. Virus was incubated in DMEM at 30°C with or without 2 M urea and NEM. (D) TN triggers isomerization. Virus in TN, pH 7.4, was incubated at 37°C with or without NEM. (E) Low pH inhibits isomerization. Virus was incubated in TN, pH 7.4, or MN, pH 5.7. (F) Ca²⁺, but not Mg²⁺, inhibits TN-induced isomerization. Virus in TN, pH 7.4, was incubated with Mg²⁺ (5 mM) or Ca²⁺ (5 mM). (G) DMEM without Ca²⁺ triggers isomerization. Mo-MLV was incubated in DMEM with or without Ca²⁺.

Receptor binding in the presence of alkylator generates an isomerization-arrested Env intermediate. (A) Induction of isomerization exposes CXXC-thiol for alkylation. Mo-MLV was adsorbed to DF-1 cells, washed two times with TN/1.8 mM Ca²⁺ and incubated for 1 h at 37°C in TN/1.8 mM Ca²⁺ (lanes 1–5), or washed two times with TN/0.05 mM EDTA and incubated in TN (lanes 6–8), with or without M135 (0.05–2.5 mM) (first inc.). The cells were washed and lysed in NP-40 buffer for 40 min at 30°C with or without alkylator (second inc.). Viral proteins were immunoprecipitated with anti-MLV pAb and analysed by nonreducing SDS–PAGE. (B) Inhibition of TN-induced isomerization by M135 and MTSET in free virus. Mo-MLV in TN was incubated for 0–3 h at 37°C in the presence of 0–1.2 mM alkylator. NEM was added, samples were lysed and viral proteins were analysed as in (A). Isomerization was estimated from the decrease of SU–TM complexes as compared to a control sample incubated in TN with 20 mM NEM and is given in %. (C) The SU–TM disulphide-bond of intact virus is DTT resistant. Mo-MLV was adsorbed to DF-1 cells and incubated for 20 or 45 min at 37°C in TN/1.8 mM Ca²⁺ with 0–30 mM DTT. NEM was added, samples were lysed and the SU–TM disulphide-bond was analysed as in (A). (D) The SU–TM disulphide-bond of isomerization-induced/M135-arrested Env is DTT sensitive. Mo-MLV was adsorbed to DF-1 cells and incubated first for 45 min at 37°C in TN with 1.5 mM M135 and then, after adding Ca²⁺ to 1.8 mM, for an additional 15 min with or without 10 mM DTT. Samples were washed, lysed without NEM and disulphide-bonds were analysed as in (A). Note that the M135 isomerization-arrested SU–TM complexes (lane 1) were reduced by DTT (lane 2). (E) Receptor binding exposes CXXC-thiol for alkylation. Mo-MLV was bound to XC or DF-1 cells and incubated for 40 min at 37°C in TN/1.8 mM Ca²⁺ with or without M135 (1.2 mM) (first inc.). The cultures were washed and lysed in NP-40 buffer for 60 min at 30°C with or without alkylator (second inc.). Viral proteins were immunoprecipitated with mAb 500 (m) or anti-MLV pAb (p) and analysed by nonreducing SDS–PAGE. Samples 5 and 6 are controls where lysates of alkylated XC or DF-1 cells have been mixed ([- -]) with nonalkylated virus–cell samples. The complete isomerization observed indicates that the alkylator has been efficiently removed after the first incubation. Note relative differences between the amounts of samples that have been applied on the gel. (F) Receptor-bound Env, arrested in isomerization by alkylation, is sensitive to SU–TM disulphide-bound reduction. Mo-MLV was bound to XC cells and incubated and analysed as in (E), but including 15 min incubation in TN/1.8 mM Ca²⁺ with or without 5 mM DTT before incubation with NP-40. Note that SU of cleaved complexes is not seen by mAb 500 (lane 3).

Isomerization induction enhances MLV fusion activity and infectivity. (A) Effect of Ca²⁺ on Mo-MLV and SFV^rec fusion. Mo-MLV was fused to XC cells (left axis) for 15 min in TN, containing 0–25 mM Ca²⁺. Fusion at control conditions, 1.8 mM Ca²⁺, is highlighted by an arrow. In some experiments, the virus was first incubated in high Ca²⁺ (10 or 25 mM) for 15 min and then under control (1.8 mM) or Ca²⁺-free (0.0 mM) conditions for another 25 min. SFV^rec fusions (right axis) were carried out for 10 min in MN, pH 5.6, in the presence of 0–25 mM Ca²⁺. (B) Effect of Ca²⁺ and Mg²⁺ on isomerization kinetics. Virus was taken up into TN containing Ca²⁺ or Mg²⁺ as indicated by dialysis and incubated at 37°C for 0–3 h. Isomerization was analysed as in (6H-inset). (C) Effect of alkylators, high Ca²⁺ and low pH on the specific SU–receptor and the unspecific virus–cell interactions. [³⁵S]Cys-labelled Mo-MLV and accompanying free SU were produced in MOV-3 cells. Part of the culture supernatant was freed from virus by centrifugation and used for SU binding analyses. The unfractionated part was used for virus analyses. Virus and SU were bound to receptor-positive XC and receptor-negative DF-1 cells at 4°C for 1 h and then washed and incubated in control conditions (DMEM or TN/1.8 mM Ca²⁺), TN/1.8 mM Ca²⁺/0.8 mM M135, TN/1.8 mM Ca²⁺/0.35 mM MTSET, MN/pH 5.7/1.8 mM Ca²⁺ or TN/25 mM Ca²⁺ at 0°C (lane 1) or 37°C (lanes 2–7) for 15 min. Cell-bound SU or virus were immunoprecipitated with anti-SU or anti-MLV pAb and analysed by nonreducing SDS–PAGE. Note that only the CA band of cell-bound virus is shown. Note also that, in contrast to virus, free SU can only bind to the receptor-positive XC cells, and that alkylators, low pH and high Ca²⁺ do not significantly affect the specific SU–receptor or the unspecific virus–cell interactions. (D) Schematic model showing how the isomerization of the SU–TM disulphide-bond controls the fusion function in MLV Env. Four steps in the activation pathway (I–IV) are shown. The main events at each step are indicated. Two SU–TM complexes of the trimeric Env are depicted. Each consists of the membrane-anchored TM subunit, with the fusion peptide (arrow), and the peripheral SU subunit. The SU–TM disulphide-bond and the associated CXXC motif in SU are indicated. The target membrane at the top contains a multispanning Env-receptor protein.

MLV and HTLV-1 Env carry isomerization activity. (A) Thiol mapping of disulphide-linked SU–TM subunits. SU–TM complexes of Env^ampho were alkylated with MB, gel isolated and glycanase F treated. Analyses of deglycosylated complexes by nonreducing (lane 1) and reducing (lane 2) SDS–PAGE. Note migration difference of reduced (red) and oxidized (ox) SU. Streptavidin capture of nonreduced (lanes 3 and 5) and reduced (lanes 4 and 6) complexes. (B) Thiol mapping of SU and TM after NP-40-induced SU–TM disulphide-bond disruption. Analyses of deglycosylated SU (lane 1) and TM (lane 2) by reducing SDS–PAGE. Streptavidin capture of SU (lanes 3 and 5) and TM (lanes 4 and 6). (C) SU and TM are disulphide-linked in HTLV-1 Env. BHK-21 cells expressing the HTLV-1 env gene were labelled with [³⁵S]Cys for 30 min and chased for 5–240 min. Cells and media were lysed in the presence of NEM. Immunoprecipitated Env was analysed by nonreducing (lanes 1–6) or reducing (lanes 7–12) SDS–PAGE. (D) HTLV-1 Env can isomerize. Cells expressing HTLV-1 env at 2 h chase time were incubated in NP-40 lysis buffer for 40 min at 30°C with or without NEM. Immunoprecipitated Env was analysed by nonreducing SDS–PAGE. (E) RSV Env cannot isomerize. Cells expressing RSV env were labelled as above, chased for 2 h and lysed for 0–2 h at 30°C with or without NEM. Immunoprecipitated Env was analysed by nonreducing (lanes 1–8) or reducing (lanes 9–12) SDS–PAGE.

Isomerization results in SU dissociation. (A) Preparation of Mo-MLV with partially or completely isomerized SU–TM disulphide-bond. Mo-MLV was dialysed against TN, incubated as indicated, lysed in the presence of NEM, subjected to IP with anti-MLV pAb and analysed by nonreducing SDS–PAGE. (B,C) Isomerization results in dissociation of SU. Mo-MLV with partially (B) or completely (C) isomerized SU–TM disulphide-bond was subjected to floatation centrifugation. Fractions were immunoprecipitated and analysed by nonreducing SDS–PAGE. Note that four times less of loading zone (54%) has been analysed as compared to other fractions. (D) Conformational changes follow isomerization. Mo-MLV were incubated for 1–3 h at 37°C in TN with or without NEM, immunoprecipitated with pAb or mAb 500 and analysed by nonreducing SDS–PAGE.

Isomerization inhibition suppresses MLV fusion and infectivity. (A,B) Mo-MLV induced fusion-from-without. Methylene-blue-stained XC cells before (A) and after (B) viral fusion (40 min at 37°C) and polykaryon formation. (C) Time course of Mo-MLV fusion in TN/1.8 mM Ca²⁺. Virus was fused with XC cells for 0–40 min before inactivation. The relative fusion efficiencies using fusion in TN/1.8 mM Ca²⁺ for 40 min as control are shown. (D) Alkylation inhibits Mo-MLV but not SFV^rec fusion. Mo-MLV fusion was performed for 10 min in TN/1.8 mM Ca²⁺ with 0–0.8 mM M135 or MTSET. Fusion efficiencies are given in % of fusion in TN/1.8 mM Ca²⁺ without drug. Fusion efficiencies with prealkylated cells or virus are also shown. SFV^rec fusions were carried out by sequential incubations in TN (pH 7.4)/1.8 mM Ca²⁺ (5 min on ice), MN (pH 5.6)/1.8 mM Ca²⁺ (5 min at 37°C) and TN (pH 7.4)/1.8 mM Ca²⁺ (5 min at 37°C) with or without alkylator. (E) Rescue of M135- and MTSET-inhibited fusion by second, drug-free incubation. Mo-MLV fusion was carried out either for 10 min or 10+25 min in TN/1.8 mM Ca²⁺ with or without 0.8 mM M135 or 0.35 mM MTSET. Fusion efficiencies are given in % of the 10+25 min fusion without drug. (F) DTT rescue of M135-inhibited fusion. Mo-MLV fusion was performed for 15 min in TN/1.8 mM Ca²⁺ with M135 (0.8 mM) or DTT (5 and 25 mM), and for an additional 10 min in TN/1.8 mM Ca²⁺ with or without DTT (1.5–25 mM). (G) Alkylation inhibits infectivity of MLV. 3T3 cells were infected with Env^Mo-MLV-egfp vectors either for 10 min or for 10+25 min with or without alkylator. Infectivities (egfp-positive cells) are given in % of that of the 10+25 min infection without drug. (H) Inhibition of Mo-MLV fusion by low pH. Fusions were performed in DMEM/25 mM Tris or /MES at pH values ranging from 7.5 (control) to 5.5 for 15 min and expressed as % of control. The reversibility was shown by consecutive 15 min incubations at pH 5.7 and 7.4. (inset) Inhibition of isomerization by low pH. Isomerization was induced by incubating virus for 1–3 h at 37°C in TN or MN buffers at the indicated pH. Samples were analysed as in and isomerization is given in % of that in control samples, which received NEM before incubation.