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1.
Proc Natl Acad Sci U S A ; 114(10): 2723-2728, 2017 03 07.
Article in English | MEDLINE | ID: mdl-28223490

ABSTRACT

Viral membrane fusion proteins of class I are trimers in which the protomeric unit is a complex of a surface subunit (SU) and a fusion active transmembrane subunit (TM). Here we have studied how the protomeric units of Moloney murine leukemia virus envelope protein (Env) are activated in relation to each other, sequentially or simultaneously. We followed the isomerization of the SU-TM disulfide and subsequent SU release from Env with biochemical methods and found that this early activation step occurred sequentially in the three protomers, generating two asymmetric oligomer intermediates according to the scheme (SU-TM)3 → (SU-TM)2TM → (SU-TM)TM2 → TM3 This was the case both when activation was triggered in vitro by depleting stabilizing Ca2+ from solubilized Env and when viral Env was receptor triggered on rat XC cells. In the latter case, the activation reaction was too fast for direct observation of the intermediates, but they could be caught by alkylation of the isomerization active thiol.


Subject(s)
Moloney murine leukemia virus/chemistry , Protein Subunits/chemistry , Viral Envelope Proteins/genetics , Viral Fusion Proteins/genetics , Alkylation , Animals , Cell Line , Disulfides/chemistry , Isomerism , Mice , Moloney murine leukemia virus/genetics , Moloney murine leukemia virus/pathogenicity , Protein Subunits/genetics , Rats , Surface Properties , Viral Envelope Proteins/chemistry , Viral Fusion Proteins/chemistry , Virus Internalization
2.
Nat Methods ; 13(4): 345-51, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26950744

ABSTRACT

A limiting factor in membrane protein research is the ability to solubilize and stabilize such proteins. Detergents are used most often for solubilizing membrane proteins, but they are associated with protein instability and poor compatibility with structural and biophysical studies. Here we present a saposin-lipoprotein nanoparticle system, Salipro, which allows for the reconstitution of membrane proteins in a lipid environment that is stabilized by a scaffold of saposin proteins. We demonstrate the applicability of the method on two purified membrane protein complexes as well as by the direct solubilization and nanoparticle incorporation of a viral membrane protein complex from the virus membrane. Our approach facilitated high-resolution structural studies of the bacterial peptide transporter PeptTSo2 by single-particle cryo-electron microscopy (cryo-EM) and allowed us to stabilize the HIV envelope glycoprotein in a functional state.


Subject(s)
Bacterial Proteins/chemistry , HIV Envelope Protein gp120/chemistry , Lipids/chemistry , Membrane Proteins/chemistry , Nanoparticles/chemistry , Saposins/chemistry , Symporters/chemistry , Bacterial Proteins/metabolism , Cryoelectron Microscopy/methods , HIV Envelope Protein gp120/metabolism , Humans , Membrane Proteins/metabolism , Protein Conformation , Saposins/metabolism , Symporters/metabolism
3.
Proc Natl Acad Sci U S A ; 111(16): 6034-9, 2014 Apr 22.
Article in English | MEDLINE | ID: mdl-24711391

ABSTRACT

The trimeric Moloney murine leukemia virus Env protein matures by two proteolytic cleavages. First, furin cleaves the Env precursor into the surface (SU) and transmembrane (TM) subunits in the cell and then the viral protease cleaves the R-peptide from TM in new virus. Here we analyzed the structure of the furin precursor, by cryoelectron microscopy. We transfected 293T cells with a furin cleavage site provirus mutant, R466G/K468G, and produced the virus in the presence of amprenavir to also inhibit the R-peptide cleavage. Although Env incorporation into particles was inhibited, enough precursor could be isolated and analyzed by cryoelectron microscopy to yield a 3D structure at 22 Å resolution. This showed an open cage-like structure like that of the R-peptide precursor and the mature Env described before. However, the middle protrusion of the protomeric unit, so prominently pointing out from the side of the more mature forms of the Env, was absent. Instead, there was extra density in the top protrusion. This suggested that the C-terminal SU domain was associated alongside the receptor binding N-terminal SU domain in the furin precursor. This was supported by mapping with a SU C-terminal domain-specific antigen binding fragment. We concluded that furin cleavage not only separates the subunits and liberates the fusion peptide at the end of TM but also allows the C-terminal domain to relocate into a peripheral position. This conformational change might explain how the C-terminal domain of SU gains the potential to undergo disulfide isomerization, an event that facilitates membrane fusion.


Subject(s)
Furin/metabolism , Gene Products, env/chemistry , Gene Products, env/metabolism , Moloney murine leukemia virus/metabolism , Animals , Electrophoresis, Polyacrylamide Gel , Humans , Immunoglobulin Fab Fragments/metabolism , Mice , Models, Molecular , Molecular Weight , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Protein Multimerization , Trypsin/metabolism
4.
J Virol ; 87(12): 7000-7, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23596290

ABSTRACT

The HIV-1 spike is composed of three protomeric units, each containing a peripheral gp120 and a transmembrane gp41 subunit. Binding to the CD4 and the chemokine receptors triggers them to mediate virus entry into cells by membrane fusion. The spikes also represent the major target for neutralizing antibodies (Abs) against the virus. We have studied how two related broadly neutralizing Abs, PG9 and PG16, react with the spike. Unexpectedly, this also suggested how the functions of the individual protomers in the spike depend on each other. The Abs have been shown to bind the V1/V2 loops of gp120, located at the top of the spike. Using blue native-polyacrylamide gel electrophoresis (BN-PAGE), we show that only single Abs or antigen-binding fragments could bind to the spikes of HIV-1 virus-like particles. Apparently, binding to one gp120 sterically interferes with binding to the other two subunits in the spike top. Despite this constraint, all of the protomers of the spike became resistant to CD4 binding and subsequent formation of the coreceptor binding site. These activities were measured by monitoring the sequential complex formation of the spike first with Abs and then with soluble 2d- or 4d-CD4 or with soluble CD4 and the CD4 inducible coreceptor binding site Ab 17b in BN-PAGE. The inhibition of the spike by single-Ab binding suggested that the activation reactions of the individual protomeric units are linked to each other in a coordinated activation process.


Subject(s)
Antibodies, Monoclonal/metabolism , Antibodies, Neutralizing/metabolism , HIV Envelope Protein gp120/metabolism , HIV Envelope Protein gp41/metabolism , HIV-1/immunology , env Gene Products, Human Immunodeficiency Virus/chemistry , env Gene Products, Human Immunodeficiency Virus/metabolism , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Binding Sites , CD4 Antigens/genetics , CD4 Antigens/metabolism , HEK293 Cells , HIV Envelope Protein gp120/immunology , HIV Envelope Protein gp41/immunology , HIV-1/metabolism , Humans , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fab Fragments/metabolism , Native Polyacrylamide Gel Electrophoresis/methods , Protein Binding , Protein Multimerization , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Virus Internalization , env Gene Products, Human Immunodeficiency Virus/immunology
5.
Proc Natl Acad Sci U S A ; 109(20): 7735-40, 2012 May 15.
Article in English | MEDLINE | ID: mdl-22547812

ABSTRACT

The Env protein of murine leukemia virus matures by two cleavage events. First, cellular furin separates the receptor binding surface (SU) subunit from the fusion-active transmembrane (TM) subunit and then, in the newly assembled particle, the viral protease removes a 16-residue peptide, the R-peptide from the endodomain of the TM. Both cleavage events are required to prime the Env for receptor-triggered activation. Cryoelectron microscopy (cryo-EM) analyses have shown that the mature Env forms an open cage-like structure composed of three SU-TM complexes, where the TM subunits formed separated Env legs. Here we have studied the structure of the R-peptide precursor Env by cryo-EM. TM cleavage in Moloney murine leukemia virus was inhibited by amprenavir, and the Envs were solubilized in Triton X-100 and isolated by sedimentation in a sucrose gradient. We found that the legs of the R-peptide Env were held together by trimeric interactions at the very bottom of the Env. This suggested that the R-peptide ties the TM legs together and that this prevents the activation of the TM for fusion. The model was supported by further cryo-EM studies using an R-peptide Env mutant that was fusion-competent despite an uncleaved R-peptide. The Env legs of this mutant were found to be separated, like in the mature Env. This shows that it is the TM leg separation, normally caused by R-peptide cleavage, that primes the Env for receptor triggering.


Subject(s)
Gene Products, env/metabolism , Leukemia Virus, Murine/metabolism , Models, Molecular , Protein Conformation , Protein Precursors/metabolism , Cryoelectron Microscopy , Electrophoresis, Polyacrylamide Gel , Fractionation, Field Flow , Moloney murine leukemia virus , Octoxynol , Protein Processing, Post-Translational , Protein Subunits/metabolism
6.
J Virol ; 85(11): 5644-50, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21430054

ABSTRACT

Alphaviruses are taken up into the endosome of the cell, where acidic conditions activate the spikes for membrane fusion. This involves dissociation of the three E2-E1 heterodimers of the spike and E1 interaction with the target membrane as a homotrimer. The biosynthesis of the heterodimer as a pH-resistant p62-E1 precursor appeared to solve the problem of premature activation in the late and acidic parts of the biosynthetic transport pathway in the cell. However, p62 cleavage into E2 and E3 by furin occurs before the spike has left the acidic compartments, accentuating the problem. In this work, we used a furin-resistant Semliki Forest virus (SFV) mutant, SFV(SQL), to study the role of E3 in spike activation. The cleavage was reconstituted with proteinase K in vitro using free virus or spikes on SFV(SQL)-infected cells. We found that E3 association with the spikes was pH dependent, requiring acidic conditions, and that the bound E3 suppressed spike activation. This was shown in an in vitro spike activation assay monitoring E1 trimer formation with liposomes and a fusion-from-within assay with infected cells. Furthermore, the wild type, SFV(wt), was found to bind significant amounts of E3, especially if produced in dense cultures, which lowered the pH of the culture medium. This E3 also suppressed spike activation. The results suggest that furin-cleaved E3 continues to protect the spike from premature activation in acidic compartments of the cell and that its release in the neutral extracellular space primes the spike for low-pH activation.


Subject(s)
Membrane Glycoproteins/metabolism , Semliki forest virus/physiology , Viral Envelope Proteins/metabolism , Animals , Cell Line , Cricetinae , Endopeptidase K/metabolism , Furin/metabolism , Protein Binding
7.
J Virol ; 85(7): 3262-9, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21228228

ABSTRACT

The spike protein of murine leukemia virus, MLV, is made as a trimer of the Env precursor. This is primed for receptor-induced activation of its membrane fusion function first by cellular furin cleavage in the ectodomain and then by viral protease cleavage in the endodomain. The first cleavage separates the peripheral surface (SU) subunit from the transmembrane (TM) subunit, and the latter releases a 16-residue-long peptide (R) from the TM endodomain. Here, we have studied the distribution of R peptide cleavages in the spike TM subunits of Moloney MLV preparations with partially R-peptide-processed spikes. The spikes were solubilized as trimers and separated with an R peptide antibody. This showed that the spikes were either uncleaved or cleaved in all of its TM subunits. Further studies showed that R peptide cleavage-inhibited Env mutants, L(649)V and L(649)I, were rescued by wild-type (wt) Env in heterotrimeric spikes. These findings suggested that the R peptide cleavages in the spike are facilitated through positive allosteric cooperativity; i.e., the cleavage of the TM subunit in one Env promoted the cleavages of the TMs in the other Envs. The mechanism ensures that protease cleavage in newly released virus will generate R-peptide-cleaved homotrimers rather than heterotrimeric intermediates. However, using a cleavage site Env mutant, L(649)R, which was not rescued by wt Env, it was possible to produce virus with heterotrimers. These were shown to be less fusion active than the R-peptide-cleaved homotrimers. Therefore, the cooperative cleavage will speed up the maturation of released virus for fusion competence.


Subject(s)
Gene Products, env/metabolism , Moloney murine leukemia virus/physiology , Virus Internalization , Virus Release , Protein Processing, Post-Translational
8.
Proc Natl Acad Sci U S A ; 107(44): 18844-9, 2010 Nov 02.
Article in English | MEDLINE | ID: mdl-20956336

ABSTRACT

The HIV-1 spike is a trimer of the transmembrane gp41 and the peripheral gp120 subunit pair. It is activated for virus-cell membrane fusion by binding sequentially to CD4 and to a chemokine receptor. Here we have studied the structural transition of the trimeric spike during the activation process. We solubilized and isolated unliganded and CD4-bound spikes from virus-like particles and used cryoelectron microscopy to reconstruct their 3D structures. In order to increase the yield and stability of the spike, we used an endodomain deleted and gp120-gp41 disulfide-linked variant. The unliganded spike displayed a hollow cage-like structure where the gp120-gp41 protomeric units formed a roof and bottom, and separated lobes and legs on the sides. The tripod structure was verified by fitting the recent atomic core structure of gp120 with intact N- and C-terminal ends into the spike density map. This defined the lobe as gp120 core, showed that the legs contained the polypeptide termini, and suggested the deleted variable loops V1/V2 and V3 to occupy the roof and gp41 the bottom. CD4 binding shifted the roof density peripherally and condensed the bottom density centrally. Fitting with a V3 containing gp120 core suggested that the V1/V2 loops in the roof were displaced laterally and the V3 lifted up, while the core and leg were kept in place. The loop displacements probably prepared the spike for coreceptor interaction and roof opening so that a new fusion-active gp41 structure, assembled at the center of the cage bottom, could reach the target membrane.


Subject(s)
CD4 Antigens , HIV Envelope Protein gp120 , HIV Envelope Protein gp41 , HIV-1/ultrastructure , Imaging, Three-Dimensional , Models, Molecular , Cryoelectron Microscopy , HIV-1/chemistry , Humans
9.
EMBO J ; 27(20): 2799-808, 2008 Oct 22.
Article in English | MEDLINE | ID: mdl-18800055

ABSTRACT

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.


Subject(s)
Gene Products, env/chemistry , Gene Products, env/physiology , Leukemia Virus, Murine/metabolism , Viral Fusion Proteins/metabolism , Animals , Cell Membrane/metabolism , Cryoelectron Microscopy/methods , Gene Products, env/metabolism , Genes, env , Image Processing, Computer-Assisted , Mice , Models, Biological , Models, Molecular , Molecular Conformation , Protein Binding/genetics , Protein Structure, Tertiary
10.
J Biol Chem ; 283(39): 26452-60, 2008 Sep 26.
Article in English | MEDLINE | ID: mdl-18596032

ABSTRACT

In alphaviruses, here represented by Semliki Forest virus, infection requires an acid-responsive spike configuration to facilitate membrane fusion. The creation of this relies on the chaperone function of glycoprotein E2 precursor (p62) and its maturation cleavage into the small external E3 and the membrane-anchored E2 glycoproteins. To reveal how the E3 domain of p62 exerts its control of spike functions, we determine the structure of a p62 cleavage-impaired mutant virus particle (SQL) by electron cryomicroscopy. A comparison with the earlier solved wild type virus structure reveals that the E3 domain of p62(SQL) forms a bulky side protrusion in the spike head region. This establishes a gripper over part of domain II of the fusion protein, with a cotter-like connection downward to a hydrophobic cluster in its central beta-sheet. This finding reevaluates the role of the precursor from being only a provider of a shield over the fusion loop to a structural playmate in formation of the fusogenic architecture.


Subject(s)
Glycoproteins/metabolism , Molecular Chaperones/metabolism , Semliki forest virus/metabolism , Viral Proteins/metabolism , Alphavirus Infections/genetics , Alphavirus Infections/metabolism , Animals , Cricetinae , Glycoproteins/genetics , Molecular Chaperones/genetics , Mutation , Polyamines/metabolism , Protein Structure, Secondary/genetics , Protein Structure, Tertiary/genetics , Semliki forest virus/genetics , Viral Proteins/genetics
11.
J Virol ; 82(14): 7135-43, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18480461

ABSTRACT

Human T-cell leukemia virus (HTLV-1) Env carries a typical disulfide isomerization motif, C(225)XXC, 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 C(225) 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.


Subject(s)
Disulfides/metabolism , Gene Products, env/metabolism , Human T-lymphotropic virus 1/physiology , Virus Internalization , Amino Acid Substitution/genetics , Animals , Cell Line , Cricetinae , Dithiothreitol/pharmacology , Gene Products, env/genetics , Humans , Mutation, Missense , Rats , Reducing Agents/pharmacology
12.
J Virol ; 82(5): 2358-66, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18094169

ABSTRACT

The transmembrane subunit (TM) of the trimeric retrovirus Env complex is thought to direct virus-cell membrane fusion by refolding into a cell membrane-interacting, extended form that subsequently folds back on itself into a very stable trimer of hairpin-like TM polypeptides. However, so far there is only limited evidence for the formation of a stable TM trimer during Env activation. Here we have studied the oligomer composition and stability of an intermediate and the fully activated form of Moloney murine leukemia virus (Mo-MLV) Env. Activation of Mo-MLV Env is controlled by isomerization of its intersubunit disulfide. This results in surface subunit (SU) dissociation and TM refolding. If activation is done in the presence of an alkylator, this will modify the isomerization-active thiol in the SU of Env and arrest Env at an intermediate stage, the isomerization-arrested state (IAS) of its activation pathway. We generated IAS and fully activated Envs in vitro and in vivo and studied their states of oligomerization by two-dimensional blue native polyacrylamide gel electrophoresis (PAGE) and nonreducing sodium dodecyl sulfate (SDS)-PAGE. The IAS Env was composed of trimers of SU-TM complexes, whereas the activated Env consisted of SU monomers and TM trimers. When the oligomers were subjected to mild SDS treatment the TM trimer was found to be 3.5 times more resistant than the IAS oligomer. Thus, this demonstrates that a structural conversion of TM takes place during activation, which results in the formation of a stable TM trimer.


Subject(s)
Biopolymers/metabolism , Gene Products, env/metabolism , Moloney murine leukemia virus/metabolism , Cell Line , Disulfides/metabolism , Electrophoresis, Polyacrylamide Gel , Gene Products, env/chemistry , Isomerism , Protein Conformation , Protein Folding
13.
J Virol ; 82(5): 2594-7, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18094170

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.


Subject(s)
Disulfides/metabolism , Gene Products, env/metabolism , Membrane Fusion , Neoplasm Proteins/metabolism , Peptides/metabolism , Animals , Disulfides/chemistry , Gene Products, env/chemistry , Hydrolysis , Isomerism , Moloney murine leukemia virus , Mutation
14.
Virol J ; 4: 101, 2007 Oct 19.
Article in English | MEDLINE | ID: mdl-17949490

ABSTRACT

BACKGROUND: Viruses that are incorporating host cell proteins might trigger autoimmune diseases. It is therefore of interest to identify possible host proteins associated with viruses, especially for enveloped viruses that have been suggested to play a role in autoimmune diseases, like human herpesvirus 6A (HHV-6A) in multiple sclerosis (MS). RESULTS: We have established a method for rapid and morphology preserving purification of HHV-6A virions, which in combination with parallel analyses with background control material released from mock-infected cells facilitates qualitative and quantitative investigations of the protein content of HHV-6A virions. In our iodixanol gradient purified preparation, we detected high levels of viral DNA by real-time PCR and viral proteins by metabolic labelling, silver staining and western blots. In contrast, the background level of cellular contamination was low in the purified samples as demonstrated by the silver staining and metabolic labelling analyses. Western blot analyses showed that the cellular complement protein CD46, the receptor for HHV-6A, is associated with the purified and infectious virions. Also, the cellular proteins clathrin, ezrin and Tsg101 are associated with intact HHV-6A virions. CONCLUSION: Cellular proteins are associated with HHV-6A virions. The relevance of the association in disease and especially in autoimmunity will be further investigated.


Subject(s)
Herpesvirus 6, Human/isolation & purification , Membrane Cofactor Protein/metabolism , Multiple Sclerosis/virology , Virion/isolation & purification , Blotting, Western , Cell Line , DNA, Viral/analysis , DNA, Viral/isolation & purification , Herpesvirus 6, Human/genetics , Herpesvirus 6, Human/immunology , Humans , Membrane Cofactor Protein/isolation & purification
15.
J Med Virol ; 79(9): 1312-21, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17607782

ABSTRACT

The accumulation of complex hepatitis B virus (HBV) variants with internal in-frame deletions in the C gene in immunosuppressed renal transplant recipients is associated with a severe course of the infection leading to end-stage liver disease (ESLD). A set of six HBV C genes with internal in-frame deletions corresponding to the pattern of HBV population in immunosuppressed patients has been expressed in two different eukaryotic cell lines. Synthesis and proteasomal degradation of HBV core (HBc) protein variants were compared with those of the wild-type HBc. In all cases, the steady-state level of internally deleted HBc proteins, predominantly with longer deletions, were considerably lower and turnover was significantly higher in comparison with those of the wild-type HBc, since all deletion variants were degraded rapidly via the proteasome pathway. Involvement and consequences of the proteasomal degradation machinery in the HBc protein turnover during HBV infection with complex HBV variants in the immunosuppressed patients are discussed.


Subject(s)
Hepatitis B Core Antigens/genetics , Hepatitis B Core Antigens/metabolism , Hepatitis B virus/genetics , Hepatitis B, Chronic/virology , Proteasome Endopeptidase Complex/metabolism , Amino Acid Sequence , Animals , Cell Line , Cell Line, Tumor , Cricetinae , Epitopes , Genes, Viral , Hepatitis B Core Antigens/chemistry , Hepatitis B Core Antigens/isolation & purification , Hepatitis B virus/ultrastructure , Humans , Immunocompromised Host , Molecular Sequence Data , Sequence Deletion , Transfection
16.
Virology ; 361(1): 149-60, 2007 Apr 25.
Article in English | MEDLINE | ID: mdl-17182074

ABSTRACT

Murine leukemia virus (MLV) fusion is controlled by isomerization of the disulphide bond between the receptor-binding surface (SU) and fusion-active transmembrane subunits of the Env-complex. The bond is in SU linked to a CXXC motif. This carries a free thiol that upon receptor binding can be activated (ionized) to attack the disulphide and rearrange it into a disulphide isomer within the motif. To find out whether His8 in the conserved SPHQ sequence of Env directs thiol activation, we analyzed its ionization in MLV vectors with wtEnv and Env with His8 deleted or substituted for Tyr or Arg, which partially or completely arrests fusion. The ionization was monitored by following the pH effect on isomerization in vitro by Ca2+ depletion or in vivo by receptor binding. We found that wtEnv isomerized optimally at slightly basic pH whereas the partially active mutant required higher and the inactive mutants still higher pH. This suggests that His8 directs the ionization of the CXXC thiol.


Subject(s)
Histidine/physiology , Moloney murine leukemia virus/physiology , Protein Disulfide-Isomerases/metabolism , Viral Envelope Proteins/metabolism , Amino Acid Substitution , Animals , Cell Line , Cell Membrane/metabolism , Hydrogen-Ion Concentration , Membrane Fusion , Protein Subunits/metabolism , Receptors, Virus/metabolism , Structure-Activity Relationship , Viral Envelope Proteins/chemistry , Virus Replication
17.
J Biol Chem ; 282(9): 6752-62, 2007 Mar 02.
Article in English | MEDLINE | ID: mdl-17192272

ABSTRACT

Semliki Forest virus is among the prototypes for Class II virus fusion and targets the endosomal membrane. Fusion protein E1 and its envelope companion E2 are both anchored in the viral membrane and form an external shell with protruding spikes. In acid environments, mimicking the early endosomal milieu, surface epitopes in the virus rearrange along with exposure of the fusion loop. To visualize this transformation into a fusogenic stage, we determined the structure of the virus at gradually lower pH values. The results show that while the fusion loop is available for external interaction and the shell and stalk domains of the spike begin to deteriorate, the E1 and E2 remain in close contact in the spike head. This unexpected observation points to E1 and E2 cooperation beyond the fusion loop exposure stage and implies a more prominent role for E2 in guiding membrane close encounter than has been earlier anticipated.


Subject(s)
Cryoelectron Microscopy , Semliki forest virus/chemistry , Semliki forest virus/pathogenicity , Viral Envelope Proteins/chemistry , Viral Fusion Proteins/chemistry , Hydrogen-Ion Concentration , Membrane Fusion , Molecular Structure , Phase Transition , Protein Conformation , Viral Envelope Proteins/physiology , Viral Fusion Proteins/physiology
18.
J Virol ; 80(19): 9921-5, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16973599

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.


Subject(s)
Cell Membrane/metabolism , Gene Products, env/metabolism , Leukemia Virus, Murine/metabolism , Receptors, Virus/metabolism , Alkylation , Cell Fusion , Cell Membrane/drug effects , Gene Products, env/genetics , Leukemia Virus, Murine/drug effects , Leukemia Virus, Murine/genetics , Peptides/pharmacology
19.
J Virol ; 80(11): 5540-51, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16699035

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.


Subject(s)
Disulfides/metabolism , Furin/metabolism , Genes, env , Leukemia Virus, Murine/physiology , Viral Envelope Proteins/metabolism , Genes, env/physiology , Isomerism , Protein Isoforms , Receptors, Virus/metabolism , Viral Fusion Proteins/metabolism
20.
J Virol ; 79(22): 13856-64, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16254321

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.


Subject(s)
Moloney murine leukemia virus/physiology , 3T3 Cells , Animals , Binding Sites , Cysteine , Disulfides/metabolism , Isomerism , Kinetics , Mice , Receptors, Virus/metabolism , Viral Envelope Proteins/metabolism , Viral Envelope Proteins/physiology , Viral Fusion Proteins/metabolism
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