Sequential activation of the three protomers in the Moloney murine leukemia virus Env.

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) → (SU-TM)TM → (SU-TM)TM → TM This was the case both when activation was triggered in vitro by depleting stabilizing Ca from solubilized Env and when viral Env was receptor triggered on rat XC cells.

A saposin-lipoprotein nanoparticle system for membrane proteins.

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.

Furin cleavage of the Moloney murine leukemia virus Env precursor reorganizes the spike structure.

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.

Inhibition of the HIV-1 spike by single-PG9/16-antibody binding suggests a coordinated-activation model for its three protomeric units.

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.

Maturation cleavage of the murine leukemia virus Env precursor separates the transmembrane subunits to prime it for receptor triggering.

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.

Activation of the alphavirus spike protein is suppressed by bound E3.

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.

Cooperative cleavage of the R peptide in the Env trimer of Moloney murine leukemia virus facilitates its maturation for fusion competence.

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.

Single-particle cryoelectron microscopy analysis reveals the HIV-1 spike as a tripod structure.

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.

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.

The dynamic envelope of a fusion class II virus. E3 domain of glycoprotein E2 precursor in Semliki Forest virus provides a unique contact with the fusion protein E1.

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.

Intersubunit disulfide isomerization controls membrane fusion of human T-cell leukemia virus Env.

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.

R-Peptide cleavage potentiates fusion-controlling isomerization of the intersubunit disulfide in Moloney murine leukemia virus Env.

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.

Stabilization of TM trimer interactions during activation of moloney murine leukemia virus Env.

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.

Purification of infectious human herpesvirus 6A virions and association of host cell proteins.

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.

Proteasomal degradation of core protein variants from chronic hepatitis B patients.

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.

The dynamic envelope of a fusion class II virus. Prefusion stages of semliki forest virus revealed by electron cryomicroscopy.

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.

The conserved His8 of the Moloney murine leukemia virus Env SU subunit directs the activity of the SU-TM disulphide bond isomerase.

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.

Receptor-triggered but alkylation-arrested env of murine leukemia virus reveals the transmembrane subunit in a prehairpin conformation.

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.

Furin cleavage potentiates the membrane fusion-controlling intersubunit disulfide bond isomerization activity of leukemia virus Env.

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.

Kinetic analyses of the surface-transmembrane disulfide bond isomerization-controlled fusion activation pathway in Moloney murine leukemia virus.

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.

The fusion-controlling disulfide bond isomerase in retrovirus Env is triggered by protein destabilization.

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.

Budding of alphaviruses.

Alphaviruses are small highly ordered enveloped RNA viruses, which replicate very efficiently in the infected cell. They consist of a nucleocapsid (NC) and a surrounding membrane with glycoproteins. In the NC the positive single stranded RNA genome of the virus is enclosed by a T=4 icosahedral shell of capsid (C) proteins.

Translation of hepatitis B virus (HBV) surface proteins from the HBV pregenome and precore RNAs in Semliki Forest virus-driven expression.

Hepatitis B virus (HBV) pregenome RNA (pgRNA) serves as a translation template for the HBV core (HBc) protein and viral polymerase (Pol). HBV precore RNA (pcRNA) directs the synthesis of the precore (preC) protein, a precursor of the hepatitis B e antigen (HBeAg). pgRNA and pcRNA were expressed in the Semliki Forest virus (SFV) expression system.

Passive and active inclusion of host proteins in human immunodeficiency virus type 1 gag particles during budding at the plasma membrane.

Human immunodeficiency virus type 1 particles form by budding at the surface of most cell types. In this process, a piece of the plasma membrane is modified into an enveloped virus particle. The process is driven by the internal viral protein Pr55(gag).

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 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.