RESUMO
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 â (SU-TM)2TM â (SU-TM)TM2 â 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.
Assuntos
Vírus da Leucemia Murina de Moloney/química , Subunidades Proteicas/química , Proteínas do Envelope Viral/genética , Proteínas Virais de Fusão/genética , Alquilação , Animais , Linhagem Celular , Dissulfetos/química , Isomerismo , Camundongos , Vírus da Leucemia Murina de Moloney/genética , Vírus da Leucemia Murina de Moloney/patogenicidade , Subunidades Proteicas/genética , Ratos , Propriedades de Superfície , Proteínas do Envelope Viral/química , Proteínas Virais de Fusão/química , Internalização do VírusRESUMO
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.
Assuntos
Furina/metabolismo , Produtos do Gene env/química , Produtos do Gene env/metabolismo , Vírus da Leucemia Murina de Moloney/metabolismo , Animais , Eletroforese em Gel de Poliacrilamida , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Camundongos , Modelos Moleculares , Peso Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Multimerização Proteica , Tripsina/metabolismoRESUMO
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.
Assuntos
Produtos do Gene env/metabolismo , Vírus da Leucemia Murina/metabolismo , Modelos Moleculares , Conformação Proteica , Precursores de Proteínas/metabolismo , Microscopia Crioeletrônica , Eletroforese em Gel de Poliacrilamida , Fracionamento por Campo e Fluxo , Vírus da Leucemia Murina de Moloney , Octoxinol , Processamento de Proteína Pós-Traducional , Subunidades Proteicas/metabolismoRESUMO
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.
Assuntos
Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Sítios de Ligação , Antígenos CD4/genética , Antígenos CD4/metabolismo , Células HEK293 , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Eletroforese em Gel de Poliacrilamida Nativa/métodos , Ligação Proteica , Multimerização Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Internalização do Vírus , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologiaRESUMO
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.
Assuntos
Antígenos CD4 , Proteína gp120 do Envelope de HIV , Proteína gp41 do Envelope de HIV , HIV-1/ultraestrutura , Imageamento Tridimensional , Modelos Moleculares , Microscopia Crioeletrônica , HIV-1/química , HumanosRESUMO
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.
Assuntos
Produtos do Gene env/química , Produtos do Gene env/fisiologia , Vírus da Leucemia Murina/metabolismo , Proteínas Virais de Fusão/metabolismo , Animais , Membrana Celular/metabolismo , Microscopia Crioeletrônica/métodos , Produtos do Gene env/metabolismo , Genes env , Processamento de Imagem Assistida por Computador , Camundongos , Modelos Biológicos , Modelos Moleculares , Conformação Molecular , Ligação Proteica/genética , Estrutura Terciária de ProteínaRESUMO
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.
Assuntos
Glicoproteínas de Membrana/metabolismo , Vírus da Floresta de Semliki/fisiologia , Proteínas do Envelope Viral/metabolismo , Animais , Linhagem Celular , Cricetinae , Endopeptidase K/metabolismo , Furina/metabolismo , Ligação ProteicaRESUMO
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.
Assuntos
Produtos do Gene env/metabolismo , Vírus da Leucemia Murina de Moloney/fisiologia , Internalização do Vírus , Liberação de Vírus , Processamento de Proteína Pós-TraducionalRESUMO
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.
Assuntos
Biopolímeros/metabolismo , Produtos do Gene env/metabolismo , Vírus da Leucemia Murina de Moloney/metabolismo , Linhagem Celular , Dissulfetos/metabolismo , Eletroforese em Gel de Poliacrilamida , Produtos do Gene env/química , Isomerismo , Conformação Proteica , Dobramento de ProteínaRESUMO
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.
Assuntos
Dissulfetos/metabolismo , Produtos do Gene env/metabolismo , Fusão de Membrana , Proteínas de Neoplasias/metabolismo , Peptídeos/metabolismo , Animais , Dissulfetos/química , Produtos do Gene env/química , Hidrólise , Isomerismo , Vírus da Leucemia Murina de Moloney , MutaçãoRESUMO
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. The glycoproteins form a second shell with corresponding symmetry on the outside of the lipid membrane. These viruses mature by budding at the plasma membrane (PM) of the infected cell and enter into new cells by acid-triggered membrane fusion in endosomes. The viral glycoprotein consists of two subunits, E1, which carries the membrane fusion function, and E2, which suppresses this function until acid activation occurs. In the infected cell the RNA replication and transcription are confined to the cytoplasmic surface of endosome-derived vesicles called cytopathic vacuoles type I (CPV I). These structures are closely associated with membranes of the endoplasmic reticulum (ER), thereby creating a microenvironment for synthesis of viral proteins, assembly of the glycoproteins and formation of genome-C complexes. The budding process of the virus is initiated by C-glycoprotein interactions, possibly already before the glycoproteins arrive at the PM. This might involve a premade, ordered NC or a less ordered form of the genome-C complex. In the latter case, the interactions in the glycoprotein shell provide the major driving force for budding. The nature of the C-glycoprotein interaction has been resolved at atomic resolution by modelling. It involves hydrophobic interactions between a Tyr-X-Leu tripeptide in the internal tail of the E2 subunit and a pocket on the surface of the C protein. When the virus enters the endosome of a new cell the acid conditions trigger rearrangements in the glycoprotein shell, which result in the dissociation of the interactions that drive budding and a concomitant activation of the membrane fusion function in the E1 subunit.
Assuntos
Alphavirus/crescimento & desenvolvimento , Capsídeo/fisiologia , Proteínas do Core Viral/metabolismo , Proteínas do Envelope Viral/metabolismo , Montagem de Vírus/fisiologia , Alphavirus/genética , Alphavirus/metabolismo , Alphavirus/fisiologia , Animais , Proteínas do Core Viral/genética , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
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.
Assuntos
Glicoproteínas/metabolismo , Chaperonas Moleculares/metabolismo , Vírus da Floresta de Semliki/metabolismo , Proteínas Virais/metabolismo , Infecções por Alphavirus/genética , Infecções por Alphavirus/metabolismo , Animais , Cricetinae , Glicoproteínas/genética , Chaperonas Moleculares/genética , Mutação , Poliaminas/metabolismo , Estrutura Secundária de Proteína/genética , Estrutura Terciária de Proteína/genética , Vírus da Floresta de Semliki/genética , Proteínas Virais/genéticaRESUMO
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.
Assuntos
Dissulfetos/metabolismo , Furina/metabolismo , Genes env , Vírus da Leucemia Murina/fisiologia , Proteínas do Envelope Viral/metabolismo , Genes env/fisiologia , Isomerismo , Isoformas de Proteínas , Receptores Virais/metabolismo , Proteínas Virais de Fusão/metabolismoRESUMO
Assembly of human immunodeficiency virus type 1 (HIV-1) is directed by the viral core protein Pr55gag. Depending on the cell type, Pr55gag accumulates either at the plasma membrane or on late endosomes/multivesicular bodies. Intracellular localization of Pr55gag determines the site of virus assembly, but molecular mechanisms that define cell surface or endosomal targeting of Pr55gag are poorly characterized. We have analyzed targeting of newly synthesized Pr55gag in HeLa H1 cells by pulse-chase studies and subcellular fractionations. Our results indicated that Pr55gag was inserted into the plasma membrane and, when coexpressed with the viral accessory protein Vpu, Pr55gag remained at the plasma membrane and virions assembled at this site. In contrast, Pr55gag expressed in the absence of Vpu was initially inserted into the plasma membrane, but subsequently endocytosed, and virus assembly was partially shifted to internal membranes. This endocytosis of Pr55gag required the host protein Tsg101. These results identified a previously unknown role for Vpu and Tsg101 as regulators for the endocytic uptake of Pr55gag and suggested that the site of HIV-1 assembly is determined by factors that regulate the endocytosis of Pr55gag.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Endocitose , Produtos do Gene gag/metabolismo , Precursores de Proteínas/metabolismo , Fatores de Transcrição/fisiologia , Proteínas Virais Reguladoras e Acessórias/fisiologia , Membrana Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte , HIV-1/fisiologia , Células HeLa , Proteínas do Vírus da Imunodeficiência Humana , Humanos , Vírion/fisiologia , Montagem de VírusRESUMO
The alphavirus envelope is built by heterodimers of the membrane proteins E1 and E2. The complex is formed as a p62E1 precursor in the endoplasmic reticulum. During transit to the plasma membrane (PM), it is cleaved into mature E1-E2 heterodimers, which are oligomerized into trimeric complexes, so-called spikes that bind both to each other and, at the PM, also to nucleocapsid (NC) structures under the membrane. These interactions drive the budding of new virus particles from the cell surface. The virus enters new cells by a low-pH-induced membrane fusion event where both inter- and intraheterodimer interactions are reorganized to establish a fusion-active membrane protein complex. There are no intact heterodimers left after fusion activation; instead, an E1 homotrimer remains in the cellular (or viral) membrane. We analyzed whether these transitions depend on interactions in the transmembrane (TM) region of the heterodimer. We observed a pattern of conserved glycines in the TM region of E1 and made two mutants where either the glycines only (SFV/E1(4L)) or the whole segment around the glycines (SFV/E1(11L)) was replaced by leucines. We found that both mutations decreased the stability of the heterodimer and increased the formation of the E1 homotrimer at a suboptimal fusion pH, while the fusion activity was decreased. This suggested that TM interactions play a role in virus assembly and entry and that anomalous or uncoordinated protein reorganizations take place in the mutants. In addition, the SFV/E1(11L) mutant was completely deficient in budding, which may reflect an inability to form multivalent NC interactions at the PM.