RESUMO
The host cell factors TRIM5alpha(hu) and Fv-1 restrict N-tropic murine leukemia virus (N-MLV) infection at an early postentry step before or after reverse transcription, respectively. Interestingly, the identity of residue 110 of the MLV capsid determines susceptibility to both TRIM5alpha(hu) and Fv-1. In this study, we investigate the fate of the MLV capsid in cells expressing either the TRIM5alpha(hu) or Fv-1 restriction factor. The expression of TRIM5alpha(hu), but not Fv-1, specifically promoted the premature conversion of particulate N-MLV capsids within infected cells to soluble capsid proteins. The TRIM5alpha(hu)-mediated disassembly of particulate N-MLV capsids was dependent upon residue 110 of the viral capsid. Furthermore, the deletion or disruption of TRIM5alpha(hu) domains necessary for potent N-MLV restriction completely abrogated the disappearance of particulate N-MLV capsids observed with wild-type TRIM5alpha(hu). These results suggest that premature disassembly of the viral capsid contributes to the restriction of N-MLV infection by TRIM5alpha(hu), but not by Fv-1.
Assuntos
Proteínas de Transporte/fisiologia , Vírus da Leucemia Murina/fisiologia , Substituição de Aminoácidos , Animais , Fatores de Restrição Antivirais , Sequência de Bases , Capsídeo/fisiologia , Proteínas de Transporte/química , Proteínas de Transporte/genética , Linhagem Celular , Primers do DNA/genética , Humanos , Vírus da Leucemia Murina/genética , Vírus da Leucemia Murina/patogenicidade , Camundongos , Mutagênese Sítio-Dirigida , Células NIH 3T3 , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas com Motivo Tripartido , Ubiquitina-Proteína LigasesRESUMO
TRIM5alpha acts on several retroviruses, including human immunodeficiency virus (HIV-1), to restrict cross-species transmission. Using natural history cohorts and tissue culture systems, we examined the effect of polymorphism in human TRIM5alpha on HIV-1 infection. In African Americans, the frequencies of two non-coding SNP variant alleles in exon 1 and intron 1 of TRIM5 were elevated in HIV-1-infected persons compared with uninfected subjects. By contrast, the frequency of the variant allele encoding TRIM5alpha 136Q was relatively elevated in uninfected individuals, suggesting a possible protective effect. TRIM5alpha 136Q protein exhibited slightly better anti-HIV-1 activity in tissue culture than the TRIM5alpha R136 protein. The 43Y variant of TRIM5alpha was less efficient than the H43 variant at restricting HIV-1 and murine leukemia virus infections in cultured cells. The ancestral TRIM5 haplotype specifying no observed variant alleles appeared to be protective against infection, and the corresponding wild-type protein partially restricted HIV-1 replication in vitro. A single logistic regression model with a permutation test indicated the global corrected P value of <0.05 for both SNPs and haplotypes. Thus, polymorphism in human TRIM5 may influence susceptibility to HIV-1 infection, a possibility that merits additional evaluation in independent cohorts.
Assuntos
Proteínas de Transporte/genética , Infecções por HIV/genética , Infecções por HIV/imunologia , Polimorfismo de Nucleotídeo Único , Adolescente , Adulto , Negro ou Afro-Americano/genética , Substituição de Aminoácidos , Animais , Fatores de Restrição Antivirais , Proteínas de Transporte/fisiologia , Linhagem Celular , Criança , Pré-Escolar , Suscetibilidade a Doenças , Cães , Éxons , Frequência do Gene , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , HIV-1/crescimento & desenvolvimento , Haplótipos , Humanos , Lactente , Recém-Nascido , Íntrons , Vírus da Leucemia Murina/crescimento & desenvolvimento , Modelos Logísticos , Proteínas com Motivo Tripartido , Ubiquitina-Proteína LigasesRESUMO
Human TRIM5alpha (TRIM5alpha(hu)) potently restricts N-tropic (N-MLV), but not B-tropic, murine leukemia virus in a manner dependent upon residue 110 of the viral capsid. Rhesus monkey TRIM5alpha (TRIM5alpha(rh)) inhibits N-MLV only weakly. The study of human-monkey TRIM5alpha chimerae revealed that both the v1 and v3 variable regions of the B30.2/SPRY domain contain potency determinants for N-MLV restriction. These variable regions are predicted to be surface-exposed elements on one face of the B30.2 domain. Acidic residues in v3 complement basic residue 110 of the N-MLV capsid. The results support recognition of the retroviral capsid by the TRIM5alpha B30.2 domain.
Assuntos
Antígenos Virais/imunologia , Proteínas de Transporte/genética , Vírus da Leucemia Murina/efeitos dos fármacos , Infecções por Retroviridae/prevenção & controle , Sequência de Aminoácidos , Antígenos Virais/efeitos dos fármacos , Fatores de Restrição Antivirais , Capsídeo/fisiologia , Proteínas de Transporte/química , Proteínas de Transporte/farmacologia , Humanos , Vírus da Leucemia Murina/química , Vírus da Leucemia Murina/patogenicidade , Estrutura Terciária de Proteína , Proteínas/química , Proteínas/farmacologia , Infecções por Retroviridae/fisiopatologia , Proteínas com Motivo Tripartido , Ubiquitina-Proteína LigasesRESUMO
A central feature of the serpin inhibition mechanism is insertion of the reactive center loop into the central beta-sheet (beta-sheet A). This insertion also occurs when the reactive center loop is cleaved without protease inhibition. Using this effect, we have measured the enthalpy (DeltaH) of loop cleavage and insertion for plasminogen activator inhibitor 1 (PAI-1) as -38 kcal/mol. Because loop insertion can be blocked by incorporating a peptide into the central beta-sheet, it was possible to assign -7 kcal/mol to loop cleavage and -31 kcal/mol to loop insertion. These values are lower than values reported for the serpins alpha 1 -proteinase inhibitor and antithrombin of -53 to -63 kcal/mol, respectively, for loop insertion with negligible enthalpy for loop cleavage. A free energy difference of -9 kcal/mol has been reported between the active and spontaneously loop inserted "latent forms" of PAI-1, which is significantly smaller in magnitude than the -31 kcal/mol of enthalpy we measured for loop insertion. Because the enthalpy should relate closely to those regions of PAI-1 that have moved to lower potential energy, a difference distance matrix is presented that identifies regions of PAI-1 that move during loop insertion.
Assuntos
Inibidor 1 de Ativador de Plasminogênio/química , Estrutura Secundária de Proteína , Calorimetria , Humanos , Elastase Pancreática/metabolismo , Proteínas Recombinantes/química , TermodinâmicaRESUMO
Reovirus replication and assembly are thought to occur within cytoplasmic inclusion bodies, which we call viral factories. A strain-dependent difference in the morphology of these structures reflects more effective microtubule association by the mu2 core proteins of some viral strains, which form filamentous factories, than by those of others, which form globular factories. For this report, we identified and characterized another strain-dependent attribute of the factories, namely, the extent to which they colocalized with conjugated ubiquitin (cUb). Among 16 laboratory strains and field isolates, the extent of factory costaining for cUb paralleled factory morphology, with globular strains exhibiting higher levels by far. In reassortant viruses, factory costaining for cUb mapped primarily to the mu2-encoding M1 genome segment, although contributions by the lambda3- and lambda2-encoding L1 and L2 genome segments were also evident. Immunoprecipitations revealed that cells infected with globular strains contained higher levels of ubiquitinated mu2 (Ub-mu2). In M1-transfected cells, cUb commonly colocalized with aggregates formed by mu2 from globular strains but not with microtubules coated by mu2 from filamentous strains, and immunoprecipitations revealed that mu2 from globular strains displayed higher levels of Ub-mu2. Allelic changes at mu2 residue 208 determined these differences. Nocodazole treatment of cells infected with filamentous strains resulted in globular factories that still showed low levels of costaining for cUb, indicating that higher levels of costaining were not a direct result of decreased microtubule association. The factories of globular strains, or their mu2 proteins expressed in transfected cells, were furthermore shown to gain microtubule association and to lose colocalization with cUb when cells were grown at reduced temperature. From the sum of these findings, we propose that mu2 from globular strains is more prone to temperature-dependent misfolding and as a result displays increased aggregation, increased levels of Ub-mu2, and decreased association with microtubules. Because so few of the viral strains formed factories that were regularly associated with ubiquitinated proteins, we conclude that reovirus factories are generally distinct from cellular aggresomes.
Assuntos
Corpos de Inclusão Viral/ultraestrutura , Orthoreovirus/crescimento & desenvolvimento , Orthoreovirus/genética , Ubiquitina/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Substituição de Aminoácidos , Genes Virais , Corpos de Inclusão Viral/metabolismo , Microtúbulos/química , Microtúbulos/efeitos dos fármacos , Nocodazol/farmacologia , Orthoreovirus/metabolismo , Fenótipo , Dobramento de Proteína , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Vírus Reordenados/genética , Vírus Reordenados/crescimento & desenvolvimento , Vírus Reordenados/metabolismo , Temperatura , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismo , Replicação Viral/genéticaRESUMO
Murine leukemia viruses (MLVs) have been classified as N-tropic (N-MLV) or B-tropic (B-MLV), depending on their ability to infect particular mouse strains. The early phase of N-MLV infection is blocked in the cells of several mammalian species, including humans. This block is mediated by a dominant host factor that targets the viral capsid soon after virus entry into the cell has been achieved. A similar block to HIV-1 in rhesus monkey cells is mediated by TRIM5alpha. Here we show that human TRIM5alpha is both necessary and sufficient for the restriction of N-MLV in human cells. Rhesus monkey TRIM5alpha, which potently blocks HIV-1 infection, exhibited only modest inhibition of N-MLV infection. B-MLV was resistant to the antiviral effects of both human and rhesus monkey TRIM5alpha; susceptibility to TRIM5alpha-mediated restriction was conferred by alteration of residue 110 of the B-MLV capsid protein to the amino acid found in the N-MLV capsid. Our results demonstrate that species-specific variation in TRIM5alpha governs its ability to block infection by diverse retroviruses.
Assuntos
Proteínas de Transporte/farmacologia , Vírus da Leucemia Murina de Moloney/fisiologia , Substituição de Aminoácidos , Animais , Fatores de Restrição Antivirais , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Linhagem Celular , Ciclofilina A/metabolismo , Relação Dose-Resposta a Droga , HIV/efeitos dos fármacos , HIV/genética , HIV/fisiologia , Humanos , Controle de Infecções , Macaca mulatta , Camundongos , Vírus da Leucemia Murina de Moloney/efeitos dos fármacos , Retroviridae/efeitos dos fármacos , Retroviridae/fisiologia , Especificidade da Espécie , Transfecção , Proteínas com Motivo Tripartido , Ubiquitina-Proteína LigasesRESUMO
In cells of Old World and some New World monkeys, dominant factors restrict human immunodeficiency virus type 1 (HIV-1) infections after virus entry. The simian immunodeficiency virus SIV(mac) is less susceptible to these restrictions, a property that is determined largely by the viral capsid protein. For this study, we altered exposed amino acid residues on the surface of the HIV-1 capsid, changing them to the corresponding residues found on the SIV(mac) capsid. We identified two distinct pathways of escape from early, postentry restriction in monkey cells. One set of mutants that were altered near the base of the cyclophilin A-binding loop of the N-terminal capsid domain or in the interdomain linker exhibited a decreased ability to bind the restricting factor(s). Consistent with the location of this putative factor-binding site, cyclophilin A and the restricting factor(s) cooperated to achieve the postentry block. A second set of mutants that were altered in the ridge formed by helices 3 and 6 of the N-terminal capsid domain efficiently bound the restricting factor(s) but were resistant to the consequences of factor binding. These results imply that binding of the simian restricting factor(s) is not sufficient to mediate the postentry block to HIV-1 and that SIV(mac) capsids escape the block by decreases in both factor binding and susceptibility to the effects of the factor(s).
Assuntos
Capsídeo/química , Ciclofilina A/metabolismo , HIV-1/patogenicidade , Sequência de Aminoácidos , Animais , Capsídeo/metabolismo , Ciclosporina/farmacologia , Suscetibilidade a Doenças , Produtos do Gene gag/metabolismo , HIV-1/química , Humanos , Macaca mulatta , Dados de Sequência Molecular , Vírus da Imunodeficiência Símia/química , Vírus da Imunodeficiência Símia/patogenicidade , Relação Estrutura-AtividadeRESUMO
Plasminogen activator inhibitor-1 (PAI-1) is a 43 kDa protein involved in the regulation of fibrinolysis. PAI-1 is the principal inhibitor of tissue-type plasminogen activator (t-PA), trapping the proteinase as an acyl-enzyme covalent complex (approximately 105 kDa). Four single tryptophan mutants of PAI-1 have been constructed in which three of the four tryptophan residues (Trp86, Trp139, Trp175, and Trp262) were replaced with phenylalanine. Biosynthetic incorporation of 5-fluorotryptophan (5F-Trp) into wild-type PAI-1 (5FW wtPAI-1) and the single tryptophan mutants (5FW86, 5FW139, 5FW175, and 5FW262) was achieved, allowing a (19)F NMR spectroscopic study of PAI-1 in its active and cleaved forms and in complex with t-PA. The (19)F NMR spectrum of active 5FW wtPAI-1 shows four clearly resolved peaks at -39.20, -49.26, -50.74, and -52.57 ppm relative to trifluoroacetic acid at 0 ppm. Unequivocal assignments of these four resonances in the spectrum of 5FW wtPAI-1 to specific tryptophan residues were accomplished by measuring the chemical shifts of the (19)F resonances of the single tryptophan mutants. There was close agreement between the resonances observed in 5FW wtPAI-1 and of those in the mutants for all three protein forms. This would imply little structural perturbation in the local structures of the tryptophan residues resulting from substitution by phenylalanine. The 5FW wtPAI-1 was observed to have lower second-order rate constant (k(app)) for the inhibition of t-PA than the natural tryptophan wtPAI-1, suggesting that the decreased activity may result from a small structural effect of the fluorine substituent of the indole ring. Further alterations in the k(app) and the stoichiometry of inhibition (SI) were observed in each of the mutants indicating an effect of the three tryptophan to phenylalanine mutations. Detailed interpretation of the (19)F NMR spectra of the PAI-1 mutants provides insights into the local segmental structure of the active form of the proteins and the structural changes that occur in the cleaved and t-PA complexed forms.
Assuntos
Inibidor 1 de Ativador de Plasminogênio/química , Triptofano/análogos & derivados , Flúor/química , Humanos , Hidrólise , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Inibidor 1 de Ativador de Plasminogênio/biossíntese , Inibidor 1 de Ativador de Plasminogênio/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/genética , Relação Estrutura-Atividade , Ativador de Plasminogênio Tecidual/antagonistas & inibidores , Ativador de Plasminogênio Tecidual/química , Triptofano/química , Triptofano/genéticaRESUMO
Host cell barriers to the early phase of immunodeficiency virus replication explain the current distribution of these viruses among human and non-human primate species. Human immunodeficiency virus type 1 (HIV-1), the cause of acquired immunodeficiency syndrome (AIDS) in humans, efficiently enters the cells of Old World monkeys but encounters a block before reverse transcription. This species-specific restriction acts on the incoming HIV-1 capsid and is mediated by a dominant repressive factor. Here we identify TRIM5alpha, a component of cytoplasmic bodies, as the blocking factor. HIV-1 infection is restricted more efficiently by rhesus monkey TRIM5alpha than by human TRIM5alpha. The simian immunodeficiency virus, which naturally infects Old World monkeys, is less susceptible to the TRIM5alpha-mediated block than is HIV-1, and this difference in susceptibility is due to the viral capsid. The early block to HIV-1 infection in monkey cells is relieved by interference with TRIM5alpha expression. Our studies identify TRIM5alpha as a species-specific mediator of innate cellular resistance to HIV-1 and reveal host cell components that modulate the uncoating of a retroviral capsid.
Assuntos
Estruturas Citoplasmáticas/química , Suscetibilidade a Doenças , Infecções por HIV/metabolismo , HIV-1/fisiologia , Macaca mulatta/metabolismo , Macaca mulatta/virologia , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Clonagem Molecular , Estruturas Citoplasmáticas/metabolismo , Variação Genética/genética , Infecções por HIV/veterinária , Infecções por HIV/virologia , HIV-1/genética , Células HeLa , Humanos , Macaca mulatta/genética , Dados de Sequência Molecular , Mutação/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas/química , Proteínas/genética , Vírus da Imunodeficiência Símia/fisiologia , Especificidade da Espécie , Ubiquitina-Proteína Ligases , Replicação ViralRESUMO
We have demonstrated that interactions within the conserved serpin breach region play a direct role in the critical step of the serpin reaction in which the acyl-enzyme intermediate must first be exposed to hydrolyzing water and aqueous deacylation. Substitution of the breach tryptophan in PAI-1 (Trp175), a residue found in virtually all known serpins, with phenylalanine altered the kinetics of the reaction mechanism and impeded the ability of PAI-1 to spontaneously become latent without compromising the inherent rate of cleaved loop insertion or partitioning between the final inhibited serpin-proteinase complex and hydrolyzed serpin. Kinetic dissection of the PAI-1 inhibitory mechanism using multiple target proteinases made possible the identification of a single rate-limiting intermediate step coupled to the molecular interactions within the breach region. This step involves the initial insertion of the proximal reactive center loop hinge residue(s) into beta-sheet A and facilitates translocation of the distal P'-side of the cleaved reactive center loop from the substrate cleft of the proteinase. Substitution of the tryptophan residue raised the kinetic barrier restricting the initial loop insertion event, significantly retarding the rate-limiting step in tPA reactions in which strong exosite interactions must be overcome for the reaction to proceed.
Assuntos
Inibidor 1 de Ativador de Plasminogênio/fisiologia , Serpinas/metabolismo , Triptofano/genética , Sequência de Bases , Dicroísmo Circular , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Inibidor 1 de Ativador de Plasminogênio/química , Inibidor 1 de Ativador de Plasminogênio/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Triptofano/químicaRESUMO
Plasminogen activator inhibitor-1 (PAI-1) is a typical member of the serpin family that kinetically traps its target proteinase as a covalent complex by distortion of the proteinase domain. Incorporation of the fluorescently silent 4-fluorotryptophan analog into PAI-1 permitted us to observe changes in the intrinsic tryptophan fluorescence of two-chain tissue-type plasminogen activator (tPA) and the proteinase domain of tPA during the inhibition reaction. We demonstrated three distinct conformational changes of the proteinase that occur during complex formation and distortion. A conformational change occurred during the initial formation of the non-covalent Michaelis complex followed by a large conformational change associated with the distortion of the proteinase catalytic domain that occurs concurrently with the formation of stable proteinase-inhibitor complexes. Following distortion, a very slow structural change occurs that may be involved in the stabilization or regulation of the trapped complex. Furthermore, by comparing the inhibition rates of two-chain tPA and the proteinase domain of tPA by PAI-1, we demonstrate that the accessory domains of tPA play a prominent role in the initial formation of the non-covalent Michaelis complex.
Assuntos
Inibidor 1 de Ativador de Plasminogênio/química , Serpinas/química , Ativador de Plasminogênio Tecidual/química , Triptofano/análogos & derivados , Domínio Catalítico , Relação Dose-Resposta a Droga , Endopeptidases/química , Humanos , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Fatores de Tempo , Triptofano/químicaRESUMO
The inhibition mechanism of serpins requires a change in structure to entrap the target proteinase as a stable acyl-enzyme complex. Although it has generally been assumed that reactive center loop insertion and associated conformational change proceeds in a concerted manner, this has not been demonstrated directly. Through the substitution of tryptophan with 7-azatryptophan and an analysis of transient reaction kinetics, we have described the formation of an inhibited serpin-proteinase complex as a single concerted transition of the serpin structure. Replacement of the four tryptophans of plasminogen activator inhibitor type-1 (PAI-1) with the spectrally unique analogue 7-azatryptophan permitted observations of conformational changes in the serpin but not those of the proteinase. Formation of covalent acyl-enzyme complexes, but not noncovalent Michaelis complexes, with tissue-type plasminogen activator (t-PA) or urokinase (u-PA) resulted in rapid decreases of fluorescence coinciding with insertion of the reactive center loop and expansion of beta-sheet A. Insertion of an octapeptide consisting of the P14-P7 residues of the reactive center loop into beta-sheet A produced the same conformational change in serpin structure measured by 7-azatryptophan fluorescence, suggesting that introduction of the proximal loop residues induces the structural rearrangement of the serpin molecule. The atom specific modification of the tryptophan indole rings through analogue substitution produced a proteinase specific effect on function. The reduced inhibitory activity of PAI-1 against t-PA but not u-PA suggested that the mechanism of loop insertion is sensitive to the intramolecular interactions of one or more tryptophan residues.