RESUMEN
Porcine Mx1 is a type of interferon-induced GTPase that inhibits the replication of certain RNA viruses. However, the antiviral effects and the underlying mechanism of porcine Mx1 for porcine reproductive and respiratory syndrome virus (PRRSV) remain unknown. In this study, we demonstrated that porcine Mx1 could significantly inhibit PRRSV replication in MARC-145 cells. By Mx1 segment analysis, it was indicated that the GTPase domain (68-341aa) was the functional area to inhibit PRRSV replication and that Mx1 interacted with the PRRSV-N protein through the GTPase domain (68-341aa) in the cytoplasm. Amino acid residues K295 and K299 in the G domain of Mx1 were the key sites for Mx1-N interaction while mutant proteins Mx1(K295A) and Mx1(K299A) still partially inhibited PRRSV replication. Furthermore, we found that the GTPase activity of Mx1 was dominant for Mx1 to inhibit PRRSV replication but was not essential for Mx1-N interaction. Finally, mechanistic studies demonstrated that the GTPase activity of Mx1 played a dominant role in inhibiting the N-Nsp9 interaction and that the interaction between Mx1 and N partially inhibited the N-Nsp9 interaction. We propose that the complete anti-PRRSV mechanism of porcine Mx1 contains a two-step process: Mx1 binds to the PRRSV-N protein and subsequently disrupts the N-Nsp9 interaction by a process requiring the GTPase activity of Mx1. Taken together, the results of our experiments describe for the first time a novel mechanism by which porcine Mx1 evolves to inhibit PRRSV replication. IMPORTANCE: Mx1 protein is a key mediator of the interferon-induced antiviral response against a wide range of viruses. How porcine Mx1 affects the replication of porcine reproductive and respiratory syndrome virus (PRRSV) and its biological function has not been studied. Here, we show that Mx1 protein inhibits PRRSV replication by interfering with N-Nsp9 interaction. Furthermore, the GTPase activity of porcine Mx1 plays a dominant role and the Mx1-N interaction plays an assistant role in this interference process. This study uncovers a novel mechanism evolved by porcine Mx1 to exert anti-PRRSV activities.
Asunto(s)
Proteínas de Resistencia a Mixovirus , Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Proteínas no Estructurales Virales , Replicación Viral , Animales , Línea Celular , Interferones/inmunología , Interferones/metabolismo , Mutación , Proteínas de Resistencia a Mixovirus/química , Proteínas de Resistencia a Mixovirus/genética , Proteínas de Resistencia a Mixovirus/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/enzimología , Síndrome Respiratorio y de la Reproducción Porcina/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/crecimiento & desarrollo , Virus del Síndrome Respiratorio y Reproductivo Porcino/metabolismo , Unión Proteica , Porcinos/virología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismoRESUMEN
Accurate diagnosis is crucial for effective patient care and the containment of antimicrobial resistance outbreaks. The intricate challenge of distinguishing bacterial from viral infections, coupled with limited diagnostic tools and overlapping symptoms has driven the utilization of molecular imprinting techniques. This study focuses on developing cost-effective, chemically stable antibody analogs for the interferon-induced protein myxovirus resistance protein A (MxA). MxA is an intracellular, cytoplasmic GTPase having activity against a wide range of viruses and serves as a distinctive biomarker for viral infections. We utilized computational design to guide the polymer assembly, centering on epitope imprinting to target MxA-specific regions crucial for interaction. Molecular docking calculations, alongside a pioneering multi-monomer simultaneous docking (MMSD) protocol, efficiently elucidate cooperativity during pre-polymerization. Monomer binding affinity scores, such as for APTMS, exhibited notable increase, ranging from -3.11 to -13.03 kcal/mol across various MMSD combinations compared to a maximum of -2.78 kcal/mol in single monomer docking, highlighting the capacity of MMSD in elucidating crucial monomer-monomer interactions. This computational approach provides a theoretical alternative to labor-intensive experimental optimization, streamlining the development process for synthetic receptors. Simulations reveal unique interactions enhancing MIP-peptide complementarity, yielding optimized receptors selectively binding to MxA epitopes. The obtained MIPs demonstrated a maximum adsorption capacity of approximately 12 mg/g and captured 1.6 times more epitope and 2.6 times more epitope containing MxA protein than corresponding NIPs. A proof-of-concept study demonstrates MxA protein binding to synthetic receptors, highlighting the potential of MIPs, analogous to antibodies, in overcoming current diagnostic challenges for precise detection of viral infection.
Asunto(s)
Biomarcadores , Simulación del Acoplamiento Molecular , Impresión Molecular , Proteínas de Resistencia a Mixovirus , Proteínas de Resistencia a Mixovirus/metabolismo , Proteínas de Resistencia a Mixovirus/química , Impresión Molecular/métodos , Virosis/diagnóstico , HumanosRESUMEN
The Myxovirus resistance (Mx) proteins are critical effectors belonging to the super-family of guanidine triphosphatase, often stimulated by type I interferon (IFN) and mediates antiviral responses to restrict the replication of numerous viral genes in fishes. In teleosts, Mx proteins display diverse and complicated antiviral activity in different species. The present investigation seeks to characterize the Mx gene from Labeo catla upon induction by double-stranded (ds) RNA, polyinosinic-polycytidylic acid, (poly I: C). Molecular modeling and all-atoms molecular dynamics (MD) simulations were employed to understand the architecture of the GTPase domain and its plausible mode of GTP recognition in Mx protein. The full-length L. catla Mx (LcMx) gene sequence (1821 bp nucleotides) encodes an open reading frame of 606 amino acids. Domain search indicated conserved tripartite domain architecture of LcMx and forms a major cluster with the Mx from other teleosts. The positively charged Arginine and polar Glutamine residues from helix 3 and 4 of stalk region LcMx aid in homo-oligomerization. MD simulation portrayed the role of conserved critical residues aid in GTP recognition by the GTPase domain which perfectly corroborates with experimental findings and prior MD studies. After injection of poly I:C, the temporal mRNA profile showed that LcMx expression was significantly elevated in the spleen, brain, kidney, liver, muscle, heart, intestine, and gill tissues. Collectively, these results suggest that the elevated expression of the major innate immune defense gene Mx was able to inhibit the poly I: C mediated virulence in fish.Communicated by Ramaswamy H. Sarma.
Asunto(s)
Cyprinidae , Poli I-C , Animales , Proteínas de Resistencia a Mixovirus/genética , Proteínas de Resistencia a Mixovirus/química , Proteínas de Resistencia a Mixovirus/metabolismo , Poli I-C/farmacología , Secuencia de Aminoácidos , Cyprinidae/metabolismo , Proteínas/metabolismo , GTP Fosfohidrolasas/metabolismo , Antivirales , Guanosina TrifosfatoRESUMEN
Zoonotic avian influenza A virus (IAV) infections are rare. Sustained transmission of these IAVs between humans has not been observed, suggesting a role for host genes. We used whole-genome sequencing to compare avian IAV H7N9 patients with healthy controls and observed a strong association between H7N9 infection and rare, heterozygous single-nucleotide variants in the MX1 gene. MX1 codes for myxovirus resistance protein A (MxA), an interferon-induced antiviral guanosine triphosphatase known to control IAV infections in transgenic mice. Most of the MxA variants identified lost the ability to inhibit avian IAVs, including H7N9, in transfected human cell lines. Nearly all of the inactive MxA variants exerted a dominant-negative effect on the antiviral function of wild-type MxA, suggesting an MxA null phenotype in heterozygous carriers. Our study provides genetic evidence for a crucial role of the MX1-based antiviral defense in controlling zoonotic IAV infections in humans.
Asunto(s)
Subtipo H7N9 del Virus de la Influenza A , Gripe Humana/genética , Gripe Humana/virología , Proteínas de Resistencia a Mixovirus/genética , Enfermedades de los Trabajadores Agrícolas/genética , Enfermedades de los Trabajadores Agrícolas/virología , Animales , Línea Celular , Predisposición Genética a la Enfermedad , Variación Genética , Heterocigoto , Humanos , Subtipo H7N9 del Virus de la Influenza A/fisiología , Virus de la Influenza A/fisiología , Mutación Missense , Proteínas de Resistencia a Mixovirus/química , Proteínas de Resistencia a Mixovirus/metabolismo , Aves de Corral , Zoonosis Virales , Secuenciación Completa del GenomaRESUMEN
Membraneless organelles (MLOs) in the cytoplasm and nucleus in the form of phase-separated biomolecular condensates are increasingly viewed as critical in regulating diverse cellular functions. We summarize a paradigm shift over the last 3 years in the field of interferon (IFN)-inducible antiviral Mx-family GTPases. Expression of the 'myxovirus resistance proteins' MxA in human cells and its ortholog Mx1 in murine cells is increased 50- to 100-fold by Type I (IFN-α and -ß) and III IFNs (IFN-λ). Human MxA forms cytoplasmic structures, while murine Mx1 forms nuclear bodies. Since 2002, it has been widely thought that human (Hu) MxA is associated with the membraneous smooth endoplasmic reticulum (ER). In a paradigm shift, our recent data showed that HuMxA formed membraneless phase-separated biomolecular condensates in the cytoplasm. Some of the HuMxA condensates adhered to intermediate filaments generating a reticular pattern. Murine (Mu) Mx1, which was predominantly nuclear, was also confirmed to be in phase-separated nuclear biomolecular condensates. A subset of Huh7 cells showed association of GFP-MuMx1 with intermediate filaments in the cytoplasm. While cells with cytoplasmic GFP-HuMxA condensates and cytoplasmic GFP-MuMx1 filaments showed an antiviral phenotype towards vesicular stomatitis virus (VSV), those with only nuclear GFP-MuMx1 bodies did not. The new data bring forward the paradigm that both human MxA and murine Mx1 give rise to phase-separated biomolecular condensates, albeit in different subcellular compartments, and that differences in the subcellular localization of condensates of different Mx proteins determines the spectrum of their antiviral activity.
Asunto(s)
Condensados Biomoleculares/metabolismo , GTP Fosfohidrolasas/biosíntesis , Interferones/fisiología , Proteínas de Resistencia a Mixovirus/biosíntesis , Secuencia de Aminoácidos , Animales , GTP Fosfohidrolasas/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ratones , Proteínas de Resistencia a Mixovirus/química , Proteínas de Resistencia a Mixovirus/metabolismo , Fracciones Subcelulares/metabolismoRESUMEN
The antiviral cytokine interferon activates expression of interferon-stimulated genes to establish an antiviral state. Myxovirus resistance 2 (MX2, also known as MxB) is an interferon-stimulated gene that inhibits the nuclear import of HIV-1 and interacts with the viral capsid and cellular nuclear transport machinery. Here, we identified the myosin light chain phosphatase (MLCP) subunits myosin phosphatase target subunit 1 (MYPT1) and protein phosphatase 1 catalytic subunit-ß (PPP1CB) as positively-acting regulators of MX2, interacting with its amino-terminal domain. We demonstrated that serine phosphorylation of the N-terminal domain at positions 14, 17 and 18 suppresses MX2 antiviral function, prevents interactions with the HIV-1 capsid and nuclear transport factors, and is reversed by MLCP. Notably, serine phosphorylation of the N-terminal domain also impedes MX2-mediated inhibition of nuclear import of cellular karyophilic cargo. We also found that interferon treatment reduces levels of phosphorylation at these serine residues and outline a homeostatic regulatory mechanism in which repression of MX2 by phosphorylation, together with MLCP-mediated dephosphorylation, balances the deleterious effects of MX2 on normal cell function with innate immunity against HIV-1.
Asunto(s)
Infecciones por VIH/inmunología , VIH-1/inmunología , Inmunidad Innata , Proteínas de Resistencia a Mixovirus/química , Proteínas de Resistencia a Mixovirus/inmunología , Secuencias de Aminoácidos , Infecciones por VIH/genética , Infecciones por VIH/virología , VIH-1/genética , VIH-1/fisiología , Células HeLa , Humanos , Fosfatasa de Miosina de Cadena Ligera/genética , Fosfatasa de Miosina de Cadena Ligera/inmunología , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Proteínas de Resistencia a Mixovirus/genética , Fosforilación , Dominios Proteicos , Proteína Fosfatasa 1/genética , Proteína Fosfatasa 1/inmunología , Serina/metabolismoRESUMEN
The type one interferon induced restriction factor Myxovirus resistance B (MxB) restricts HIV-1 nuclear entry evidenced by inhibition of 2-LTR but not linear forms of viral DNA. The HIV-1 capsid is the key determinant of MxB sensitivity and cofactor binding defective HIV-1 capsid mutants P90A (defective for cyclophilin A and Nup358 recruitment) and N74D (defective for CPSF6 recruitment) have reduced dependency on nuclear transport associated cofactors, altered integration targeting preferences and are not restricted by MxB expression. This has suggested that nuclear import mechanism may determine MxB sensitivity. Here we have use genetics to separate HIV-1 nuclear import cofactor dependence from MxB sensitivity. We provide evidence that MxB sensitivity depends on HIV-1 capsid conformation, rather than cofactor recruitment. We show that depleting CPSF6 to change nuclear import pathway does not impact MxB sensitivity, but mutants that recapitulate the effect of Cyclophilin A binding on capsid conformation and dynamics strongly impact MxB sensitivity. We demonstrate that HIV-1 primary isolates have different MxB sensitivities due to cytotoxic T lymphocyte (CTL) selected differences in Gag sequence but similar cofactor dependencies. Overall our work demonstrates a complex relationship between cyclophilin dependence and MxB sensitivity likely driven by CTL escape. We propose that cyclophilin binding provides conformational flexibility to HIV-1 capsid facilitating simultaneous evasion of capsid-targeting restriction factors including TRIM5 as well as MxB.
Asunto(s)
Cápside/química , VIH-1/fisiología , Proteínas de Resistencia a Mixovirus/química , Transporte Activo de Núcleo Celular , VIH-1/química , HumanosRESUMEN
We compared the Mx expression and anti-viral function and the 3D structure of Mx protein in four species: chicken (Gallus gallus), whooper swan (Cygnus cygnus), jungle crow (Corvus macrorhynchos), and rock dove (Columba livia). We observed different mortalities associated with highly pathogenic avian influenza virus (HPAIV) infection to understand the relationship between Mx function as an immune response factor and HPAIV proliferation in bird cells. Different levels of Mx were observed among the different bird species after virus infection. Strong Mx expression was confirmed in the rock dove and whooper swan 6 hr after viral infection. The lowest virus copy numbers were observed in rock dove. The virus infectivity was significantly reduced in the BALB/3T3 cells expressing rock dove and jungle crow Mx. These results suggested that high Mx expression and significant Mx-induced anti-viral effects might result in the rock dove primary cells having the lowest virus copy number. Comparison of the expected 3D structure of Mx protein in all four bird species demonstrated that the structure of loop L4 varied among the investigated species. It was reported that differences in amino acid sequence in loop L4 affect antiviral activity in human and mouse cells, and a significant anti-viral effect was observed in the rock dove Mx. Thus, the amino acid sequence of loop L4 in rock dove might represent relatively high anti-viral activity.
Asunto(s)
Subtipo H5N1 del Virus de la Influenza A/inmunología , Gripe Aviar/inmunología , Proteínas de Resistencia a Mixovirus/química , Proteínas de Resistencia a Mixovirus/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Aves , Células Cultivadas , Subtipo H5N1 del Virus de la Influenza A/crecimiento & desarrollo , Gripe Aviar/virología , Ratones , Proteínas de Resistencia a Mixovirus/genética , Conformación Proteica , ARN Viral , Especificidad de la EspecieRESUMEN
Membraneless organelles (MLOs) in the cytoplasm and nucleus in the form of 2D and 3D phase-separated biomolecular condensates are increasingly viewed as critical in regulating diverse cellular functions. These functions include cell signaling, immune synapse function, nuclear transcription, RNA splicing and processing, mRNA storage and translation, virus replication and maturation, antiviral mechanisms, DNA sensing, synaptic transmission, protein turnover and mitosis. Components comprising MLOs often associate with low affinity; thus cell integrity can be critical to the maintenance of the full complement of respective MLO components. Phase-separated condensates are typically metastable (shape-changing) and can undergo dramatic, rapid and reversible assembly and disassembly in response to cell signaling events, cell stress, during mitosis, and after changes in cytoplasmic "crowding" (as observed with condensates of the human myxovirus resistance protein MxA). Increasing evidence suggests that neuron-specific aberrations in phase-separation properties of RNA-binding proteins (e.g. FUS and TDP-43) and others (such as the microtubule-binding protein tau) contribute to the development of degenerative neurological diseases (e.g. amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and Alzheimer's disease). Thus, studies of liquid-like phase separation (LLPS) and the formation, structure and function of MLOs are of considerable importance in understanding basic cell biology and the pathogenesis of human diseases.
Asunto(s)
Núcleo Celular/química , Citoplasma/química , Proteínas de Resistencia a Mixovirus/aislamiento & purificación , Orgánulos/química , Biología Celular , Núcleo Celular/virología , Citoplasma/virología , Humanos , Proteínas de Resistencia a Mixovirus/química , Orgánulos/virologíaRESUMEN
The interferon-induced GTP-binding protein Mx is responsible for a specific antiviral state against a broad spectrum of viral infections that are induced by type-I interferons (IFN α/ß) in different vertebrates. In this study, the Mx gene was isolated from the constructed mullet cDNA database. Structural features of mullet Mx (MuMx) were analyzed using different in-silico tools. The pairwise comparison revealed that the MuMx sequence was related to Stegastes partitus Mx with an 83.7% sequence identity, whereas MuMx was clustered into the teleost category in the phylogentic analysis. Sequence alignment showed that the dynamin-type guanine nucleotide-binding domain (G_DYNAMIN_2), central interactive domain (CID), and GTPase effector domain (GED) were conserved among Mx counterparts. The transcriptional expression of MuMx was the highest in blood cells from unchallenged fish. The temporal mRNA profile showed that MuMx expression was significantly elevated in all tissues, including blood, spleen, head kidney, liver, and gills after the injection of polyinosinic-polycytidylic acid (poly I:C) at many time points. Moreover, MuMx expression increased slightly, in the blood, spleen, and head kidney at a few time points after the injection of lipopolysaccharide (LPS) and Lactococcus garvieae (L. garvieae). Results of the subcellular localization analysis confirmed that the MuMx protein was highly expressed in the cytoplasm. The analysis of the gene expression of the viral hemorrhagic septicemia virus (VHSV) under conditions of MuMx overexpression confirmed the significant inhibition of viral transcripts. The cell viability (MTT) assay and VHSV titer quantification with the presence of MuMx indicated a significant reduction in virus replication. Collectively, these findings suggest that Mx is a specific immune-related gene that elicits crucial antiviral functions against viral antigens in the mullet fish.
Asunto(s)
Enfermedades de los Peces/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Proteínas de Resistencia a Mixovirus/genética , Proteínas de Resistencia a Mixovirus/inmunología , Smegmamorpha/genética , Smegmamorpha/inmunología , Secuencia de Aminoácidos , Animales , Proteínas de Peces/química , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Expresión Génica , Perfilación de la Expresión Génica/veterinaria , Infecciones por Bacterias Grampositivas/inmunología , Infecciones por Bacterias Grampositivas/veterinaria , Lactococcus/fisiología , Lipopolisacáridos/farmacología , Proteínas de Resistencia a Mixovirus/química , Novirhabdovirus/fisiología , Filogenia , Poli I-C/farmacología , Infecciones por Rhabdoviridae/inmunología , Infecciones por Rhabdoviridae/veterinaria , Alineación de Secuencia/veterinariaRESUMEN
Myxovirus resistance (Mx) proteins represents the subclass of the dynamin superfamily of large Guanosine triphosphates (GTPases), play esential role in intracellular vesicle trafficking, endocytosis, organelle homeostasis and mitochondria distribution. These proteins are key players of the vertebrate immune system, induced by type-I and type-III interferons (IFN) of infected host and inhibit viral replication by sequestering its nucleoprotein. In the present study, we report the sequencing and characterization of Cirrhinus mrigala Mx protein (CmMx) for the first time and observed its constitutive expression in different tissues for a period of fourteen days. The synthetic peptide, LSGVALPRGTGI, was dissolved in PBS and injected into a rabbit and the antibody raised against CmMx was used to study the level of its expression. The full length of the CmMx cDNA is 2244â¯bp with a molecular mass of 70.9â¯kDa and a predicted isoelectric point of 8.25. The 627 amino acids polypeptide formed of three main functional domains: N-terminal GTPase domain (GD), a middle domain (MD) and GTPase effector domain (GED) with carboxy terminal leucine zipper motif. The 3D models of CmMx protein was modeled based on available close structural homologs and further validated through molecular dynamics (MD) simulations. MD study revealed the importance of G-domain responsible for recognition of GTP, which perfectly corroborate with earlier studies. MM/PBSA binding free energy analysis displayed that van der Waals and electrostatic energy were the key driving force behind molecular recognition of GTP by CmMx protein. The results from this study will illuminate more lights into the ongoing research on myxovirus resistance protein and its role in inhibition of viral replication in other eukaryotic system as well.
Asunto(s)
Cipriniformes/genética , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Regulación de la Expresión Génica , Guanosina Trifosfato/metabolismo , Proteínas de Resistencia a Mixovirus/genética , Proteínas de Resistencia a Mixovirus/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN Complementario/genética , Proteínas de Peces/química , Cinética , Simulación de Dinámica Molecular , Proteínas de Resistencia a Mixovirus/química , Filogenia , Unión Proteica , Dominios Proteicos , Estabilidad Proteica , ARN Mensajero/genética , TermodinámicaRESUMEN
The human antiviral protein MxB is a restriction factor that fights HIV infection. Previous experiments have demonstrated that MxB targets the HIV capsid, a protein shell that protects the viral genome. To make the conical-shaped capsid, HIV CA proteins are organized into a lattice composed of hexamer and pentamer building blocks, providing many interfaces for host proteins to recognize. Through extensive biochemical and biophysical studies and molecular dynamics simulations, we show that MxB is targeting the HIV capsid by recognizing the region created at the intersection of three CA hexamers. We are further able to map this interaction to a few CA residues, located in a negatively charged well at the interface between the three CA hexamers. This work provides detailed residue-level mapping of the targeted capsid interface and how MxB interacts. This information could inspire the development of capsid-targeting therapies for HIV.
Asunto(s)
Cápside/química , Cápside/metabolismo , VIH-1/metabolismo , Proteínas de Resistencia a Mixovirus/química , Proteínas de Resistencia a Mixovirus/metabolismo , Sitios de Unión , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/genética , Interacciones Huésped-Patógeno , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Mutación , Proteínas de Resistencia a Mixovirus/genética , Unión Proteica , Conformación Proteica , Multimerización de ProteínaRESUMEN
Myxovirus resistance (Mx) proteins are interferon (IFN)-inducible Dynamin-like GTPases, which play an important role in antiviral immunity. Three Mx genes (Mx1-3) have been cloned previously in rainbow trout. In this study, an additional six Mx genes were cloned that reside in four chromosomal loci. Further bioinformatics analysis suggests the presence of three teleost Mx groups (TMG) each with a characteristic gene organisation. Salmonid Mx belong to TMG1 and TMG2. The increased salmonid Mx gene copies are due mainly to local gene duplications that happened before and after salmonid speciation, in a lineage/species specific manner. Trout Mx molecules have been diversified in the loop 1 and 4 regions, and in the nuclear localisation signal in loop 4. The trout Mx genes were shown to be differentially expressed in tissues, with high levels of expression of TMG1 (Mx1-4) in blood and TMG2 (Mx5-9) in intestine. The expression of the majority of the trout Mx genes was induced by poly IC in vitro and in vivo, and increased during development. In addition, induction by antiviral (IFN) and proinflammatory cytokines was studied, and showed that type I IFN, IFNγ and IL-1ß can induce Mx gene expression in an Mx gene-, cytokine- and cell line-dependent manner. These results show that salmonids possess a large number Mx genes as well as complex regulatory pathways, which may contribute to their success in an anadromous life style.
Asunto(s)
Enfermedades de los Peces/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Proteínas de Resistencia a Mixovirus/genética , Proteínas de Resistencia a Mixovirus/inmunología , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/inmunología , Secuencia de Aminoácidos , Animales , Citocinas/genética , Citocinas/inmunología , Citocinas/metabolismo , Proteínas de Peces/química , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Perfilación de la Expresión Génica/veterinaria , Familia de Multigenes/inmunología , Proteínas de Resistencia a Mixovirus/química , Filogenia , Poli I-C/farmacología , Alineación de Secuencia/veterinariaRESUMEN
A highly specific transport and sorting machinery directing secretory cargo to the apical or basolateral plasma membrane maintains the characteristic polarized architecture of epithelial cells. This machinery comprises a defined set of transport carriers, which are crucial for cargo delivery to the correct membrane domain. Each carrier is composed of a distinct set of proteins to verify precise routing and cargo selection. Among these components, the dynamin-related GTPase Mx1 was identified on post-Golgi vesicles destined for the apical membrane of MDCK cells. In addition to the presence on late secretory compartments, Mx1 was also detected on compartments of the early secretory pathway. Vesicular structures positive for this GTPase are highly dynamic, and we have studied the influence of the microtubule cytoskeleton on this motility. Live-cell microscopy indicated that microtubule disruption using nocodazole inhibits long-range trafficking of these structures. Mx1 directly or indirectly interacts with α-tubulin and the kinesin motor Kif5B as assessed by coimmunoprecipitation. In agreement with these observations knock out of Mx1 or a mutation in the unstructured L4 loop of Mx1 decreases the efficiency of apical cargo delivery. Interestingly, the L4 loop mutant still interacts with Kif5B; however, it causes vesicle elongation. This suggests that Mx1 aids in vesicle fission and stabilizes the interaction between Kif5B, microtubules and apical transport carriers.
Asunto(s)
Cinesinas/metabolismo , Microtúbulos/metabolismo , Proteínas de Resistencia a Mixovirus/metabolismo , Animales , Sitios de Unión , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Perros , Células de Riñón Canino Madin Darby , Proteínas de Resistencia a Mixovirus/química , Unión Proteica , Señales de Clasificación de Proteína , Transporte de Proteínas , Vesículas Secretoras/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
Interferons limit viral replication by inducing intracellular restriction factors, such as the GTPase MxB (also designated MX2), which inhibits HIV-1 and, as recently shown, herpesviruses. Inhibition of these viruses occurs at ill-defined steps after viral entry and requires formation of MxB dimers or oligomers, but GTP hydrolysis is needed only for blocking herpesviruses. Together with previous findings on related MxA, the new research on MxB highlights the mechanistic diversity by which MX proteins interfere with viral replication.
Asunto(s)
VIH-1/fisiología , Herpesviridae/fisiología , Interferones/farmacología , Proteínas de Resistencia a Mixovirus/metabolismo , VIH-1/efectos de los fármacos , Herpesviridae/efectos de los fármacos , Humanos , Modelos Moleculares , Proteínas de Resistencia a Mixovirus/química , Conformación Proteica , Multimerización de Proteína , Regulación hacia Arriba , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
The capsid-binding assay is an in vitro experiment used to determine whether cellular proteins interact with the HIV-1 core. In vitro assembled HIV-1 capsids recapitulate the surface of the HIV-1 core. The assay involves the incubation of in vitro assembled HIV-1 capsid-nucleocapsid (CA-NC) complexes with the protein in question. Subsequently, the mixture is spun through a sucrose cushion using an ultracentrifuge, and the pellet is analyzed for the presence of the protein in question. Although this binding assay is reliable, it is labor intensive and does not contain washing steps. Here we have developed a simpler and faster assay to measure whether a cellular protein is binding to capsid. More importantly, this novel capsid-binding assay contains washing steps. In this assay, we took advantage of the HIV-1 capsid mutant A14C/E45C protein, which is stabilized by disulfide bonds, and is resistant to washing steps. We validated the reliability and specificity of this novel assay by testing the capsid binding ability of TRIMCyp, CPSF6 and MxB with their corresponding controls. Overall, this novel assay provides a reliable and fast methodology to search for novel capsid binders.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Bioensayo , Proteínas de la Cápside/química , VIH-1/química , Proteínas de la Membrana/química , Proteínas de Resistencia a Mixovirus/química , Factores de Escisión y Poliadenilación de ARNm/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , VIH-1/metabolismo , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Mutación , Proteínas de Resistencia a Mixovirus/genética , Proteínas de Resistencia a Mixovirus/metabolismo , Nucleocápside/química , Nucleocápside/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genética , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
The Tembusu virus (TMUV) is an avian pathogenic flavivirus that causes a highly contagious disease and catastrophic losses to the poultry industry. The myxovirus resistance protein (Mx) of innate immune effectors is a key antiviral “workhorse" of the interferon (IFN) system. Although mammalian Mx resistance against myxovirus and retrovirus was witnessed for decades, whether or not bird Mx has anti-flavivirus activity remains unknown. In this study, we found that the transcription of goose Mx (goMx) was obviously driven by TMUV infection, both in vivo and in vitro, and that the titers and copies of TMUV were significantly reduced by goMx overexpression. In both primary (goose embryo fibroblasts, GEFs) and passaged cells (baby hamster kidney cells, BHK21, and human fetal kidney cells, HEK 293T), it was shown that goMx was mainly located in the cytoplasm, and sporadically distributed in the nucleus. The intracellular localization of this protein is attributed to the predicted bipartite nuclear localization signal (NLS; 30 residues: the 441st471st amino acids of goMx). Intuitively, it seems that the cells with a higher level of goMx expression tend to have lower TMUV loads in the cytoplasm, as determined by an immunofluorescence assay. To further explore the antiviral determinants, a panel of variants was constructed. Two amino acids at the 125th (Lys) and 145th (Thr) positions in GTP-binding elements, not in the L4 loop (40 residues: the 532nd572nd amino acids of goMx), were vital for the antiviral function of goMx against TMUV in vitro. These findings will contribute to our understanding of the functional significance of the antiviral system in aquatic birds, and the development of goMx could be a valuable therapeutic agent against TMUV.
Asunto(s)
GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/metabolismo , Lisina , Proteínas de Resistencia a Mixovirus/química , Proteínas de Resistencia a Mixovirus/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Supervivencia Celular , Fibroblastos/metabolismo , Flavivirus/efectos de los fármacos , Flavivirus/fisiología , Gansos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Espacio Intracelular/metabolismo , Lisina/química , Proteínas de Resistencia a Mixovirus/genética , Proteínas de Resistencia a Mixovirus/farmacología , Transporte de Proteínas , Replicación ViralRESUMEN
Human dynamin-like, interferon-induced myxovirus resistance 2 (Mx2 or MxB) is a potent HIV-1 inhibitor. Antiviral activity requires both the amino-terminal region of MxB and protein oligomerization, each of which has eluded structural determination due to difficulties in protein preparation. We report that maltose binding protein-fused, full-length wild-type MxB purifies as oligomers and further self-assembles into helical arrays in physiological salt. Guanosine triphosphate (GTP), but not guanosine diphosphate, binding results in array disassembly, whereas subsequent GTP hydrolysis allows its reformation. Using cryo-electron microscopy (cryoEM), we determined the MxB assembly structure at 4.6 Å resolution, representing the first near-atomic resolution structure in the mammalian dynamin superfamily. The structure revealed previously described and novel MxB assembly interfaces. Mutational analyses demonstrated a critical role for one of the novel interfaces in HIV-1 restriction.
Asunto(s)
Fármacos Anti-VIH/química , Microscopía por Crioelectrón , Proteínas de Resistencia a Mixovirus/química , Multimerización de Proteína , Fármacos Anti-VIH/aislamiento & purificación , Resistencia a la Enfermedad , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Interacciones Huésped-Patógeno , Humanos , Modelos Moleculares , Proteínas de Resistencia a Mixovirus/genética , Proteínas de Resistencia a Mixovirus/aislamiento & purificación , Proteínas de Resistencia a Mixovirus/ultraestructura , Unión Proteica , Conformación Proteica , Proteínas Recombinantes de Fusión , Relación Estructura-ActividadRESUMEN
Human myxovirus resistance protein 1 (MxA) restricts a wide range of viruses and is closely related to the membrane-remodelling GTPase dynamin. The functions of MxA rely on domain rearrangements coupled with GTP hydrolysis cycles. To gain insight into this process, we studied real-time domain dynamics of MxA by single-molecule fluorescence resonance energy transfer. We find that the GTPase domain-bundle-signalling-element (BSE) region can adopt either an 'open' or a 'closed' conformation in all nucleotide-loading conditions. Whereas the open conformation is preferred in nucleotide-free, GDP·AlF4--bound and GDP-bound forms, loading of GTP activates the relative movement between the two domains and alters the conformational preference to the 'closed' state. Moreover, frequent relative movement was observed between BSE and stalk via hinge 1. On the basis of these results, we suggest how MxA molecules within a helical polymer collectively generate a stable torque through random GTP hydrolysis cycles. Our study provides mechanistic insights into fundamental cellular events such as viral resistance and endocytosis.