RESUMEN
Perturbation of the endoplasmic reticulum (ER), a central organelle of the cell, can have critical consequences for cellular homeostasis. An elaborate surveillance system known as ER quality control ensures that cells can respond and adapt to stress via the unfolded protein response (UPR) and that only correctly assembled proteins reach their destination. Interestingly, several bacterial pathogens hijack the ER to establish an infection. However, it remains poorly understood how bacterial pathogens exploit ER quality-control functions to complete their intracellular cycle. Brucella spp. replicate extensively within an ER-derived niche, which evolves into specialized vacuoles suited for exit from infected cells. Here we present Brucella-secreted protein L (BspL), a Brucella abortus effector that interacts with Herp, a central component of the ER-associated degradation (ERAD) machinery. We found that BspL enhances ERAD at the late stages of the infection. BspL targeting of Herp and ERAD allows tight control of the kinetics of autophagic Brucella-containing vacuole formation, delaying the last step of its intracellular cycle and cell-to-cell spread. This study highlights a mechanism by which a bacterial pathogen hijacks ERAD components for fine regulation of its intracellular trafficking.
Asunto(s)
Proteínas Bacterianas/metabolismo , Brucella abortus/patogenicidad , Brucelosis/metabolismo , Animales , Proteínas Bacterianas/genética , Brucella abortus/metabolismo , Brucelosis/microbiología , Retículo Endoplásmico/metabolismo , Degradación Asociada con el Retículo Endoplásmico , Células HeLa , Interacciones Huésped-Patógeno/fisiología , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Factor de Transcripción CHOP/genética , Sistemas de Secreción Tipo IV/metabolismo , Proteína 1 de Unión a la X-Box/genéticaRESUMEN
The phosphoprotein (P) of the nonsegmented negative-sense RNA viruses is a multimeric modular protein that is essential for RNA transcription and replication. Despite great variability in length and sequence, the architecture of this protein is conserved among the different viral families, with a long N-terminal intrinsically disordered region comprising a nucleoprotein chaperone module, a central multimerization domain (PMD), connected by a disordered linker to a C-terminal nucleocapsid-binding domain. The P protein of vesicular stomatitis virus (VSV) forms dimers, and here we investigate the importance of its dimerization domain, PMD, for viral gene expression and virus growth. A truncated P protein lacking the central dimerization domain (PΔMD) loses its ability to form dimers both in vitro and in a yeast two-hybrid system but conserves its ability to bind N. In a minireplicon system, the truncated monomeric protein performs almost as well as the full-length dimeric protein, while a recombinant virus harboring the same truncation in the P protein has been rescued and follows replication kinetics similar to those seen with the wild-type virus, showing that the dimerization domain of P is dispensable for viral gene expression and virus replication in cell culture. Because RNA viruses have high mutation rates, it is unlikely that a structured domain such as a VSV dimerization domain would persist in the absence of a function(s), but our work indicates that it is not required for the functioning of the RNA polymerase machinery or for the assembly of new viruses.IMPORTANCE The phosphoprotein (P) is an essential and conserved component of all nonsegmented negative-sense RNA viruses, including some major human pathogens (e.g., rabies virus, measles virus, respiratory syncytial virus [RSV], Ebola virus, and Nipah virus). P is a modular protein with intrinsically disordered regions and folded domains that plays specific and similar roles in the replication of the different viruses and, in some cases, hijacks cell components to the advantage of the virus and is involved in immune evasion. All P proteins are multimeric, but the role of this multimerization is still unclear. Here, we demonstrate that the dimerization domain of VSV P is dispensable for the expression of virally encoded proteins and for virus growth in cell culture. This provides new insights into and raises questions about the functioning of the RNA-synthesizing machinery of the nonsegmented negative-sense RNA viruses.
Asunto(s)
Fosfoproteínas/química , Dominios Proteicos , Multimerización de Proteína , Virus de la Estomatitis Vesicular Indiana/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Dimerización , Modelos Moleculares , Nucleocápside/metabolismo , Nucleoproteínas/metabolismo , Fosfoproteínas/genética , Unión Proteica , Conformación Proteica , Multimerización de Proteína/genética , ARN Viral/genética , Alineación de Secuencia , Estomatitis Vesicular/virología , Virus de la Estomatitis Vesicular Indiana/genética , Virus de la Estomatitis Vesicular Indiana/crecimiento & desarrollo , Replicación ViralRESUMEN
Type I interferons (IFN-I) are key innate immune effectors predominantly produced by activated plasmacytoid dendritic cells (pDCs). By modulating immune responses at their foundation, IFNs can widely reshape immunity to control infectious diseases and malignancies. Nevertheless, their biological activities can also be detrimental to surrounding healthy cells, as prolonged IFN-I signaling is associated with excessive inflammation and immune dysfunction. The interaction of the human pDC receptor immunoglobulin-like transcript 7 (ILT7) with its IFN-I-regulated ligand, bone marrow stromal cell antigen 2 (BST2) plays a key role in controlling the IFN-I amounts produced by pDCs in response to Toll-like receptor (TLR) activation. However, the structural determinants and molecular features of BST2 that govern ILT7 engagement and activation are largely undefined. Using two functional assays to measure BST2-stimulated ILT7 activation as well as biophysical studies, here we identified two structurally-distinct regions of the BST2 ectodomain that play divergent roles during ILT7 activation. We found that although the coiled-coil region contains a newly defined ILT7-binding surface, the N-terminal region appears to suppress ILT7 activation. We further show that a stable BST2 homodimer binds to ILT7, but post-binding events associated with the unique BST2 coiled-coil plasticity are required to trigger receptor signaling. Hence, BST2 with an unstable or a rigid coiled-coil fails to activate ILT7, whereas substitutions in its N-terminal region enhance activation. Importantly, the biological relevance of these newly defined domains of BST2 is underscored by the identification of substitutions having opposing potentials to activate ILT7 in pathological malignant conditions.
Asunto(s)
Antígeno 2 del Estroma de la Médula Ósea/metabolismo , Receptores Inmunológicos/metabolismo , Secuencia de Aminoácidos , Antígeno 2 del Estroma de la Médula Ósea/química , Antígeno 2 del Estroma de la Médula Ósea/genética , Línea Celular , Dimerización , Humanos , Mutagénesis , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios Proteicos , Alineación de SecuenciaRESUMEN
The cell nucleus is a primary target for intracellular bacterial pathogens to counteract immune responses and hijack host signalling pathways to cause disease. Here we identify two Brucella abortus effectors, NyxA and NyxB, that interfere with host protease SENP3, and this facilitates intracellular replication of the pathogen. The translocated Nyx effectors directly interact with SENP3 via a defined acidic patch (identified from the crystal structure of NyxB), preventing nucleolar localisation of SENP3 at late stages of infection. By sequestering SENP3, the effectors promote cytoplasmic accumulation of nucleolar AAA-ATPase NVL and ribosomal protein L5 (RPL5) in effector-enriched structures in the vicinity of replicating bacteria. The shuttling of ribosomal biogenesis-associated nucleolar proteins is inhibited by SENP3 and requires the autophagy-initiation protein Beclin1 and the SUMO-E3 ligase PIAS3. Our results highlight a nucleomodulatory function of two Brucella effectors and reveal that SENP3 is a crucial regulator of the subcellular localisation of nucleolar proteins during Brucella infection, promoting intracellular replication of the pathogen.
Asunto(s)
Brucelosis , Proteínas Nucleares , Humanos , Proteínas Nucleares/metabolismo , Núcleo Celular/metabolismo , Brucella abortus/metabolismo , Nucléolo Celular/metabolismo , Brucelosis/microbiología , Chaperonas Moleculares/metabolismo , Proteínas Inhibidoras de STAT Activados/metabolismo , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismoRESUMEN
The LEAFY (LFY) protein is a key regulator of flower development in angiosperms. Its gradually increased expression governs the sharp floral transition, and LFY subsequently controls the patterning of flower meristems by inducing the expression of floral homeotic genes. Despite a wealth of genetic data, how LFY functions at the molecular level is poorly understood. Here, we report crystal structures for the DNA-binding domain of Arabidopsis thaliana LFY bound to two target promoter elements. LFY adopts a novel seven-helix fold that binds DNA as a cooperative dimer, forming base-specific contacts in both the major and minor grooves. Cooperativity is mediated by two basic residues and plausibly accounts for LFY's effectiveness in triggering sharp developmental transitions. Our structure reveals an unexpected similarity between LFY and helix-turn-helix proteins, including homeodomain proteins known to regulate morphogenesis in higher eukaryotes. The appearance of flowering plants has been linked to the molecular evolution of LFY. Our study provides a unique framework to elucidate the molecular mechanisms underlying floral development and the evolutionary history of flowering plants.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Flores/fisiología , Secuencias Hélice-Giro-Hélice , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Arabidopsis/anatomía & histología , Proteínas de Arabidopsis/genética , Cristalografía por Rayos X , ADN/metabolismo , Dimerización , Sustancias Macromoleculares/química , Sustancias Macromoleculares/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , Unión Proteica , Conformación Proteica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores de Transcripción/genéticaRESUMEN
HIV-1 viral protein R (Vpr) from laboratory-adapted virus strains activates the DNA damage/stress sensor ATR kinase and induces cell cycle arrest at the G(2)/M phase through a process that requires Vpr to engage the DDB1-CUL4A (VprBP/DCAF-1) E3 ligase complex. Activation of this DNA damage/stress checkpoint in G(2) by Vpr was shown to modulate NKG2D-dependent NK cell effector functions via enhancing expression of NKG2D ligands, notably ULBP2. However, it is unknown whether Vpr from HIV-1 primary isolates (groups M, N, O, and P) could modulate NKG2D-mediated cytotoxic functions of NK cells. Here, we report that Vpr from most HIV-1 primary isolates can upregulate ULBP2 expression and induce NKG2D-dependent NK cell killing. Importantly, these activities were always accompanied by an active G(2) cell cycle arrest function. Interestingly, Vpr variants from group P and a clade D isolate of group M were defective at enhancing NKG2D-mediated NK cell lysis owing to their inability to augment ULBP2 expression. However, distinct mechanisms were responsible for their failure to do so. While Vpr from group P was deficient in its ability to engage the DDB1-CUL4A (VprBP/DCAF-1) E3 ligase complex, the Vpr variant from group D was unable to properly localize to the nucleus, underlining the importance of these biological properties in Vpr function. In conclusion, the ability of Vpr from HIV-1 primary isolates to regulate NK cell effector function underscores the importance of this HIV-1 accessory protein in the modulation of the host's innate immune responses.
Asunto(s)
Proliferación Celular , VIH-1/aislamiento & purificación , VIH-1/patogenicidad , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/patología , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencia de Aminoácidos , Western Blotting , Puntos de Control del Ciclo Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Daño del ADN , Técnica del Anticuerpo Fluorescente , Fase G2 , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/genética , Células HeLa , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Datos de Secuencia Molecular , Subfamilia K de Receptores Similares a Lectina de Células NK/genética , Complejo de la Endopetidasa Proteasomal , Homología de Secuencia de Aminoácido , Replicación Viral , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
HIV-1 Viral protein R (Vpr) induces a cell cycle arrest at the G2/M phase by activating the ATR DNA damage/stress checkpoint. Recently, we and several other groups showed that Vpr performs this activity by recruiting the DDB1-CUL4A (VPRBP) E3 ubiquitin ligase. While recruitment of this E3 ubiquitin ligase complex has been shown to be required for G2 arrest, the subcellular compartment where this complex forms and functionally acts is unknown. Herein, using immunofluorescence and confocal microscopy, we show that Vpr forms nuclear foci in several cell types including HeLa cells and primary CD4+ T-lymphocytes. These nuclear foci contain VPRBP and partially overlap with DNA repair foci components such as gamma-H2AX, 53BP1 and RPA32. While treatment with the non-specific ATR inhibitor caffeine or depletion of VPRBP by siRNA did not inhibit formation of Vpr nuclear foci, mutations in the C-terminal domain of Vpr and cytoplasmic sequestration of Vpr by overexpression of Gag-Pol resulted in impaired formation of these nuclear structures and defective G2 arrest. Consistently, we observed that G2 arrest-competent sooty mangabey Vpr could form these foci but not its G2 arrest-defective paralog Vpx, suggesting that formation of Vpr nuclear foci represents a critical early event in the induction of G2 arrest. Indeed, we found that Vpr could associate to chromatin via its C-terminal domain and that it could form a complex with VPRBP on chromatin. Finally, analysis of Vpr nuclear foci by time-lapse microscopy showed that they were highly mobile and stable structures. Overall, our results suggest that Vpr recruits the DDB1-CUL4A (VPRBP) E3 ligase to these nuclear foci and uses these mobile structures to target a chromatin-bound cellular substrate for ubiquitination in order to induce DNA damage/replication stress, ultimately leading to ATR activation and G2 cell cycle arrest.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/fisiología , Cromatina/metabolismo , Fase G2/fisiología , Complejo de la Endopetidasa Proteasomal/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismo , Adulto , Proteínas de la Ataxia Telangiectasia Mutada , Western Blotting , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Cromatina/genética , Reparación del ADN , Células HeLa , Humanos , Inmunoprecipitación , Proteínas Serina-Treonina Quinasas/genética , Ubiquitina-Proteína Ligasas , Ubiquitinación , Replicación Viral , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
As for all non-segmented negative RNA viruses, rabies virus has its genome packaged in a linear assembly of nucleoprotein (N), named nucleocapsid. The formation of new nucleocapsids during virus replication in cells requires the production of soluble N protein in complex with its phosphoprotein (P) chaperone. In this study, we reconstituted a soluble heterodimeric complex between an armless N protein of rabies virus (RABV), lacking its N-terminal subdomain (NNT-ARM), and a peptide encompassing the N0 chaperon module of the P protein. We showed that the chaperone module undergoes a disordered-order transition when it assembles with N0 and measured an affinity in the low nanomolar range using a competition assay. We solved the crystal structure of the complex at a resolution of 2.3 Å, unveiling the details of the conserved interfaces. MD simulations showed that both the chaperon module of P and RNA-mediated polymerization reduced the ability of the RNA binding cavity to open and close. Finally, by reconstituting a complex with full-length P protein, we demonstrated that each P dimer could independently chaperon two N0 molecules.
Asunto(s)
Virus de la Rabia , Virus de la Rabia/genética , Nucleoproteínas/metabolismo , Unión Proteica , Proteínas de la Nucleocápside/genética , Chaperonas Moleculares/metabolismo , Fosfoproteínas/genética , ARN/metabolismo , ARN Viral/metabolismoRESUMEN
To understand the dynamic interactions between the phosphoprotein (P) and the nucleoprotein (N) within the transcription/replication complex of the Paramyxoviridae and to decipher their roles in regulating viral multiplication, we characterized the structural properties of the C-terminal X domain (PXD) of Nipah (NiV) and Hendra virus (HeV) P protein. In crystals, isolated NiV PXD adopted a two-helix dimeric conformation, which was incompetent for binding its partners, but in complex with the C-terminal intrinsically disordered tail of the N protein (NTAIL), it folded into a canonical 3H bundle conformation. In solution, SEC-MALLS, SAXS and NMR spectroscopy experiments indicated that both NiV and HeV PXD were larger in size than expected for compact proteins of the same molecular mass and were in conformational exchange between a compact three-helix (3H) bundle and partially unfolded conformations, where helix α3 is detached from the other two. Some measurements also provided strong evidence for dimerization of NiV PXD in solution but not for HeV PXD. Ensemble modeling of experimental SAXS data and statistical-dynamical modeling reconciled all these data, yielding a model where NiV and HeV PXD exchanged between different conformations, and where NiV but not HeV PXD formed dimers. Finally, recombinant NiV comprising a chimeric P carrying HeV PXD was rescued and compared with parental NiV. Experiments carried out in cellula demonstrated that the replacement of PXD did not significantly affect the replication dynamics while caused a slight virus attenuation, suggesting a possible role of the dimerization of NiV PXD in viral replication.
Asunto(s)
Virus Hendra , Virus Nipah , Proteínas de la Nucleocápside , Fosfoproteínas , Proteínas Virales , Replicación Viral , Virus Hendra/genética , Virus Hendra/fisiología , Humanos , Virus Nipah/genética , Virus Nipah/fisiología , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Dominios Proteicos , Pliegue de Proteína , Multimerización de Proteína , Dispersión del Ángulo Pequeño , Proteínas Virales/química , Proteínas Virales/genética , Difracción de Rayos XRESUMEN
Influenza viruses are negative single-stranded RNA viruses with nuclear transcription and replication. They enter the nucleus by using the cellular importin-α/-ß nuclear import machinery. Influenza nucleoproteins from influenza A, B, C and D viruses possess a nuclear localization signal (NLS) localized on an intrinsically disordered extremity (NPTAIL). In this paper, using size exclusion chromatography (SEC), SEC-multi-angle laser light scattering (SEC-MALLS) analysis, surface plasmon resonance (SPR) and fluorescence anisotropy, we provide the first comparative study designed to dissect the interaction between the four NPTAILs and four importins-α identified as partners. All interactions between NPTAILs and importins-α have high association and dissociation rates and present a distinct and specific behaviour. D/NPTAIL interacts strongly with all importins-α while B/NPTAIL shows weak affinity for importins-α. A/NPTAIL and C/NPTAIL present preferential importin-α partners. Mutations in B/NPTAIL and D/NPTAIL show a loss of importin-α binding, confirming key NLS residues. Taken together, our results provide essential highlights of this complex translocation mechanism.
Asunto(s)
Interacciones Microbiota-Huesped , Proteínas de la Nucleocápside/metabolismo , Orthomyxoviridae/metabolismo , alfa Carioferinas/metabolismo , Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Polarización de Fluorescencia , Humanos , Mutación , Señales de Localización Nuclear , Proteínas de la Nucleocápside/genética , Orthomyxoviridae/genética , Unión Proteica , Resonancia por Plasmón de SuperficieRESUMEN
The rabies and Ebola viruses recruit the highly conserved host protein LC8 for their own reproductive success. In vivo knockouts of the LC8 recognition motif within the rabies virus phosphoprotein (RavP) result in completely nonlethal viral infections. In this work, we examine the molecular role LC8 plays in viral lethality. We show that RavP and LC8 colocalize in rabies infected cells, and that LC8 interactions are essential for efficient viral polymerase functionality. NMR, SAXS, and molecular modeling demonstrate that LC8 binding to a disordered linker adjacent to an endogenous dimerization domain results in restrictions in RavP domain orientations. The resulting ensemble structure of RavP-LC8 tetrameric complex is similar to that of a related virus phosphoprotein that does not bind LC8, suggesting that with RavP, LC8 binding acts as a switch to induce a more active conformation. The high conservation of the LC8 motif in Lyssavirus phosphoproteins and its presence in other analogous proteins such as the Ebola virus VP35 evinces a broader purpose for LC8 in regulating downstream phosphoprotein functions vital for viral replication.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/química , Proteínas de Drosophila/química , Dineínas/química , Lyssavirus/enzimología , Fosfoproteínas/química , Proteínas Virales/química , Secuencia Conservada , ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas de Drosophila/metabolismo , Dineínas/metabolismo , Activación Enzimática , Interacciones Huésped-Patógeno/inmunología , Modelos Biológicos , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Fosfoproteínas/metabolismo , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Virus de la Rabia/metabolismo , Factor de Transcripción STAT1/metabolismo , Relación Estructura-Actividad , Proteínas Virales/metabolismoRESUMEN
This paper focuses on the nucleoprotein (NP) of the newly identified member of the Orthomyxoviridae family, Influenza D virus. To date several X-ray structures of NP of Influenza A (A/NP) and B (B/NP) viruses and of infectious salmon anemia (ISA/NP) virus have been solved. Here we purified, characterized and solved the X-ray structure of the tetrameric D/NP at 2.4 Å resolution. The crystal structure of its core is similar to NP of other Influenza viruses. However, unlike A/NP and B/NP which possess a flexible amino-terminal tail containing nuclear localization signals (NLS) for their nuclear import, D/NP possesses a carboxy-terminal tail (D/NPTAIL). We show that D/NPTAIL harbors a bipartite NLS and designed C-terminal truncated mutants to demonstrate the role of D/NPTAIL for nuclear transport.
Asunto(s)
Núcleo Celular/metabolismo , Nucleoproteínas/química , Orthomyxoviridae/metabolismo , Thogotovirus/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Cristalografía por Rayos X , Células HEK293 , Humanos , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Alineación de Secuencia , alfa Carioferinas/metabolismoRESUMEN
Nucleocapsid assembly is an essential process in the replication of the non-segmented, negative-sense RNA viruses (NNVs). Unassembled nucleoprotein (N(0)) is maintained in an RNA-free and monomeric form by its viral chaperone, the phosphoprotein (P), forming the N(0)-P complex. Our earlier work solved the structure of vesicular stomatitis virus complex formed between an N-terminally truncated N (NΔ21) and a peptide of P (P60) encompassing the N(0)-binding site, but how the full-length P interacts with N(0) remained unknown. Here, we combine several experimental biophysical methods including size exclusion chromatography with detection by light scattering and refractometry, small-angle X-ray and neutron scattering and nuclear magnetic resonance spectroscopy with molecular dynamics simulation and computational modeling to characterize the NΔ21(0)-PFL complex formed with dimeric full-length P. We show that for multi-molecular complexes, simultaneous multiple-curve fitting using small-angle neutron scattering data collected at varying contrast levels provides additional information and can help refine structural ensembles. We demonstrate that (a) vesicular stomatitis virus PFL conserves its high flexibility within the NΔ21(0)-PFL complex and interacts with NΔ21(0) only through its N-terminal extremity; (b) each protomer of P can chaperone one N(0) client protein, leading to the formation of complexes with stoichiometries 1N:P2 and 2N:P2; and (c) phosphorylation of residues Ser60, Thr62 and Ser64 provides no additional interactions with N(0) but creates a metal binding site in PNTR. A comparison with the structures of Nipah virus and Ebola virus N(0)-P core complex suggests a mechanism for the control of nucleocapsid assembly that is common to all NNVs.
Asunto(s)
Chaperonas Moleculares/metabolismo , Nucleoproteínas/metabolismo , Fosfoproteínas/metabolismo , Virus de la Estomatitis Vesicular Indiana/metabolismo , Proteínas Estructurales Virales/metabolismo , Sitios de Unión , Nucleocápside/metabolismo , Unión Proteica/genética , ARN Viral/genética , Estomatitis Vesicular/virologíaRESUMEN
HIV viral protein R (Vpr) induces a cell cycle arrest at the G2/M phase by activating the ATR DNA damage/replication stress signalling pathway through engagement of the DDB1-CUL4A-DCAF1 E3 ubiquitin ligase via a direct binding to the substrate specificity receptor DCAF1. Since no high resolution structures of the DDB1-DCAF1-Vpr substrate recognition module currently exist, we used a mutagenesis approach to better define motifs in DCAF1 that are crucial for Vpr and DDB1 binding. Herein, we show that the minimal domain of DCAF1 that retained the ability to bind Vpr and DDB1 was mapped to residues 1041 to 1393 (DCAF1 WD). Mutagenic analyses identified an α-helical H-box motif and F/YxxF/Y motifs located in the N-terminal domain of DCAF1 WD that are involved in exclusive binding to DDB1. While we could not identify elements specifically involved in Vpr binding, overall, the mutagenesis data suggest that the predicted ß-propeller conformation of DCAF1 is likely to be critical for Vpr association. Importantly, we provide evidence that binding of Vpr to DCAF1 appears to modulate the formation of a DDB1/DCAF1 complex. Lastly, we show that expression of DCAF1 WD in the absence of endogenous DCAF1 was not sufficient to enable Vpr-mediated G2 arrest activity. Overall, our results reveal that Vpr and DDB1 binding on DCAF1 can be genetically separated and further suggest that DCAF1 contains determinants in addition to the Vpr and DDB1 minimal binding domain, which are required for Vpr to enable the induction of a G2 arrest.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Puntos de Control de la Fase G2 del Ciclo Celular/fisiología , Infecciones por VIH/metabolismo , Complejos Multiproteicos/metabolismo , Transducción de Señal/fisiología , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencia de Aminoácidos , Western Blotting , Proteínas Portadoras/genética , Proteínas Cullin/metabolismo , Proteínas de Unión al ADN/genética , Células HEK293 , Células HeLa , Humanos , Inmunoprecipitación , Microscopía Fluorescente , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plásmidos/genética , Unión Proteica , Proteínas Serina-Treonina Quinasas , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The phosphoprotein (P) is an essential component of the viral replication machinery of non-segmented negative-strand RNA viruses, connecting the viral polymerase to its nucleoprotein-RNA template and acting as a chaperone of the nucleoprotein by preventing nonspecific encapsidation of cellular RNAs. The phosphoprotein of vesicular stomatitis virus (VSV) forms homodimers and possesses a modular organization comprising two stable, well-structured domains concatenated with two intrinsically disordered regions. Here, we used a combination of nuclear magnetic resonance spectroscopy and small-angle X-ray scattering to depict VSV P as an ensemble of continuously exchanging conformers that captures the dynamic character of this protein. We discuss the implications of the dynamics and the large conformational space sampled by VSV P in the assembly and functioning of the viral transcription/replication machinery.
Asunto(s)
Fosfoproteínas/química , Virus de la Estomatitis Vesicular Indiana/metabolismo , Proteínas Estructurales Virales/química , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Fosfoproteínas/metabolismo , Conformación Proteica , ARN Viral/química , ARN Viral/metabolismo , Proteínas Estructurales Virales/metabolismo , Replicación ViralRESUMEN
The phosphoprotein (P) of vesicular stomatitis virus (VSV) interacts with nascent nucleoprotein (N), forming the N(0)-P complex that is indispensable for the correct encapsidation of newly synthesized viral RNA genome. In this complex, the N-terminal region (P(NTR)) of P prevents N from binding to cellular RNA and keeps it available for encapsidating viral RNA genomes. Here, using nuclear magnetic resonance (NMR) spectroscopy and small-angle X-ray scattering (SAXS), we show that an isolated peptide corresponding to the 60 first N-terminal residues of VSV P (P(60)) and encompassing P(NTR) has overall molecular dimensions and a dynamic behavior characteristic of a disordered protein but transiently populates conformers containing α-helices. The modeling of P(60) as a conformational ensemble by the ensemble optimization method using SAXS data correctly reproduces the α-helical content detected by NMR spectroscopy and suggests the coexistence of subensembles of different compactness. The populations and overall dimensions of these subensembles are affected by the addition of stabilizing (1M trimethylamine-N-oxide) or destabilizing (6M guanidinium chloride) cosolvents. Our results are interpreted in the context of a scenario whereby VSV P(NTR) constitutes a molecular recognition element undergoing a disorder-to-order transition upon binding to its partner when forming the N(0)-P complex.
Asunto(s)
Proteínas de la Cápside/química , Fosfoproteínas/química , Estructura Secundaria de Proteína , Vesiculovirus/química , Sitios de Unión , Cápside/química , Proteínas de la Cápside/genética , Complejos Multiproteicos/química , Resonancia Magnética Nuclear Biomolecular , Nucleoproteínas/química , Nucleoproteínas/genética , Fosfoproteínas/genética , Unión Proteica , Dispersión del Ángulo PequeñoRESUMEN
Rhabdoviridae are single stranded negative sense RNA viruses. The viral RNA condensed by the nucleoprotein (N), the phosphoprotein (P) and the large subunit (L) of the RNA-dependent RNA polymerase are the viral components of the transcription/replication machineries. Both P and N contain intrinsically disordered regions (IDRs) that play different roles in the virus life cycle. Here, we describe the modular organization of P based on recent structural, biophysical and bioinformatics data. We show how flexible loops in N participate in the attachment of P to the N-RNA template by an induced-fit mechanism. Finally, we discuss the roles of IDRs in the mechanism of replication/transcription, and propose a new model for the interaction of the L subunit with its N-RNA template.
Asunto(s)
Nucleoproteínas/química , Fosfoproteínas/química , Rhabdoviridae/química , Rhabdoviridae/fisiología , Replicación Viral , Genoma Viral , Modelos Moleculares , Estructura Terciaria de Proteína , Rhabdoviridae/genética , Transcripción GenéticaRESUMEN
A phosphoprotein (P) is found in all viruses of the Mononegavirales order. These proteins form homo-oligomers, fulfil similar roles in the replication cycles of the various viruses, but differ in their length and oligomerization state. Sequence alignments reveal no sequence similarity among proteins from viruses belonging to the same family. Sequence analysis and experimental data show that phosphoproteins from viruses of the Paramyxoviridae contain structured domains alternating with intrinsically disordered regions. Here, we used predictions of disorder of secondary structure, and an analysis of sequence conservation to predict the domain organization of the phosphoprotein from Sendai virus, vesicular stomatitis virus (VSV) and rabies virus (RV P). We devised a new procedure for combining the results from multiple prediction methods and locating the boundaries between disordered regions and structured domains. To validate the proposed modular organization predicted for RV P and to confirm that the putative structured domains correspond to autonomous folding units, we used two-hybrid and biochemical approaches to characterize the properties of several fragments of RV P. We found that both central and C-terminal domains can fold in isolation, that the central domain is the oligomerization domain, and that the C-terminal domain binds to nucleocapsids. Our results suggest a conserved organization of P proteins in the Rhabdoviridae family in concatenated functional domains resembling that of the P proteins in the Paramyxoviridae family.
Asunto(s)
Fosfoproteínas/química , Conformación Proteica , Virus de la Rabia/química , Proteínas Estructurales Virales/química , Secuencia de Aminoácidos , Chaperonas Moleculares , Datos de Secuencia Molecular , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de Proteína , Técnicas del Sistema de Dos Híbridos , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/metabolismoRESUMEN
In rabies virus, the attachment of the L polymerase (L) to the viral nucleocapsids (NCs)-a nucleoprotein (N)-RNA complex that serves as template for RNA transcription and replication-is mediated by the polymerase cofactor, the phosphoprotein (P). P forms dimers (P(2)) that bind through their C-terminal domains (P(CTD)) to the C-terminal region of the N. Recombinant circular N(m)-RNA complexes containing 9 to 12 protomers of N (hereafter, the subscript m denotes the number of N protomers) served here as model systems for studying the binding of P to NC-like N(m)-RNA complexes. Titration experiments show that there are only two equivalent and independent binding sites for P dimers on the N(m)-RNA rings and that each P dimer binds through a single P(CTD). A dissociation constant in the nanomolar range (160+/-20 nM) was measured by surface plasmon resonance, indicating a strong interaction between the two partners. Small-angle X-ray scattering (SAXS) data and small-angle neutron scattering data showed that binding of two P(CTD) had almost no effect on the size and shape of the N(m)-RNA rings, whereas binding of two P(2) significantly increased the size of the complexes. SAXS data and molecular modeling were used to add flexible loops (N(NTD) loop, amino acids 105-118; N(CTD) loop, amino acids 376-397) missing in the recently solved crystal structure of the circular N(11)-RNA complex and to build a model for the N(10)-RNA complex. Structural models for the N(m)-RNA-(P(CTD))(2) complexes were then built by docking the known P(CTD) structure onto the completed structures of the circular N(10)-RNA and N(11)-RNA complexes. A multiple-stage flexible docking procedure was used to generate decoys, and SAXS and biochemical data were used for filtering the models. In the refined model, the P(CTD) is bound to the C-terminal top of one N protomer (N(i)), with the C-terminal helix (alpha(6)) of P(CTD) lying on helix alpha(14) of N(i). By an induced-fit mechanism, the N(CTD) loop of the same protomer (N(i)) and that of the adjacent one (N(i)(-1)) mold around the P(CTD), making extensive protein-protein contacts that could explain the strong affinity of P for its template. The structural model is in agreement with available biochemical data and provides new insights on the mechanism of attachment of the polymerase complex to the NC template.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas de la Nucleocápside/metabolismo , Virus de la Rabia/metabolismo , Proteínas Virales/metabolismo , Sitios de Unión , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , Dimerización , Sustancias Macromoleculares , Modelos Moleculares , Difracción de Neutrones , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/genética , Paramyxoviridae/genética , Paramyxoviridae/metabolismo , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , ARN Viral/química , ARN Viral/genética , ARN Viral/metabolismo , Virus de la Rabia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhabdoviridae/genética , Rhabdoviridae/metabolismo , Dispersión del Ángulo Pequeño , Especificidad de la Especie , Electricidad Estática , Resonancia por Plasmón de Superficie , Termodinámica , Proteínas Virales/química , Proteínas Virales/genética , Difracción de Rayos XRESUMEN
Beyond common features in their genome organization and replication mechanisms, the evolutionary relationships among viruses of the Rhabdoviridae family are difficult to decipher because of the great variability in the amino acid sequence of their proteins. The phosphoprotein (P) of vesicular stomatitis virus (VSV) is an essential component of the RNA transcription and replication machinery; in particular, it contains binding sites for the RNA-dependent RNA polymerase and for the nucleoprotein. Here, we devised a new method for defining boundaries of structured domains from multiple disorder prediction algorithms, and we identified an autonomous folding C-terminal domain in VSV P (P(CTD)). We show that, like the C-terminal domain of rabies virus (RV) P, VSV P(CTD) binds to the viral nucleocapsid (nucleoprotein-RNA complex). We solved the three-dimensional structure of VSV P(CTD) by NMR spectroscopy and found that the topology of its polypeptide chain resembles that of RV P(CTD). The common part of both proteins could be superimposed with a backbone RMSD from mean atomic coordinates of 2.6 A. VSV P(CTD) has a shorter N-terminal helix (alpha(1)) than RV P(CTD); it lacks two alpha-helices (helices alpha(3) and alpha(6) of RV P), and the loop between strands beta(1) and beta(2) is longer than that in RV. Dynamical properties measured by NMR relaxation revealed the presence of fast motions (below the nanosecond timescale) in loop regions (amino acids 209-214) and slower conformational exchange in the N- and C-terminal helices. Characterization of a longer construct indicated that P(CTD) is preceded by a flexible linker. The results presented here support a modular organization of VSV P, with independent folded domains separated by flexible linkers, which is conserved among different genera of Rhabdoviridae and is similar to that proposed for the P proteins of the Paramyxoviridae.