RESUMEN
The establishment of a virus infection is the result of the pathogen's ability to replicate in a hostile environment generated by the host's immune system. Here, we found that ISG15 restricts Dengue and Zika viruses' replication through the stabilization of its binding partner USP18. ISG15 expression was necessary to control DV replication driven by both autocrine and paracrine type one interferon (IFN-I) signaling. Moreover, USP18 competes with NS5-mediated STAT2 degradation, a major mechanism for establishment of flavivirus infection. Strikingly, reconstitution of USP18 in ISG15-deficient cells was sufficient to restore the STAT2's stability and restrict virus growth, suggesting that the IFNAR-mediated ISG15 activity is also antiviral. Our results add a novel layer of complexity in the virus/host interaction interface and suggest that NS5 has a narrow window of opportunity to degrade STAT2, therefore suppressing host's IFN-I mediated response and promoting virus replication.
Asunto(s)
Dengue , Interferón Tipo I , Infección por el Virus Zika , Virus Zika , Humanos , Interferón Tipo I/metabolismo , Infección por el Virus Zika/genética , Replicación Viral , Dengue/genética , Ubiquitinas/metabolismo , Citocinas/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Factor de Transcripción STAT2/genética , Factor de Transcripción STAT2/metabolismoRESUMEN
Zika virus (ZIKV) can infect human developing brain (HDB) progenitors resulting in epidemic microcephaly, whereas analogous cellular tropism offers treatment potential for the adult brain cancer, glioblastoma (GBM). We compared productive ZIKV infection in HDB and GBM primary tissue explants that both contain SOX2+ neural progenitors. Strikingly, although the HDB proved uniformly vulnerable to ZIKV infection, GBM was more refractory, and this correlated with an innate immune expression signature. Indeed, GBM-derived CD11b+ microglia/macrophages were necessary and sufficient to protect progenitors against ZIKV infection in a non-cell autonomous manner. Using SOX2+ GBM cell lines, we found that CD11b+-conditioned medium containing type 1 interferon beta (IFNß) promoted progenitor resistance to ZIKV, whereas inhibition of JAK1/2 signaling restored productive infection. Additionally, CD11b+ conditioned medium, and IFNß treatment rendered HDB progenitor lines and explants refractory to ZIKV. These findings provide insight into neuroprotection for HDB progenitors as well as enhanced GBM oncolytic therapies.
Asunto(s)
Infección por el Virus Zika , Virus Zika , Humanos , Células Mieloides , Células Madre , InterferonesRESUMEN
Translation initiation on structured mammalian mRNAs requires DHX29, a DExH protein that comprises a unique 534-aa-long N-terminal region (NTR) and a common catalytic DExH core. DHX29 binds to 40S subunits and possesses 40S-stimulated NTPase activity essential for its function. In the cryo-EM structure of DHX29-bound 43S preinitiation complexes, the main DHX29 density resides around the tip of helix 16 of 18S rRNA, from which it extends through a linker to the subunit interface forming an intersubunit domain next to the eIF1A binding site. Although a DExH core model can be fitted to the main density, the correlation between the remaining density and the NTR is unknown. Here, we present a model of 40S-bound DHX29, supported by directed hydroxyl radical cleavage data, showing that the intersubunit domain comprises a dsRNA-binding domain (dsRBD, aa 377-448) whereas linker corresponds to the long α-helix (aa 460-512) that follows the dsRBD. We also demonstrate that the N-terminal α-helix and the following UBA-like domain form a four-helix bundle (aa 90-166) that constitutes a previously unassigned section of the main density and resides between DHX29's C-terminal α-helix and the linker. In vitro reconstitution experiments revealed the critical and specific roles of these NTR elements for DHX29's function.
Asunto(s)
Iniciación de la Cadena Peptídica Traduccional/genética , Biosíntesis de Proteínas/genética , ARN Helicasas/genética , ARN Mensajero/genética , Ribosomas/genética , Regiones no Traducidas 3'/genética , Animales , Secuencia de Bases , Sitios de Unión/genética , Microscopía por Crioelectrón , Humanos , Mamíferos/genética , Mamíferos/metabolismo , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica , ARN Helicasas/química , ARN Helicasas/metabolismo , ARN Mensajero/metabolismo , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/metabolismo , Ribosomas/metabolismo , Ribosomas/ultraestructuraRESUMEN
Flaviviruses, including dengue virus and Zika virus, contain a single-stranded positive sense RNA genome that encodes viral proteins essential for replication and also serves as the template for new genome synthesis. As these processes move in opposite directions along the genome, translation must be inhibited at a defined point following infection to clear the template of ribosomes to allow efficient replication. Here, we demonstrate in vitro and in cell-based assays that the viral RNA polymerase, NS5, inhibits translation of the viral genome. By reconstituting translation in vitro using highly purified components, we show that this translation block occurs at the initiation stage and that translation inhibition depends on NS5-RNA interaction, primarily through association with the 5' replication promoter region. This work supports a model whereby expression of a viral protein signals successful translation of the infecting genome, prompting a switch to a ribosome depleted replication-competent form.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Genoma Viral , Biosíntesis de Proteínas , ARN Viral/metabolismo , Proteínas no Estructurales Virales/metabolismo , Animales , Chlorocebus aethiops , Virus del Dengue/enzimología , Iniciación de la Cadena Peptídica Traduccional , ARN Viral/química , Células Vero , Replicación Viral , Virus Zika/enzimología , Virus Zika/fisiologíaRESUMEN
Knockout mouse models have been extensively used to study the antiviral activity of IFIT (interferon-induced protein with tetratricopeptide repeats). Human IFIT1 binds to cap0 (m7GpppN) RNA, which lacks methylation on the first and second cap-proximal nucleotides (cap1, m7GpppNm, and cap2, m7GpppNmNm, respectively). These modifications are signatures of "self" in higher eukaryotes, whereas unmodified cap0-RNA is recognized as foreign and, therefore, potentially harmful to the host cell. IFIT1 inhibits translation at the initiation stage by competing with the cap-binding initiation factor complex, eIF4F, restricting infection by certain viruses that possess "nonself" cap0-mRNAs. However, in mice and other rodents, the IFIT1 orthologue has been lost, and the closely related Ifit1b has been duplicated twice, yielding three paralogues: Ifit1, Ifit1b, and Ifit1c. Although murine Ifit1 is similar to human IFIT1 in its cap0-RNA-binding selectivity, the roles of Ifit1b and Ifit1c are unknown. Here, we found that Ifit1b preferentially binds to cap1-RNA, whereas binding is much weaker to cap0- and cap2-RNA. In murine cells, we show that Ifit1b can modulate host translation and restrict WT mouse coronavirus infection. We found that Ifit1c acts as a stimulatory cofactor for both Ifit1 and Ifit1b, promoting their translation inhibition. In this way, Ifit1c acts in an analogous fashion to human IFIT3, which is a cofactor to human IFIT1. This work clarifies similarities and differences between the human and murine IFIT families to facilitate better design and interpretation of mouse models of human infection and sheds light on the evolutionary plasticity of the IFIT family.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Coronavirus/crecimiento & desarrollo , Coronavirus/genética , Biosíntesis de Proteínas , Proteínas de Unión a Caperuzas de ARN/metabolismo , Caperuzas de ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Coronavirus/metabolismo , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Modelos Moleculares , Mutación , Unión Proteica , Células RAW 264.7 , Proteínas de Unión al ARN/genéticaRESUMEN
Members of the Flaviviridae family, including dengue virus (DENV) and yellow fever virus, cause serious disease in humans, whilst maternal infection with Zika virus (ZIKV) can induce microcephaly in newborns. Following infection, flaviviral RNA genomes are translated to produce the viral replication machinery but must then serve as a template for the transcription of new genomes. However, the ribosome and viral polymerase proceed in opposite directions along the RNA, risking collisions and abortive replication. Whilst generally linear, flavivirus genomes can adopt a circular conformation facilitated by long-range RNA-RNA interactions, shown to be essential for replication. Using an in vitro reconstitution approach, we demonstrate that circularization inhibits de novo translation initiation on ZIKV and DENV RNA, whilst the linear conformation is translation-competent. Our results provide a mechanism to clear the viral RNA of ribosomes in order to promote efficient replication and, therefore, define opposing roles for linear and circular conformations of the flavivirus genome.
Asunto(s)
Virus del Dengue/genética , Flavivirus/genética , Biosíntesis de Proteínas , Virus Zika/genética , Virus del Dengue/patogenicidad , Flavivirus/patogenicidad , Genoma Viral/genética , Genómica , Humanos , Recién Nacido , ARN Viral/genética , Replicación Viral/genética , Virus de la Fiebre Amarilla/genética , Virus de la Fiebre Amarilla/patogenicidad , Virus Zika/patogenicidad , Infección por el Virus Zika/genética , Infección por el Virus Zika/virologíaRESUMEN
Background: Norovirus, also known as the winter vomiting bug, is the predominant cause of non-bacterial gastroenteritis worldwide. Disease control is predicated on a robust innate immune response during the early stages of infection. Double-stranded RNA intermediates generated during viral genome replication are recognised by host innate immune sensors in the cytoplasm, activating the strongly antiviral interferon gene programme. Ifit proteins (interferon induced proteins with tetratricopeptide repeats), which are highly expressed during the interferon response, have been shown to directly inhibit viral protein synthesis as well as regulate innate immune signalling pathways. Ifit1 is well-characterised to inhibit viral translation by sequestration of eukaryotic initiation factors or by directly binding to the 5' terminus of foreign RNA, particularly those with non-self cap structures. However, noroviruses have a viral protein, VPg, covalently linked to the 5' end of the genomic RNA, which acts as a cap substitute to recruit the translation initiation machinery. Methods: Ifit1 knockout RAW264.7 murine macrophage-like cells were generated using CRISPR-Cas9 gene editing. These cells were analysed for their ability to support murine norovirus infection, determined by virus yield, and respond to different immune stimuli, assayed by quantitative PCR. The effect of Ifit proteins on norovirus translation was also tested in vitro. Results: Here, we show that VPg-dependent translation is completely refractory to Ifit1-mediated translation inhibition in vitro and Ifit1 cannot bind the 5' end of VPg-linked RNA. Nevertheless, knockout of Ifit1 promoted viral replication in murine norovirus infected cells. We then demonstrate that Ifit1 promoted interferon-beta expression following transfection of synthetic double-stranded RNA but had little effect on toll-like receptor 3 and 4 signalling. Conclusions: Ifit1 is an antiviral factor during norovirus infection but cannot directly inhibit viral translation. Instead, Ifit1 stimulates the antiviral state following cytoplasmic RNA sensing, contributing to restriction of norovirus replication.
RESUMEN
The interferon-induced proteins with tetratricopeptide repeats (IFITs) are a family of antiviral proteins conserved throughout all vertebrates. IFIT1 binds tightly to non-self RNA, particularly capped transcripts lacking methylation on the first cap-proximal nucleotide, and inhibits their translation by out-competing the cellular translation initiation apparatus. This exerts immense selection pressure on cytoplasmic RNA viruses to maintain mechanisms that protect their messenger RNA from IFIT1 recognition. However, it is becoming increasingly clear that protein-protein interactions are necessary for optimal IFIT function. Recently, IFIT1, IFIT2 and IFIT3 have been shown to form a functional complex in which IFIT3 serves as a central scaffold to regulate and/or enhance the antiviral functions of the other two components. Moreover, IFITs interact with other cellular proteins to expand their contribution to regulation of the host antiviral response by modulating innate immune signalling and apoptosis. Here, we summarize recent advances in our understanding of the IFIT complex and review how this impacts on the greater role of IFIT proteins in the innate antiviral response.
Asunto(s)
Inmunidad Innata , Factores Inmunológicos/metabolismo , Mapas de Interacción de Proteínas , Multimerización de Proteína , Proteínas/metabolismo , Virus ARN/inmunología , Repeticiones de Tetratricopéptidos , Animales , Humanos , Factores Inmunológicos/química , Unión Proteica , Proteínas/química , VertebradosRESUMEN
Interferon-induced proteins with tetratricopeptide repeats (IFITs) are highly expressed during the cell-intrinsic immune response to viral infection. IFIT1 inhibits translation by binding directly to the 5' end of foreign RNAs, particularly those with non-self cap structures, precluding the recruitment of the cap-binding eukaryotic translation initiation factor 4F and ribosome recruitment. The presence of IFIT1 imposes a requirement on viruses that replicate in the cytoplasm to maintain mechanisms to avoid its restrictive effects. Interaction of different IFIT family members is well described, but little is known of the molecular basis of IFIT association or its impact on function. Here, we reconstituted different complexes of IFIT1, IFIT2 and IFIT3 in vitro, which enabled us to reveal critical aspects of IFIT complex assembly. IFIT1 and IFIT3 interact via a YxxxL motif present in the C-terminus of each protein. IFIT2 and IFIT3 homodimers dissociate to form a more stable heterodimer that also associates with IFIT1. We show for the first time that IFIT3 stabilizes IFIT1 protein expression, promotes IFIT1 binding to a cap0 Zika virus reporter mRNA and enhances IFIT1 translation inhibition. This work reveals molecular aspects of IFIT interaction and provides an important missing link between IFIT assembly and function.
Asunto(s)
Proteínas Portadoras/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Biosíntesis de Proteínas , Proteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Reguladoras de la Apoptosis , Proteínas Portadoras/genética , Cromatografía en Gel , Genes Reporteros , Células HEK293 , Interacciones Huésped-Patógeno/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteínas/genética , Caperuzas de ARN/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN , Virus Zika/genéticaRESUMEN
A recent outbreak of Zika virus in Brazil has led to a simultaneous increase in reports of neonatal microcephaly. Zika targets cerebral neural precursors, a cell population essential for cortical development, but the cause of this neurotropism remains obscure. Here we report that the neural RNA-binding protein Musashi-1 (MSI1) interacts with the Zika genome and enables viral replication. Zika infection disrupts the binding of MSI1 to its endogenous targets, thereby deregulating expression of factors implicated in neural stem cell function. We further show that MSI1 is highly expressed in neural progenitors of the human embryonic brain and is mutated in individuals with autosomal recessive primary microcephaly. Selective MSI1 expression in neural precursors could therefore explain the exceptional vulnerability of these cells to Zika infection.
Asunto(s)
Genoma Viral , Microcefalia/metabolismo , Microcefalia/virología , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Unión al ARN/metabolismo , Replicación Viral , Infección por el Virus Zika/virología , Virus Zika/fisiología , Animales , Encéfalo/anomalías , Encéfalo/metabolismo , Encéfalo/virología , Niño , Chlorocebus aethiops , Femenino , Células HEK293 , Humanos , Masculino , Microcefalia/genética , Mutación , Células-Madre Neurales/metabolismo , Células-Madre Neurales/fisiología , Células-Madre Neurales/virología , Células Vero , Virus Zika/genéticaRESUMEN
The eukaryotic initiation factor 4A (eIF4A) is a DEAD box helicase that unwinds RNA structure in the 5' untranslated region (UTR) of mRNAs. Here, we investigated the role of eIF4A in porcine sapovirus VPg-dependent translation. Using inhibitors and dominant-negative mutants, we found that eIF4A is required for viral translation and infectivity, suggesting that despite the presence of a very short 5' UTR, eIF4A is required to unwind RNA structure in the sapovirus genome to facilitate virus translation.
Asunto(s)
Factor 4A Eucariótico de Iniciación/metabolismo , Genoma Viral , Sapovirus/genética , Proteínas Virales/biosíntesis , Regiones no Traducidas 5' , Animales , Factor 4A Eucariótico de Iniciación/genética , Mutación , Unión Proteica , ARN Viral/metabolismo , Conejos , Reticulocitos/metabolismo , Sapovirus/fisiología , Esteroles/farmacología , Porcinos , Proteínas Virales/genética , Replicación ViralRESUMEN
Vaccinia virus (VACV) is the prototypic orthopoxvirus and the vaccine used to eradicate smallpox. Here we show that VACV strain Western Reserve protein 169 is a cytoplasmic polypeptide expressed early during infection that is excluded from virus factories and inhibits the initiation of cap-dependent and cap-independent translation. Ectopic expression of protein 169 causes the accumulation of 80S ribosomes, a reduction of polysomes, and inhibition of protein expression deriving from activation of multiple innate immune signaling pathways. A virus lacking 169 (vΔ169) replicates and spreads normally in cell culture but is more virulent than parental and revertant control viruses in intranasal and intradermal murine models of infection. Intranasal infection by vΔ169 caused increased pro-inflammatory cytokines and chemokines, infiltration of pulmonary leukocytes, and lung weight. These alterations in innate immunity resulted in a stronger CD8+ T-cell memory response and better protection against virus challenge. This work illustrates how inhibition of host protein synthesis can be a strategy for virus suppression of innate and adaptive immunity.
Asunto(s)
Inmunidad Adaptativa , Interacciones Huésped-Patógeno , Inmunidad Innata , Iniciación de la Cadena Peptídica Traduccional , Virus Vaccinia/fisiología , Vaccinia/virología , Proteínas Virales/metabolismo , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Linfocitos T CD8-positivos/virología , Línea Celular , Femenino , Eliminación de Gen , Regulación de la Expresión Génica , Humanos , Memoria Inmunológica , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Vaccinia/inmunología , Vaccinia/metabolismo , Vaccinia/patología , Virus Vaccinia/inmunología , Virus Vaccinia/patogenicidad , Proteínas Virales/genética , VirulenciaRESUMEN
The viral protease represents a key drug target for the development of antiviral therapeutics. Because many protease inhibitors mimic protease substrates, differences in substrate recognition between proteases may affect their sensitivity to a given inhibitor. Here we use a cell-based FRET sensor to investigate the activity of different norovirus proteases upon cleavage of various norovirus cleavage sites inserted into a linker region separating cyan fluorescent protein and yellow fluorescent protein. Using this system, we demonstrate that differences in substrate processing exist between proteases from human noroviruses (genogroups I (GI) and II) and the commonly used murine norovirus (MNV, genogroup V) model. These altered the cleavage efficiency of specific cleavage sites both within and between genogroups. The differences observed between these proteases may affect sensitivity to protease inhibitors and the suitability of MNV as a model system for testing such molecules against the human norovirus protease. Finally, we demonstrate that replacement of MNV polyprotein cleavage sites with the GI or GII equivalents, with the exception of the NS6-7 junction, leads to the production of infectious virus when the MNV NS6 protease, but not the GI or GII proteases, are present.
Asunto(s)
Norovirus/enzimología , Péptido Hidrolasas/metabolismo , Poliproteínas/metabolismo , Proteolisis , Proteínas no Estructurales Virales/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Línea Celular , Transferencia Resonante de Energía de Fluorescencia , Humanos , Ratones , Datos de Secuencia Molecular , Norovirus/genética , Péptido Hidrolasas/química , Péptido Hidrolasas/genética , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genéticaRESUMEN
Foot-and-mouth disease virus (FMDV) causes a highly infectious and economically devastating disease of livestock. The FMDV genome is translated as a single polypeptide precursor that is cleaved into functional proteins predominantly by the highly conserved viral 3C protease, making this enzyme an attractive target for antiviral drugs. A peptide corresponding to an optimal substrate has been modified at the C-terminus, by the addition of a warhead, to produce irreversible inhibitors that react as Michael acceptors with the enzyme active site. Further investigation highlighted key structural determinants for inhibition, with a positively charged P2 being particularly important for potency.
Asunto(s)
Antivirales/síntesis química , Antivirales/farmacología , Cisteína Endopeptidasas/química , Diseño de Fármacos , Virus de la Fiebre Aftosa/efectos de los fármacos , Virus de la Fiebre Aftosa/enzimología , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/química , Proteasas Virales 3C , Animales , Cisteína Endopeptidasas/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Virales/metabolismoRESUMEN
Picornavirus Type 1 IRESs comprise five principal domains (dII-dVI). Whereas dV binds eIF4G, a conserved AUG in dVI was suggested to stimulate attachment of 43S ribosomal preinitiation complexes, which then scan to the initiation codon. Initiation on Type 1 IRESs also requires IRES trans-acting factors (ITAFs), and several candidates have been proposed. Here, we report the in vitro reconstitution of initiation on three Type 1 IRESs: poliovirus (PV), enterovirus 71 (EV71), and bovine enterovirus (BEV). All of them require eIF2, eIF3, eIF4A, eIF4G, eIF4B, eIF1A, and a single ITAF, poly(C) binding protein 2 (PCBP2). In each instance, initiation starts with binding of eIF4G/eIF4A. Subsequent recruitment of 43S complexes strictly requires direct interaction of their eIF3 constituent with eIF4G. The following events can differ between IRESs, depending on the stability of dVI. If it is unstructured (BEV), all ribosomes scan through dVI to the initiation codon, requiring eIF1 to bypass its AUG. If it is structured (PV, EV71), most initiation events occur without inspection of dVI, implying that its AUG does not determine ribosomal attachment.
Asunto(s)
Enterovirus Humano A/fisiología , Enterovirus Bovino/fisiología , Iniciación de la Cadena Peptídica Traduccional , Poliovirus/fisiología , Codón Iniciador/metabolismo , Enterovirus Humano A/genética , Enterovirus Humano A/metabolismo , Enterovirus Bovino/genética , Enterovirus Bovino/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Poliovirus/genética , Poliovirus/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribosomas/metabolismoRESUMEN
Ribosomal recruitment of cellular mRNAs depends on binding of eIF4F to the mRNA's 5'-terminal 'cap'. The minimal 'cap0' consists of N7-methylguanosine linked to the first nucleotide via a 5'-5' triphosphate (ppp) bridge. Cap0 is further modified by 2'-O-methylation of the next two riboses, yielding 'cap1' (m7GpppNmN) and 'cap2' (m7GpppNmNm). However, some viral RNAs lack 2'-O-methylation, whereas others contain only ppp- at their 5'-end. Interferon-induced proteins with tetratricopeptide repeats (IFITs) are highly expressed effectors of innate immunity that inhibit viral replication by incompletely understood mechanisms. Here, we investigated the ability of IFIT family members to interact with cap1-, cap0- and 5'ppp- mRNAs and inhibit their translation. IFIT1 and IFIT1B showed very high affinity to cap-proximal regions of cap0-mRNAs (K1/2,app â¼9 to 23 nM). The 2'-O-methylation abrogated IFIT1/mRNA interaction, whereas IFIT1B retained the ability to bind cap1-mRNA, albeit with reduced affinity (K1/2,app â¼450 nM). The 5'-terminal regions of 5'ppp-mRNAs were recognized by IFIT5 (K1/2,app â¼400 nM). The activity of individual IFITs in inhibiting initiation on a specific mRNA was determined by their ability to interact with its 5'-terminal region: IFIT1 and IFIT1B efficiently outcompeted eIF4F and abrogated initiation on cap0-mRNAs, whereas inhibition on cap1- and 5'ppp- mRNAs by IFIT1B and IFIT5 was weaker and required higher protein concentrations.
Asunto(s)
Proteínas Portadoras/metabolismo , Iniciación de la Cadena Peptídica Traduccional , Caperuzas de ARN/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Reguladoras de la Apoptosis , Unión Competitiva , Proteínas Portadoras/química , Proteínas Portadoras/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Factor 4F Eucariótico de Iniciación/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas/genética , Proteínas/metabolismo , Caperuzas de ARN/química , ARN de Transferencia/metabolismo , Proteínas de Unión al ARN , Proteínas Recombinantes/metabolismo , Ribosomas/metabolismoRESUMEN
On most eukaryotic mRNAs, initiation codon selection involves base-by-base inspection of 5' UTRs by scanning ribosomal complexes. Although the eukaryotic initiation factors 4A/4B/4G can mediate scanning through medium-stability hairpins, scanning through more stable structures additionally requires DHX29, a member of the superfamily 2 DEAH/RNA helicase A (RHA) helicase family that binds to 40S subunits and possesses 40S-stimulated nucleoside triphosphatase (NTPase) activity. Here, sequence alignment and structural modeling indicated that DHX29 comprises a unique 534-aa-long N-terminal region (NTR), central catalytic RecA1/RecA2 domains containing a large insert in the RecA2 domain, and the C-terminal part, which includes winged-helix, ratchet, and oligonucleotide/oligosaccharide-binding (OB) domains that are characteristic of DEAH/RHA helicases. Functional characterization revealed that specific ribosomal targeting is required for DHX29's activity in initiation and is determined by elements that map to the NTR and to the N-terminal half of the winged-helix domain. The ribosome-binding determinant located in the NTR was identified as a putative double-stranded RNA-binding domain. Mutational analyses of RecA1/RecA2 domains confirmed the essential role of NTP hydrolysis for DHX29's function in initiation and validated the significance of a ß-hairpin protruding from RecA2. The large RecA2 insert played an autoinhibitory role in suppressing DHX29's intrinsic NTPase activity but was not essential for its 40S-stimulated NTPase activity and function in initiation. Deletion of the OB domain also increased DHX29's basal NTPase activity, but more importantly, abrogated the responsiveness of the NTPase activity to stimulation, which abolished DHX29's function in initiation. This finding suggests that the OB domain, which is specific for DEAH/RHA helicases, plays an important role in their NTPase cycle.
Asunto(s)
Biosíntesis de Proteínas/fisiología , ARN Helicasas/química , Regiones no Traducidas 5'/fisiología , Sustitución de Aminoácidos , Humanos , Mutación Missense , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , ARN Helicasas/genética , ARN Helicasas/metabolismo , Alineación de Secuencia , Homología de Secuencia , Relación Estructura-ActividadRESUMEN
The 5'-untranslated regions (5' UTRs) of picornavirus genomes contain an internal ribosomal entry site (IRES) that promotes the end-independent initiation of translation. Picornavirus IRESs are classified into four structurally distinct groups, each with different initiation factor requirements. Here, we identify a fifth IRES class in members of Kobuvirus, Salivirus, and Paraturdivirus genera of Picornaviridae: Aichi virus (AV), bovine kobuvirus (BKV), canine kobuvirus (CKoV), mouse kobuvirus (MKoV), sheep kobuvirus (SKV), salivirus A (SV-A), turdivirus 2 (TV2), and TV3. The 410-nucleotide (nt)-long AV IRES comprises four domains (I to L), including a hairpin (L) that overlaps a Yn-Xm-AUG (pyrimidine tract/spacer/initiation codon) motif. SV-A, CKoV, and MKoV also contain these four domains, whereas BKV, SKV, and TV2/TV3 5' UTRs contain domains that are related to domain I and equivalent to domains J and K but lack an AV-like domain L. These IRESs are located at different relative positions between a conserved 5'-terminal origin of replication and divergent coding sequences. Elements in these IRESs also occur elsewhere: domain J's apical subdomain, which contains a GNRA tetraloop, matches an element in type 1 IRESs, and eIF4G-binding motifs in domain K and in type 2 IRESs are identical. Other elements are unique, and their presence leads to unique initiation factor requirements. In vitro reconstitution experiments showed that like AV, but in contrast to other currently characterized IRESs, SV-A requires the DExH-box protein DHX29 during initiation, which likely ensures that the initiation codon sequestered in domain L is properly accommodated in the ribosomal mRNA-binding cleft.
Asunto(s)
Picornaviridae/fisiología , Ribosomas/metabolismo , Regiones no Traducidas 5' , Animales , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Picornaviridae/genética , Biosíntesis de Proteínas , ARN Viral/química , ARN Viral/genéticaRESUMEN
Picornavirus mRNAs contain IRESs that sustain their translation during infection, when host protein synthesis is shut off. The major classes of picornavirus IRESs (Types 1 and 2) have distinct structures and sequences, but initiation on both is determined by their specific interaction with eIF4G. We report here that Aichivirus (AV), a member of the Kobuvirus genus of Picornaviridae, contains an IRES that differs structurally from Type 1 and Type 2 IRESs. Its function similarly involves interaction with eIF4G, but its eIF4G-interacting domain is structurally distinct, although it contains an apical eIF4G-interacting motif similar to that in Type 2 IRESs. Like Type 1 and Type 2 IRESs, AV IRES function is enhanced by pyrimidine tract-binding protein (PTB), but the pattern of PTB's interaction with each of these IRESs is distinct. Unlike all known IRESs, the AV IRES is absolutely dependent on DHX29, a requirement imposed by sequestration of its initiation codon in a stable hairpin.
Asunto(s)
Kobuvirus/genética , Iniciación de la Cadena Peptídica Traduccional/genética , Picornaviridae/genética , ARN Viral/genética , Elementos Reguladores de la Transcripción/fisiología , Regiones no Traducidas 5'/genética , Secuencia de Bases , Sitios de Unión/genética , Regulación Viral de la Expresión Génica , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Picornaviridae/metabolismo , Biosíntesis de Proteínas/genética , Biosíntesis de Proteínas/fisiología , ARN Viral/química , ARN Viral/metabolismo , Elementos Reguladores de la Transcripción/genética , Ribosomas/metabolismoRESUMEN
Foot-and-mouth disease virus (FMDV), a positive sense, single-stranded RNA virus, causes a highly contagious disease in cloven-hoofed livestock. Like other picornaviruses, FMDV has a conserved 2C protein assigned to the superfamily 3 helicases a group of AAA+ ATPases that has a predicted N-terminal membrane-binding amphipathic helix attached to the main ATPase domain. In infected cells, 2C is involved in the formation of membrane vesicles, where it co-localizes with viral RNA replication complexes, but its precise role in virus replication has not been elucidated. We show here that deletion of the predicted N-terminal amphipathic helix enables overexpression in Escherichia coli of a highly soluble truncated protein, 2C(34-318), that has ATPase and RNA binding activity. ATPase activity was abrogated by point mutations in the Walker A (K116A) and B (D160A) motifs and Motif C (N207A) in the active site. Unliganded 2C(34-318) exhibits concentration-dependent self-association to yield oligomeric forms, the largest of which is tetrameric. Strikingly, in the presence of ATP and RNA, FMDV 2C(34-318) containing the N207A mutation, which binds but does not hydrolyze ATP, was found to oligomerize specifically into hexamers. Visualization of FMDV 2C-ATP-RNA complexes by negative stain electron microscopy revealed hexameric ring structures with 6-fold symmetry that are characteristic of AAA+ ATPases. ATPase assays performed by mixing purified active and inactive 2C(34-318) subunits revealed a coordinated mechanism of ATP hydrolysis. Our results provide new insights into the structure and mechanism of picornavirus 2C proteins that will facilitate new investigations of their roles in infection.