RESUMEN
BACKGROUND: RNA helicases are emerging as key factors regulating host-virus interactions. The DEAD-box ATP-dependent RNA helicase DDX5, which plays an important role in many aspects of cellular RNA biology, was also found to either promote or inhibit viral replication upon infection with several RNA viruses. Here, our aim is to examine the impact of DDX5 on Sindbis virus (SINV) infection. METHODS: We analysed the interaction between DDX5 and the viral RNA using imaging and RNA-immunoprecipitation approaches. The interactome of DDX5 in mock- and SINV-infected cells was determined by mass spectrometry. We validated the interaction between DDX17 and the viral capsid by co- immunoprecipitation in the presence or absence of an RNase treatment. We determined the subcellular localization of DDX5, its cofactor DDX17 and the viral capsid protein by co-immunofluorescence. Finally, we investigated the impact of DDX5 depletion and overexpression on SINV infection at the viral protein, RNA and infectious particle accumulation level. The contribution of DDX17 was also tested by knockdown experiments. RESULTS: In this study we demonstrate that DDX5 interacts with the SINV RNA during infection. Furthermore, the proteomic analysis of the DDX5 interactome in mock and SINV-infected HCT116 cells identified new cellular and viral partners and confirmed the interaction between DDX5 and DDX17. Both DDX5 and DDX17 re-localize from the nucleus to the cytoplasm upon SINV infection and interact with the viral capsid protein. We also show that DDX5 depletion negatively impacts the viral replication cycle, while its overexpression has a pro-viral effect. Finally, we observed that DDX17 depletion reduces SINV infection, an effect which is even more pronounced in a DDX5-depleted background, suggesting a synergistic pro-viral effect of the DDX5 and DDX17 proteins on SINV. CONCLUSIONS: These results not only shed light on DDX5 as a novel and important host factor to the SINV life cycle, but also expand our understanding of the roles played by DDX5 and DDX17 as regulators of viral infections.
Asunto(s)
Infecciones por Alphavirus , Proteínas de la Cápside , Humanos , Proteómica , Replicación Viral , ARN , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Virus Sindbis/metabolismoRESUMEN
Viruses are obligate intracellular parasites, which depend on the host cellular machineries to replicate their genome and complete their infectious cycle. Long double-stranded (ds)RNA is a common viral by-product originating during RNA virus replication and is universally sensed as a danger signal to trigger the antiviral response. As a result, viruses hide dsRNA intermediates into viral replication factories and have evolved strategies to hijack cellular proteins for their benefit. The characterization of the host factors associated with viral dsRNA and involved in viral replication remains a major challenge to develop new antiviral drugs against RNA viruses. Here, we performed anti-dsRNA immunoprecipitation followed by mass spectrometry analysis to fully characterize the dsRNA interactome in Sindbis virus (SINV) infected human cells. Among the identified proteins, we characterized SFPQ (splicing factor, proline-glutamine rich) as a new dsRNA-associated proviral factor upon SINV infection. We showed that SFPQ depletion reduces SINV infection in human HCT116 and SK-N-BE(2) cells, suggesting that SFPQ enhances viral production. We demonstrated that the cytoplasmic fraction of SFPQ partially colocalizes with dsRNA upon SINV infection. In agreement, we proved by RNA-IP that SFPQ can bind dsRNA and viral RNA. Furthermore, we showed that overexpression of a wild-type, but not an RNA binding mutant SFPQ, increased viral infection, suggesting that RNA binding is essential for its positive effect on the virus. Overall, this study provides the community with a compendium of dsRNA-associated factors during viral infection and identifies SFPQ as a new proviral dsRNA binding protein.
Asunto(s)
Virus ARN , ARN Bicatenario , Humanos , ARN Bicatenario/genética , Proteómica , Virus Sindbis/genética , Virus Sindbis/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Virus ARN/genética , Replicación Viral/genéticaRESUMEN
Many viruses encode ion channel proteins that oligomerize to form hydrophilic pores in membranes of virus-infected cells and the viral membrane in some enveloped viruses. Alphavirus 6K, human immunodeficiency virus type 1 Vpu (HIV-Vpu), influenza A virus M2 (IAV-M2), and hepatitis C virus P7 (HCV-P7) are transmembrane ion channel proteins that play essential roles in virus assembly, budding, and entry. While the oligomeric structures and mechanisms of ion channel activity are well-established for M2 and P7, these remain unknown for 6K. Here we investigated the functional role of the ion channel activity of 6K in alphavirus assembly by utilizing a series of Sindbis virus (SINV) ion channel chimeras expressing the ion channel helix from Vpu or M2 or substituting the entire 6K protein with full-length P7, in cis. We demonstrate that the Vpu helix efficiently complements 6K, whereas M2 and P7 are less efficient. Our results indicate that while SINV is primarily insensitive to the M2 ion channel inhibitor amantadine, the Vpu inhibitor 5-N, N-Hexamethylene amiloride (HMA), significantly reduces SINV release, suggesting that the ion channel activity of 6K similar to Vpu, promotes virus budding. Using live-cell imaging of SINV with a miniSOG-tagged 6K and mCherry-tagged E2, we further demonstrate that 6K and E2 colocalize with the Golgi apparatus in the secretory pathway. To contextualize the localization of 6K in the Golgi, we analyzed cells infected with SINV and SINV-ion channel chimeras using transmission electron microscopy. Our results provide evidence for the first time for the functional role of 6K in type II cytopathic vacuoles (CPV-II) formation. We demonstrate that in the absence of 6K, CPV-II, which originates from the Golgi apparatus, is not detected in infected cells, with a concomitant reduction in the glycoprotein transport to the plasma membrane. Substituting a functional ion channel, M2 or Vpu localizing to Golgi, restores CPV-II production, whereas P7, retained in the ER, is inadequate to induce CPV-II formation. Altogether our results indicate that ion channel activity of 6K is required for the formation of CPV-II from the Golgi apparatus, promoting glycoprotein spike transport to the plasma membrane and efficient virus budding.
Asunto(s)
Virus Sindbis , Liberación del Virus , Amantadina/farmacología , Glicoproteínas/metabolismo , Humanos , Canales Iónicos/genética , Canales Iónicos/metabolismo , Virus Sindbis/genética , Virus Sindbis/metabolismoRESUMEN
The innate immune response controls the acute phase of virus infections; critical to this response is the induction of type I interferon (IFN) and resultant IFN-stimulated genes to establish an antiviral environment. One such gene, zinc finger antiviral protein (ZAP), is a potent antiviral factor that inhibits replication of diverse RNA and DNA viruses by binding preferentially to CpG-rich viral RNA. ZAP restricts alphaviruses and the flavivirus Japanese encephalitis virus (JEV) by inhibiting translation of their positive-sense RNA genomes. While ZAP residues important for RNA binding and CpG specificity have been identified by recent structural studies, their role in viral translation inhibition has yet to be characterized. Additionally, the ubiquitin E3 ligase tripartite motif-containing protein 25 (TRIM25) has recently been uncovered as a critical co-factor for ZAP's suppression of alphavirus translation. While TRIM25 RNA binding is required for efficient TRIM25 ligase activity, its importance in the context of ZAP translation inhibition remains unclear. Here, we characterized the effects of ZAP and TRIM25 RNA binding on translation inhibition in the context of the prototype alphavirus Sindbis virus (SINV) and JEV. To do so, we generated a series of ZAP and TRIM25 RNA binding mutants, characterized loss of their binding to SINV genomic RNA, and assessed their ability to interact with each other and to suppress SINV replication, SINV translation, and JEV translation. We found that mutations compromising general RNA binding of ZAP and TRIM25 impact their ability to restrict SINV replication, but mutations specifically targeting ZAP CpG-mediated RNA binding have a greater effect on SINV and JEV translation inhibition. Interestingly, ZAP-TRIM25 interaction is a critical determinant of JEV translation inhibition. Taken together, these findings illuminate the contribution of RNA binding and co-factor interaction to the synergistic inhibition of viral translation by ZAP and TRIM25.
Asunto(s)
Virus de la Encefalitis Japonesa (Especie) , Proteínas de Unión al ARN , Antivirales/farmacología , Virus de la Encefalitis Japonesa (Especie)/genética , ARN Viral/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Virus Sindbis/genética , Virus Sindbis/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/farmacología , Replicación ViralRESUMEN
Arboviruses are transmitted by specific vectors, and the reasons for this specificity are not fully understood. One contributing factor is the existence of tissue barriers within the vector such as the midgut escape barrier. We used microRNA (miRNA) targeting of Sindbis virus (SINV) to study how replication in midgut cells contributes to overcoming this barrier in the mosquito Aedes aegypti. SINV constructs were designed to be attenuated specifically in midgut cells by inserting binding sites for midgut-specific miRNAs into either the 3' untranslated region (MRE3'miRT) or the structural open reading frame (MRE-ORFmiRT) of the SINV genome. Both miRNA-targeted viruses replicated less efficiently than control viruses in the presence of these miRNAs. When mosquitoes were given infectious blood meals containing miRNA-targeted viruses, only around 20% (MRE3'miRT) or 40% (MRE-ORFmiRT) of mosquitoes developed disseminated infection. In contrast, dissemination occurred in almost all mosquitoes fed control viruses. Deep sequencing of virus populations from individual mosquitoes ruled out selection for mutations in the inserted target sequences as the cause for dissemination in these mosquitoes. In mosquitoes that became infected with miRNA-targeted viruses, titers were equivalent to those of mosquitoes infected with control virus in both the midgut and the carcass, and there was no evidence of a threshold titer necessary for dissemination. Instead, it appeared that if infection was successfully established in the midgut, replication and dissemination were largely normal. Our results support the hypothesis that replication is an important factor in allowing SINV to overcome the midgut escape barrier but hint that other factors are also likely involved. IMPORTANCE When a mosquito ingests an arbovirus during a blood meal, the arbovirus must escape from the midgut of the vector and infect the salivary glands in order to be transmitted to a new host. We used tissue-specific miRNA targeting to examine the requirement for Sindbis virus (SINV) to replicate in midgut epithelium in order to cause disseminated infection in the mosquito Aedes aegypti. Our results indicate that specifically reducing the ability of SINV to replicate in the mosquito midgut reduces its overall ability to establish infection in the mosquito, but if infection is established, replication and dissemination occur normally. These results are consistent with an importance for replication in the midgut epithelium in aiding arboviruses in crossing the midgut barrier.
Asunto(s)
Aedes/virología , Tracto Gastrointestinal/virología , MicroARNs/genética , Virus Sindbis/crecimiento & desarrollo , Replicación Viral/genética , Animales , Línea Celular , Cricetinae , Mosquitos Vectores/virología , Especificidad de Órganos , Glándulas Salivales/virología , Virus Sindbis/genética , Virus Sindbis/metabolismoRESUMEN
Viruses maximize their genetic coding capacity through a variety of biochemical mechanisms, including programmed ribosomal frameshifting (PRF), which facilitates the production of multiple proteins from a single mRNA transcript. PRF is typically stimulated by structural elements within the mRNA that generate mechanical tension between the transcript and ribosome. However, in this work, we show that the forces generated by the cotranslational folding of the nascent polypeptide chain can also enhance PRF. Using an array of biochemical, cellular, and computational techniques, we first demonstrate that the Sindbis virus structural polyprotein forms two competing topological isomers during its biosynthesis at the ribosome-translocon complex. We then show that the formation of one of these topological isomers is linked to PRF. Coarse-grained molecular dynamics simulations reveal that the translocon-mediated membrane integration of a transmembrane domain upstream from the ribosomal slip site generates a force on the nascent polypeptide chain that scales with observed frameshifting. Together, our results indicate that cotranslational folding of this viral protein generates a tension that stimulates PRF. To our knowledge, this constitutes the first example in which the conformational state of the nascent polypeptide chain has been linked to PRF. These findings raise the possibility that, in addition to RNA-mediated translational recoding, a variety of cotranslational folding or binding events may also stimulate PRF.
Asunto(s)
Alphavirus/clasificación , Sistema de Lectura Ribosómico , Poliproteínas/biosíntesis , Biosíntesis de Proteínas , Pliegue de Proteína , Virus Sindbis/metabolismo , Proteínas Virales/biosíntesis , Alphavirus/química , Células HEK293 , Humanos , Virus Sindbis/genéticaRESUMEN
Sindbis virus (SINV) produces the small membrane protein TF from the 6K gene via a (-1) programmed ribosomal frameshifting. While several groups have shown that TF-deficient virus exhibits reduced virulence, the mechanism(s) by which this occurs remain unknown. Here, we demonstrate a role for TF in antagonizing the host interferon response. Using wild-type and type 1 interferon receptor-deficient mice and primary cells derived from these animals, we show that TF controls the induction of the host interferon response at early times during infection. Loss of TF production leads to elevated interferon and a concurrent reduction in viral loads with a loss of pathogenicity. Palmitoylation of TF has been shown to be important for particle assembly and morphology. We find that palmitoylation of TF also contributes to the ability of TF to antagonize host interferon responses as dysregulated palmitoylation of TF reduces virulence in a manner similar to loss of TF.
Asunto(s)
Interferón Tipo I/biosíntesis , Virus Sindbis/inmunología , Virus Sindbis/metabolismo , Proteínas Virales/inmunología , Proteínas Virales/metabolismo , Infecciones por Alphavirus/genética , Infecciones por Alphavirus/inmunología , Infecciones por Alphavirus/virología , Animales , Femenino , Genes Virales , Interacciones Microbiota-Huesped/genética , Interacciones Microbiota-Huesped/inmunología , Interacciones Microbiota-Huesped/fisiología , Lipoilación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Receptor de Interferón alfa y beta/deficiencia , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/inmunología , Virus Sindbis/genética , Proteínas Virales/genética , Virulencia/genética , Virulencia/inmunología , Virulencia/fisiologíaRESUMEN
Sindbis virus particles contain the viral proteins capsid, E1 and E2, and low levels of a small membrane protein called TF. TF is produced during a (-1) programmed ribosomal frameshifting event during the translation of the structural polyprotein. TF from Sindbis virus-infected cells is present in two palmitoylated states, basal and maximal; unpalmitoylated TF is not detectable. Mutagenesis studies demonstrated that without palmitoylation, TF is not incorporated into released virions, suggesting palmitoylation of TF is a regulated step in virus assembly. In this work, we identified Domains within the TF protein that regulate its palmitoylation state. Mutations and insertions in Domain III, a region proposed to be in the cytoplasmic loop of TF, increase levels of unpalmitoylated TF found during an infection but still unpalmitoylated TF was not incorporated into virions. Mutations in Domain IV, the TF unique region, are likely to impact the balance between basal and maximal palmitoylation.
Asunto(s)
Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Virus Sindbis/metabolismo , Proteínas de la Cápside/genética , Lipoilación , Mutagénesis , Dominios Proteicos , Virus Sindbis/química , Virus Sindbis/genética , Virión/química , Virión/genética , Virión/metabolismo , Replicación ViralRESUMEN
The compendium of RNA-binding proteins (RBPs) has been greatly expanded by the development of RNA-interactome capture (RIC). However, it remained unknown if the complement of RBPs changes in response to environmental perturbations and whether these rearrangements are important. To answer these questions, we developed "comparative RIC" and applied it to cells challenged with an RNA virus called sindbis (SINV). Over 200 RBPs display differential interaction with RNA upon SINV infection. These alterations are mainly driven by the loss of cellular mRNAs and the emergence of viral RNA. RBPs stimulated by the infection redistribute to viral replication factories and regulate the capacity of the virus to infect. For example, ablation of XRN1 causes cells to be refractory to SINV, while GEMIN5 moonlights as a regulator of SINV gene expression. In summary, RNA availability controls RBP localization and function in SINV-infected cells.
Asunto(s)
Células Epiteliales/virología , Perfilación de la Expresión Génica/métodos , ARN Viral/genética , Proteínas de Unión al ARN/genética , Virus Sindbis/genética , Transcriptoma , Neoplasias del Cuello Uterino/virología , Regiones no Traducidas 5' , Sitios de Unión , Células Epiteliales/metabolismo , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Femenino , Regulación Viral de la Expresión Génica , Células HEK293 , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Unión Proteica , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/genética , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Proteínas del Complejo SMN , Virus Sindbis/crecimiento & desarrollo , Virus Sindbis/metabolismo , Virus Sindbis/patogenicidad , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/metabolismo , Replicación ViralRESUMEN
Noncanonical translation, and particularly initiation on non-AUG codons, are frequently used by viral and cellular mRNAs during virus infection and disease. The Sindbis virus (SINV) subgenomic mRNA (sgRNA) constitutes a unique model system to analyze the translation of a capped viral mRNA without the participation of several initiation factors. Moreover, sgRNA can initiate translation even when the AUG initiation codon is replaced by other codons. Using SINV replicons, we examined the efficacy of different codons in place of AUG to direct the synthesis of the SINV capsid protein. The substitution of AUG by CUG was particularly efficient in promoting the incorporation of leucine or methionine in similar percentages at the amino terminus of the capsid protein. Additionally, valine could initiate translation when the AUG is replaced by GUG. The ability of sgRNA to initiate translation on non-AUG codons was dependent on the integrity of a downstream stable hairpin (DSH) structure located in the coding region. The structural requirements of this hairpin to signal the initiation site on the sgRNA were examined in detail. Of interest, a virus bearing CUG in place of AUG in the sgRNA was able to infect cells and synthesize significant amounts of capsid protein. This virus infects the human haploid cell line HAP1 and the double knockout variant that lacks eIF2A and eIF2D. Collectively, these findings indicate that leucine-tRNA or valine-tRNA can participate in the initiation of translation of sgRNA by a mechanism dependent on the DSH. This mechanism does not involve the action of eIF2, eIF2A, or eIF2D.
Asunto(s)
Codón Iniciador/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Viral/genética , Transducción de Señal/genética , Virus Sindbis/genética , Proteínas de la Cápside/biosíntesis , Proteínas de la Cápside/genética , Línea Celular Tumoral , Codón Iniciador/metabolismo , Factor 2 Eucariótico de Iniciación/deficiencia , Factor 2 Eucariótico de Iniciación/genética , Fibroblastos/metabolismo , Fibroblastos/virología , Regulación de la Expresión Génica , Haploidia , Interacciones Huésped-Patógeno/genética , Humanos , Secuencias Invertidas Repetidas , Leucina/genética , Leucina/metabolismo , Metionina/genética , Metionina/metabolismo , Conformación de Ácido Nucleico , ARN Mensajero/metabolismo , ARN de Transferencia de Leucina/genética , ARN de Transferencia de Leucina/metabolismo , ARN de Transferencia de Valina/genética , ARN de Transferencia de Valina/metabolismo , ARN Viral/metabolismo , Replicón , Virus Sindbis/metabolismo , Valina/genética , Valina/metabolismoRESUMEN
Recent advances in biological research reveal that the majority of the experiments strive for comprehensive exploration of the biological system rather than targeting specific biological entities. The qualitative and quantitative findings of the investigations are often exclusively available in the form of figures in published papers. There is no denying that such findings have been instrumental in intensive understanding of biological processes and pathways. However, data as such is unacknowledged by machines as the descriptions in the figure captions comprise of sumptuous information in an ambiguous manner. The abbreviated term 'SIN' exemplifies such issue as it may stand for Sindbis virus or the sex-lethal interactor gene (Drosophila melanogaster). To overcome this ambiguity, entities should be identified by linking them to the respective entries in notable biological databases. Among all entity types, the task of identifying species plays a pivotal role in disambiguating related entities in the text. In this study, we present our species identification tool SPRENO (Species Recognition and Normalization), which is established for recognizing organism terms mentioned in figure captions and linking them to the NCBI taxonomy database by exploiting the contextual information from both the figure caption and the corresponding full text. To determine the ID of ambiguous organism mentions, two disambiguation methods have been developed. One is based on the majority rule to select the ID that has been successfully linked to previously mentioned organism terms. The other is a convolutional neural network (CNN) model trained by learning both the context and the distance information of the target organism mention. As a system based on the majority rule, SPRENO was one of the top-ranked systems in the BioCreative VI BioID track and achieved micro F-scores of 0.776 (entity recognition) and 0.755 (entity normalization) on the official test set, respectively. Additionally, the SPRENO-CNN exhibited better precisions with lower recalls and F-scores (0.720/0.711 for entity recognition/normalization). SPRENO is freely available at https://bigodatamining.github.io/software/201801/.Database URL: https://bigodatamining.github.io/software/201801/.
Asunto(s)
Clasificación , Minería de Datos/métodos , Bases de Datos Factuales , Redes Neurales de la Computación , Animales , Drosophila melanogaster , Genes de Insecto , Genes Virales , Virus Sindbis/genética , Virus Sindbis/metabolismoRESUMEN
Mosquitoes are hematophagous insects that carry-on and transmit many human viruses. However, little information is available regarding the common mechanisms underlying the infection of mosquitoes by these viruses. In this study, we reveal that the hematophagous nature of mosquitoes contributes to arboviral infection after a blood meal, which suppresses antiviral innate immunity by activating the GABAergic pathway. dsRNA-mediated interruption of the GABA signaling and blockage of the GABAA receptor by the specific inhibitors both significantly impaired arbovirus replication. Consistently, inoculation of GABA enhanced arboviral infection, indicating that GABA signaling facilitates the arboviral infection of mosquitoes. The ingestion of blood by mosquitoes resulted in robust GABA production from glutamic acid derived from blood protein digestion. The oral introduction of glutamic acid increased virus acquisition by mosquitoes via activation of the GABAergic system. Our study reveals that blood meals enhance arbovirus replication in mosquitoes through activation of the GABAergic system.
Asunto(s)
Aedes/inmunología , Arbovirus/metabolismo , Sangre/inmunología , Culex/inmunología , Inmunidad Innata/inmunología , Replicación Viral/inmunología , Ácido gamma-Aminobutírico/inmunología , Animales , Virus Bunyamwera/metabolismo , Virus del Dengue/metabolismo , Virus de la Encefalitis de California/metabolismo , Virus de la Encefalitis Japonesa (Especie)/metabolismo , Antagonistas de Receptores de GABA-A/farmacología , Humanos , Mosquitos Vectores/inmunología , ARN Bicatenario/metabolismo , Receptores de GABA-A/metabolismo , Virus de los Bosques Semliki/metabolismo , Transducción de Señal , Virus Sindbis/metabolismo , Replicación Viral/efectos de los fármacos , Replicación Viral/fisiología , Ácido gamma-Aminobutírico/metabolismo , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Alphaviruses are arthropod-borne viruses that represent a significant threat to public health at a global level. While the formation of alphaviral nucleocapsid cores, consisting of cargo nucleic acid and the viral capsid protein, is an essential molecular process of infection, the precise interactions between the two partners are ill-defined. A CLIP-seq approach was used to screen for candidate sites of interaction between the viral Capsid protein and genomic RNA of Sindbis virus (SINV), a model alphavirus. The data presented in this report indicates that the SINV capsid protein binds to specific viral RNA sequences in the cytoplasm of infected cells, but its interaction with genomic RNA in mature extracellular viral particles is largely non-specific in terms of nucleotide sequence. Mutational analyses of the cytoplasmic viral RNA-capsid interaction sites revealed a functional role for capsid binding early in infection. Interaction site mutants exhibited decreased viral growth kinetics; however, this defect was not a function of decreased particle production. Rather mutation of the cytoplasmic capsid-RNA interaction sites negatively affected the functional capacity of the incoming viral genomic RNAs leading to decreased infectivity. Furthermore, cytoplasmic capsid interaction site mutants are attenuated in a murine model of neurotropic alphavirus infection. Collectively, the findings of this study indicate that the identified cytoplasmic interactions of the viral capsid protein and genomic RNA, while not essential for particle formation, are necessary for genomic RNA function early during infection. This previously unappreciated role of capsid protein during the alphaviral replication cycle also constitutes a novel virulence determinant.
Asunto(s)
Proteínas de la Cápside/metabolismo , ARN Viral/metabolismo , Virus Sindbis/metabolismo , Animales , Cápside/metabolismo , Citoplasma/metabolismo , Genoma Viral/genética , Virus Sindbis/genética , Virus Sindbis/patogenicidad , Proteínas del Envoltorio Viral/metabolismo , Virión/metabolismo , Virulencia/fisiología , Ensamble de Virus/fisiologíaRESUMEN
The function of a neural circuit is determined by the details of its synaptic connections. At present, the only available method for determining a neural wiring diagram with single synapse precision-a 'connectome'-is based on imaging methods that are slow, labor-intensive and expensive. Here, we present SYNseq, a method for converting the connectome into a form that can exploit the speed and low cost of modern high-throughput DNA sequencing. In SYNseq, each neuron is labeled with a unique random nucleotide sequence-an RNA 'barcode'-which is targeted to the synapse using engineered proteins. Barcodes in pre- and postsynaptic neurons are then associated through protein-protein crosslinking across the synapse, extracted from the tissue, and joined into a form suitable for sequencing. Although our failure to develop an efficient barcode joining scheme precludes the widespread application of this approach, we expect that with further development SYNseq will enable tracing of complex circuits at high speed and low cost.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/genética , Conectoma/métodos , Hipocampo/metabolismo , Moléculas de Adhesión de Célula Nerviosa/genética , Neuronas/metabolismo , ARN/genética , Animales , Proteínas de Unión al Calcio , Moléculas de Adhesión Celular Neuronal/metabolismo , Embrión de Mamíferos , Regulación de la Expresión Génica , Genes Reporteros , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Hipocampo/citología , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuronas/citología , Plásmidos/química , Plásmidos/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Cultivo Primario de Células , ARN/metabolismo , Virus Sindbis/genética , Virus Sindbis/metabolismo , Sinapsis/metabolismo , Transmisión Sináptica , Transfección , Proteína Fluorescente RojaRESUMEN
Introduction of selectivity/specificity into viral-based gene delivery systems, such as lentiviral vectors (LVs), is crucial in their systemic administration for cancer gene therapy. The pivotal role of tumor-associated endothelial cells (TAECs) in tumor angiogenesis and overexpression of vascular endothelial growth factor receptor-2 (VEGFR2 or KDR) in TAECs makes them a potent target in cancer treatment. Herein, we report the development of VEGFR2-targeted LVs pseudotyped with chimeric sindbis virus E2 glycoprotein (cSVE2s). For this purpose, either sequence of a VEGFR2-specific nanobody or its natural ligand (VEGF121) was inserted into the binding site of sindbis virus E2 glycoprotein. In silico modeling data suggested that the inserted targeting motifs were exposed in the context of cSVE2s. Western blot analysis of LVs indicated the incorporation of cSVE2s into viral particles. Capture ELISA demonstrated the specificity/functionality of the incorporated cSVE2s. Transduction of 293/KDR (expressing VEGFR2) or 293T cells (negative control) by constructed LVs followed by fluorescent microscopy and flow cytometric analyses indicated selective transduction of 293/KDR cells (30 %) by both targeting motifs compared to 293T control cells (1-2 %). These results implied similar targeting properties of VEGFR2-specific nanobody compared to the VEGF121 and indicated the potential for transductional targeting of tumor vasculature by the nanobody displaying LVs.
Asunto(s)
Lentivirus/genética , Virus Sindbis/metabolismo , Anticuerpos de Dominio Único/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/inmunología , Proteínas del Envoltorio Viral/genética , Simulación por Computador , Marcación de Gen , Vectores Genéticos , Células HEK293 , Humanos , Modelos Moleculares , Virus Sindbis/genética , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/metabolismo , Transducción Genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Previously, we reported that salicylate-based analogs of bryostatin protect cells from chikungunya virus (CHIKV)-induced cell death. Interestingly, 'capping' the hydroxyl group at C26 of a lead bryostatin analog, a position known to be crucial for binding to and modulation of protein kinase C (PKC), did not abrogate the anti-CHIKV activity of the scaffold, putatively indicating the involvement of a pathway independent of PKC. The work detailed in this study demonstrates that salicylate-derived analog 1 and two capped analogs (2 and 3) are not merely cytoprotective compounds, but act as selective and specific inhibitors of CHIKV replication. Further, a detailed comparative analysis of the effect of the non-capped versus the two capped analogs revealed that compound 1 acts both at early and late stages in the chikungunya virus replication cycle, while the capped analogs only interfere with a later stage process. Co-dosing with the PKC inhibitors sotrastaurin and Gö6976 counteracts the antiviral activity of compound 1 without affecting that of capped analogs 2 and 3, providing further evidence that the latter elicit their anti-CHIKV activity independently of PKC. Remarkably, treatment of CHIKV-infected cells with a combination of compound 1 and a capped analog resulted in a pronounced synergistic antiviral effect. Thus, these salicylate-based bryostatin analogs can inhibit CHIKV replication through a novel, yet still elusive, non-PKC dependent pathway.
Asunto(s)
Antivirales/farmacología , Brioestatinas/farmacología , Virus Chikungunya/efectos de los fármacos , Diseño de Fármacos , Proteína Quinasa C/metabolismo , Proteínas Virales/metabolismo , Acetilación , Animales , Antivirales/agonistas , Antivirales/antagonistas & inhibidores , Antivirales/química , Brioestatinas/agonistas , Brioestatinas/antagonistas & inhibidores , Brioestatinas/química , Carbazoles/química , Carbazoles/farmacología , Línea Celular , Virus Chikungunya/crecimiento & desarrollo , Virus Chikungunya/metabolismo , Chlorocebus aethiops , Sinergismo Farmacológico , Regulación Viral de la Expresión Génica/efectos de los fármacos , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Metilación , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/química , Proteína Quinasa C/genética , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Pirroles/química , Pirroles/farmacología , Quinazolinas/química , Quinazolinas/farmacología , Virus de los Bosques Semliki/efectos de los fármacos , Virus de los Bosques Semliki/crecimiento & desarrollo , Virus de los Bosques Semliki/metabolismo , Virus Sindbis/efectos de los fármacos , Virus Sindbis/crecimiento & desarrollo , Virus Sindbis/metabolismo , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/química , Proteínas Virales/genética , Replicación Viral/efectos de los fármacosRESUMEN
Fanconi anemia (FA) pathway genes are important tumor suppressors whose best-characterized function is repair of damaged nuclear DNA. Here, we describe an essential role for FA genes in two forms of selective autophagy. Genetic deletion of Fancc blocks the autophagic clearance of viruses (virophagy) and increases susceptibility to lethal viral encephalitis. Fanconi anemia complementation group C (FANCC) protein interacts with Parkin, is required in vitro and in vivo for clearance of damaged mitochondria, and decreases mitochondrial reactive oxygen species (ROS) production and inflammasome activation. The mitophagy function of FANCC is genetically distinct from its role in genomic DNA damage repair. Moreover, additional genes in the FA pathway, including FANCA, FANCF, FANCL, FANCD2, BRCA1, and BRCA2, are required for mitophagy. Thus, members of the FA pathway represent a previously undescribed class of selective autophagy genes that function in immunity and organellar homeostasis. These findings have implications for understanding the pathogenesis of FA and cancers associated with mutations in FA genes.
Asunto(s)
Proteína del Grupo de Complementación C de la Anemia de Fanconi/metabolismo , Animales , Autofagia , Embrión de Mamíferos/citología , Proteína del Grupo de Complementación C de la Anemia de Fanconi/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Fibroblastos/metabolismo , Células HeLa , Herpesvirus Humano 1/metabolismo , Humanos , Inflamasomas/metabolismo , Ratones , Mitofagia , Especies Reactivas de Oxígeno/metabolismo , Virus Sindbis/metabolismoRESUMEN
Flaviviruses are 5' capped positive-stranded RNA viruses that replicate their genomes within endoplasmic reticulum-derived vesicles. Flaviviruses are well known to induce oxidative stress late in infection but it is unknown if oxidative stress plays a positive role in the viral RNA replication cycle. We therefore examined how oxidation affects flavivirus RNA replication. We found that antioxidant treatment reduced virus production, reduced the viral positive-to-negative strand RNA ratio, and resulted in the accumulation of uncapped positive-sense viral RNAs. Treatment of the NS5 RNA capping enzyme in vitro with oxidizing agents enhanced guanylyltransferase activity, indicating that the guanylyltransferase function of the flavivirus NS5 RNA capping enzyme is activated by oxidative conditions. Antioxidant treatment also reduced alphavirus RNA replication and protein expression while enhancing nsP1 capping activity. These findings suggest that RNA viruses may utilize oxidative stress induced during infection to help temporally control genome RNA capping and genome replication.
Asunto(s)
Genoma Viral/fisiología , Estrés Oxidativo/fisiología , Caperuzas de ARN/metabolismo , Virus Sindbis/metabolismo , Virus del Nilo Occidental/genética , Aedes , Animales , Línea Celular , Cricetinae , Regulación Viral de la Expresión Génica/fisiología , Humanos , Nucleotidiltransferasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Virus Sindbis/genética , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The AR86 strain of Sindbis virus causes lethal neurologic disease in adult mice. Previous studies have identified a virulence determinant at nonstructural protein (nsP) 1 position 538 that regulates neurovirulence, modulates clearance from the CNS, and interferes with the type I interferon pathway. The studies herein demonstrate that in the absence of type I interferon signaling, the attenuated mutant exhibited equivalent virulence to S300 virus. Furthermore, both S300 and nsP1 T538I viruses displayed similar neurovirulence and replication kinetics in IPS-1-/- mice. TRIF dependent signaling played a modest role in protecting against disease by both S300 and nsP1 T538I, but did not contribute to control of nsP1 T538I replication within the CNS, while MyD88 played no role in the disease process. These results indicate that the control of the nsP1 T538I mutant virus is largely mediated by IPS-1-dependent RLR signaling, with TRIF-dependent TLR signaling also contributing to protection from virus-induced neurologic disease.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Mutación , Virus Sindbis/genética , Virus Sindbis/patogenicidad , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Infecciones por Alphavirus/mortalidad , Infecciones por Alphavirus/virología , Animales , Línea Celular , Enfermedades Virales del Sistema Nervioso Central , Femenino , Regulación de la Expresión Génica , Interferón Tipo I/metabolismo , Ratones , Ratones Endogámicos C57BL , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Transducción de Señal , Virus Sindbis/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Virulencia/genética , Replicación ViralRESUMEN
Sindbis viral vectors target and inhibit the growth of various solid tumors in mouse models. However, their efficacy against blood cancer has not been well established. Here, we show that Sindbis vectors infect and efficiently trigger apoptosis in mouse BW5147 malignant hematopoietic T-cells, but only at low levels in human lymphoma and leukemia cells (Jurkat, Karpas, CEM, DHL and JB). The Mr 37/67 kD laminin receptor (LAMR) has been suggested to be the receptor for Sindbis virus. However, JB cells, which are infected by Sindbis at low efficiency, express high levels of LAMR, revealing that additional factors are involved in Sindbis tropism. To test the infectivity and therapeutic efficacy of Sindbis vectors against malignant hematopoietic cells in vivo, we injected BW5147 cells intraperitoneally into (C3HXAKR) F1 hybrid mice. We found that Sindbis vectors targeted the tumors and significantly prolonged survival of tumor-bearing mice. We also tested the Sindbis vectors in a transgenic CD4-Rgr model, which spontaneously develop thymic lymphomas. However, infectivity in this model was less efficient. Taken together, these results demonstrate that Sindbis vectors have the potential to target and kill hematopoietic malignancies in mice, but further research is needed to evaluate the mechanism underlining the susceptibility of human lymphoid malignancies to Sindbis therapy.