RESUMEN
Parainfluenza virus type 5 (PIV5) can either have a persistent or a lytic phenotype in cultured cells. We have previously shown that the phenotype is determined by the phosphorylation status of the phosphoprotein (P). Single amino acid substitutions at critical residues, including a serine-to-phenylalanine substitution at position 157 on P, result in a switch between persistent and lytic phenotypes. Here, using PIV5 vectors expressing either mCherry or GFP with persistent or lytic phenotypes, we show that in co-infections the persistent phenotype is dominant. Thus, in contrast to the cell death observed with cells infected solely with the lytic variant, in co-infected cells persistence is immediately established and both lytic and persistent genotypes persist. Furthermore, 10-20â% of virus released from dually infected cells contains both genotypes, indicating that PIV5 particles can package more than one genome. Co-infected cells continue to maintain both genotypes/phenotypes during cell passage, as do individual colonies of cells derived from a culture of persistently infected cells. A refinement of our model on how the dynamics of virus selection may occur in vivo is presented.
Asunto(s)
Coinfección , Virus de la Parainfluenza 5 , Paramyxovirinae , Infecciones por Respirovirus , Humanos , Virus de la Parainfluenza 5/genética , FenotipoRESUMEN
Infected hosts possess two alternative strategies to protect themselves against the negative impact of virus infections: resistance, used to abrogate virus replication, and disease tolerance, used to avoid tissue damage without controlling viral burden. The principles governing pathogen resistance are well understood, while less is known about those involved in disease tolerance. Here, we studied bluetongue virus (BTV), the cause of bluetongue disease of ruminants, as a model system to investigate the mechanisms of virus-host interactions correlating with disease tolerance. BTV induces clinical disease mainly in sheep, while cattle are considered reservoirs of infection, rarely exhibiting clinical symptoms despite sustained viremia. Using primary cells from multiple donors, we show that BTV consistently reaches higher titers in ovine cells than cells from cattle. The variable replication kinetics of BTV in sheep and cow cells were mostly abolished by abrogating the cell type I interferon (IFN) response. We identified restriction factors blocking BTV replication, but both the sheep and cow orthologues of these antiviral genes possess anti-BTV properties. Importantly, we demonstrate that BTV induces a faster host cell protein synthesis shutoff in primary sheep cells than cow cells, which results in an earlier downregulation of antiviral proteins. Moreover, by using RNA sequencing (RNA-seq), we also show a more pronounced expression of interferon-stimulated genes (ISGs) in BTV-infected cow cells than sheep cells. Our data provide a new perspective on how the type I IFN response in reservoir species can have overall positive effects on both virus and host evolution. IMPORTANCE The host immune response usually aims to inhibit virus replication in order to avoid cell damage and disease. In some cases, however, the infected host avoids the deleterious effects of infection despite high levels of viral replication. This strategy is known as disease tolerance, and it is used by animal reservoirs of some zoonotic viruses. Here, using a virus of ruminants (bluetongue virus [BTV]) as an experimental system, we dissected virus-host interactions in cells collected from species that are susceptible (sheep) or tolerant (cow) to disease. We show that (i) virus modulation of the host antiviral type I interferon (IFN) responses, (ii) viral replication kinetics, and (iii) virus-induced cell damage differ in tolerant and susceptible BTV-infected cells. Understanding the complex virus-host interactions in disease tolerance can allow us to disentangle the critical balance between protective and damaging host immune responses.
Asunto(s)
Lengua Azul , Interferón Tipo I , Femenino , Ovinos , Animales , Bovinos , Interferón Tipo I/genética , Lengua Azul/metabolismo , Viremia , AntiviralesRESUMEN
Auditory Scene Analysis (ASA) refers to the grouping of acoustic signals into auditory objects. Previously, we have shown that perceived musicality of auditory sequences varies with high-level organizational features. Here, we explore the neural mechanisms mediating ASA and auditory object perception. Participants performed musicality judgments on randomly generated pure-tone sequences and manipulated versions of each sequence containing low-level changes (amplitude; timbre). Low-level manipulations affected auditory object perception as evidenced by changes in musicality ratings. fMRI was used to measure neural activation to sequences rated most and least musical, and the altered versions of each sequence. Next, we generated two partially overlapping networks: (i) a music processing network (music localizer) and (ii) an ASA network (base sequences vs. ASA manipulated sequences). Using Representational Similarity Analysis, we correlated the functional profiles of each ROI to a model generated from behavioral musicality ratings as well as models corresponding to low-level feature processing and music perception. Within overlapping regions, areas near primary auditory cortex correlated with low-level ASA models, whereas right IPS was correlated with musicality ratings. Shared neural mechanisms that correlate with behavior and underlie both ASA and music perception suggests that low-level features of auditory stimuli play a role in auditory object perception.
Asunto(s)
Corteza Auditiva , Música , Humanos , Percepción Auditiva/fisiología , Imagen por Resonancia Magnética , Mapeo Encefálico , Neuroimagen , Estimulación Acústica , Corteza Auditiva/diagnóstico por imagen , Corteza Auditiva/fisiologíaRESUMEN
Immunodiagnostics have been widely used in the detection of disease biomarkers. The conventional immunological tests in central laboratories require expensive equipment and, for non-specialists, the tests are technically demanding and time-consuming, which has prevented their use by the public. Thus, point-of-care tests (POCT), such as lateral flow immunoassays, are being, or have been, developed as more convenient and low-cost methods for immunodiagnostics. However, the sensitivity of such tests is often a concern. Here, a fluorescence-linked immunosorbent assay (FLISA) using organic light-emitting diodes (OLEDs) as excitation light sources was investigated as a way forward for the development of compact and sensitive POCTs. Phycoerythrin (PE) was selected as the fluorescent dye, and OLEDs were designed with different emission spectra. The leakage light of different OLEDs for exciting PE was then investigated to reduce the background noise and improve the sensitivity of the system. Finally, as proof-of-principle that OLED-based technology can be successfully further developed for POCT, antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human serum was detected by OLED-FLISA.
Asunto(s)
COVID-19 , Inmunoadsorbentes , Humanos , SARS-CoV-2 , Fluorescencia , COVID-19/diagnóstico , Anticuerpos AntiviralesRESUMEN
Purpose: Janus kinase-1 (JAK1) tyrosine kinase mediates signaling from multiple cytokine receptors, including interferon alpha/beta and gamma (IFN-α/ß and IFN-γ), which are important for viral and mycobacterial protection respectively. We previously reported autosomal recessive (AR) hypomorphic JAK1 mutations in a patient with recurrent atypical mycobacterial infections and relatively minor viral infections. This study tests the impact of partial JAK1 deficiency on cellular responses to IFNs and pathogen control. Methods: We investigated the role of partial JAK1 deficiency using patient cells and cell models generated with lentiviral vectors expressing shRNA. Results: Partial JAK1 deficiency impairs IFN-γ-dependent responses in multiple cell types including THP-1 macrophages, Epstein-Barr Virus (EBV)-transformed B cells and primary dermal fibroblasts. In THP-1 myeloid cells, partial JAK1 deficiency reduced phagosome acidification and apoptosis and resulted in defective control of mycobacterial infection with enhanced intracellular survival. Although both EBV-B cells and primary dermal fibroblasts with partial JAK1 deficiency demonstrate reduced IFN-α responses, control of viral infection was impaired only in patient EBV-B cells and surprisingly intact in patient primary dermal fibroblasts. Conclusion: Our data suggests that partial JAK1 deficiency predominantly affects susceptibility to mycobacterial infection through impact on the IFN-γ responsive pathway in myeloid cells. Susceptibility to viral infections as a result of reduced IFN-α responses is variable depending on cell type. Description of additional patients with inherited JAK1 deficiency will further clarify the spectrum of bacterial and viral susceptibility in this condition. Our results have broader relevance for anticipating infectious complications from the increasing use of selective JAK1 inhibitors.
Asunto(s)
Infecciones por Virus de Epstein-Barr , Infecciones por Mycobacterium , Mycobacterium , Herpesvirus Humano 4/genética , Humanos , Interferón-alfa/farmacología , Interferón beta , Interferón gamma/genética , Janus Quinasa 1/genética , Mycobacterium/genética , Infecciones por Mycobacterium/genética , ARN Interferente Pequeño , Receptores de CitocinasRESUMEN
A central challenge of auditory processing involves the segregation, analysis, and integration of acoustic information into auditory perceptual objects for processing by higher order cognitive operations. This study explores the influence of low-level features on auditory object perception. Participants provided perceived musicality ratings in response to randomly generated pure tone sequences. Previous work has shown that music perception relies on the integration of discrete sounds into a holistic structure. Hence, high (versus low) ratings were viewed as indicative of strong (versus weak) object formation. Additionally, participants rated sequences in which random subsets of tones were manipulated along one of three low-level dimensions (timbre, amplitude, or fade-in) at one of three strengths (low, medium, or high). Our primary findings demonstrate how low-level acoustic features modulate the perception of auditory objects, as measured by changes in musicality ratings for manipulated sequences. Secondarily, we used principal component analysis to categorize participants into subgroups based on differential sensitivities to low-level auditory dimensions, thereby highlighting the importance of individual differences in auditory perception. Finally, we report asymmetries regarding the effects of low-level dimensions; specifically, the perceptual significance of timbre. Together, these data contribute to our understanding of how low-level auditory features modulate auditory object perception.
Asunto(s)
Percepción Auditiva , Música , Estimulación Acústica , Acústica , Humanos , SonidoRESUMEN
The concept that type I interferons (IFN-I) are essential to antiviral immunity derives from studies on animal models and cell lines. Virtually all pathogenic viruses have evolved countermeasures to IFN-I restriction, and genetic loss of viral IFN-I antagonists leads to virus attenuation. But just how important is IFN-I to antiviral defence in humans? The recent discovery of genetic defects of IFN-I signalling illuminates this and other questions of IFN biology, including the role of the mucosa-restricted type III IFNs (IFN-III), informing our understanding of the place of the IFN system within the concerted antiviral response. Here we review monogenic lesions of IFN-I signalling pathways and summarise the organising principles which emerge.
Asunto(s)
Antivirales/inmunología , Inmunidad Innata/inmunología , Interferón Tipo I/antagonistas & inhibidores , Virus/inmunología , Animales , Antivirales/farmacología , Humanos , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Transducción de Señal , Virus/efectos de los fármacosRESUMEN
IFNs, produced during viral infections, induce the expression of hundreds of IFN-stimulated genes (ISGs). Some ISGs have specific antiviral activity, whereas others regulate the cellular response. Besides functioning as an antiviral effector, ISG15 is a negative regulator of IFN signaling, and inherited ISG15 deficiency leads to autoinflammatory IFNopathies, in which individuals exhibit elevated ISG expression in the absence of pathogenic infection. We have recapitulated these effects in cultured human A549-ISG15-/- cells and (using A549-UBA7-/- cells) confirmed that posttranslational modification by ISG15 (ISGylation) is not required for regulation of the type I IFN response. ISG15-deficient cells pretreated with IFN-α were resistant to paramyxovirus infection. We also showed that IFN-α treatment of ISG15-deficient cells led to significant inhibition of global protein synthesis, leading us to ask whether resistance was due to the direct antiviral activity of ISGs or whether cells were nonpermissive because of translation defects. We took advantage of the knowledge that IFN-induced protein with tetratricopeptide repeats 1 (IFIT1) is the principal antiviral ISG for parainfluenza virus 5. Knockdown of IFIT1 restored parainfluenza virus 5 infection in IFN-α-pretreated, ISG15-deficient cells, confirming that resistance was due to the direct antiviral activity of the IFN response. However, resistance could be induced if cells were pretreated with IFN-α for longer times, presumably because of inhibition of protein synthesis. These data show that the cause of virus resistance is 2-fold; ISG15 deficiency leads to the early overexpression of specific antiviral ISGs, but the later response is dominated by an unanticipated, ISG15-dependent loss of translational control.
Asunto(s)
Citocinas/deficiencia , Resistencia a la Enfermedad/genética , Interferón-alfa/metabolismo , Infecciones por Paramyxoviridae/inmunología , Transducción de Señal/inmunología , Ubiquitinas/deficiencia , Células A549 , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Chlorocebus aethiops , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Humanos , Virus de la Parainfluenza 2 Humana/inmunología , Virus de la Parainfluenza 3 Humana/inmunología , Virus de la Parainfluenza 5/inmunología , Infecciones por Paramyxoviridae/virología , Procesamiento Proteico-Postraduccional/inmunología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transducción de Señal/genética , Enzimas Activadoras de Ubiquitina/genética , Células VeroRESUMEN
During the replication of parainfluenza virus 5 (PIV5), copyback defective virus genomes (DVGs) are erroneously produced and are packaged into "infectious" virus particles. Copyback DVGs are the primary inducers of innate intracellular responses, including the interferon (IFN) response. While DVGs can interfere with the replication of nondefective (ND) virus genomes and activate the IFN-induction cascade before ND PIV5 can block the production of IFN, we demonstrate that the converse is also true, i.e., high levels of ND virus can block the ability of DVGs to activate the IFN-induction cascade. By following the replication and amplification of DVGs in A549 cells that are deficient in a variety of innate intracellular antiviral responses, we show that DVGs induce an uncharacterized IFN-independent innate response(s) that limits their replication. High-throughput sequencing was used to characterize the molecular structure of copyback DVGs. While there appears to be no sequence-specific break or rejoining points for the generation of copyback DVGs, our findings suggest there are region, size, and/or structural preferences selected for during for their amplification.IMPORTANCE Copyback defective virus genomes (DVGs) are powerful inducers of innate immune responses both in vitro and in vivo They impact the outcome of natural infections, may help drive virus-host coevolution, and promote virus persistence. Due to their potent interfering and immunostimulatory properties, DVGs may also be used therapeutically as antivirals and vaccine adjuvants. However, little is known of the host cell restrictions which limit their amplification. We show here that the generation of copyback DVGs readily occurs during parainfluenza virus 5 (PIV5) replication, but that their subsequent amplification is restricted by the induction of innate intracellular responses. Molecular characterization of PIV5 copyback DVGs suggests that while there are no genome sequence-specific breaks or rejoin points for the generation of copyback DVGs, genome region, size, and structural preferences are selected for during their evolution and amplification.
Asunto(s)
Inmunidad Innata/inmunología , Virus de la Parainfluenza 5/genética , Virus de la Parainfluenza 5/inmunología , Células A549 , Animales , Secuencia de Bases/genética , Línea Celular , Chlorocebus aethiops , Citoplasma , Virus Defectuosos/genética , Genoma Viral/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Interferones/genética , ARN Viral/genética , Células Vero , Virión/genética , Virosis/genética , Replicación Viral/genéticaRESUMEN
Excessive type I interferon (IFNα/ß) activity is implicated in a spectrum of human disease, yet its direct role remains to be conclusively proven. We investigated two siblings with severe early-onset autoinflammatory disease and an elevated IFN signature. Whole-exome sequencing revealed a shared homozygous missense Arg148Trp variant in STAT2, a transcription factor that functions exclusively downstream of innate IFNs. Cells bearing STAT2R148W in homozygosity (but not heterozygosity) were hypersensitive to IFNα/ß, which manifest as prolonged Janus kinase-signal transducers and activators of transcription (STAT) signaling and transcriptional activation. We show that this gain of IFN activity results from the failure of mutant STAT2R148W to interact with ubiquitin-specific protease 18, a key STAT2-dependent negative regulator of IFNα/ß signaling. These observations reveal an essential in vivo function of STAT2 in the regulation of human IFNα/ß signaling, providing concrete evidence of the serious pathological consequences of unrestrained IFNα/ß activity and supporting efforts to target this pathway therapeutically in IFN-associated disease.
Asunto(s)
Enfermedades del Sistema Inmune/genética , Interferón Tipo I/inmunología , Factor de Transcripción STAT2/genética , Mutación de Línea Germinal , Humanos , Enfermedades del Sistema Inmune/inmunología , Lactante , Masculino , Transducción de SeñalRESUMEN
In reverse genetic experiments we have isolated recombinant mumps viruses (rMuV) that carry large numbers of mutations clustered in small parts of their genome, which are not caused by biased hyper-mutation. In two separate experiments we obtained such recombinant viruses: one virus had 11 mutations in the V/P region of the genome; the other, which also contained an extra transcription unit encoding green fluorescent protein (EGFP), had 32 mutations in the N gene. These specific sets of mutations have not been observed in naturally occurring MuV isolates. Unusually, the vast majority of the mutations (48/51) were synonymous. On passage in Vero cells and human B-LCL cells, a B lymphocyte-like cell line, these mutations appear stable as no reversion occurred to the original consensus sequence, although mutations in other parts of the genome occurred and changed in frequency during passage. Defective interfering RNAs accumulate in passage in Vero cells but not in B-LCL cells. Interestingly, in all passaged samples the level of variation in the EGFP gene is the same as in the viral genes, though it is unlikely that this gene is under any functionality constraint. What mechanism gave rise to these viruses with clustered mutations and their stability remains an open question, which is likely of interest to a wider field than mumps reverse genetics.
Asunto(s)
ADN Complementario/genética , Virus de la Parotiditis/fisiología , Mutación , Proteínas Virales/genética , Animales , Línea Celular Tumoral , Chlorocebus aethiops , Proteínas Fluorescentes Verdes/genética , Humanos , Virus de la Parotiditis/genética , Genética Inversa/métodos , Pase Seriado , Células Vero , Replicación ViralRESUMEN
We have developed a high-throughput sequencing (HTS) workflow for investigating paramyxovirus transcription and replication. We show that sequencing of oligo(dT)-selected polyadenylated mRNAs, without considering the orientation of the RNAs from which they had been generated, cannot accurately be used to analyze the abundance of viral mRNAs because genomic RNA copurifies with the viral mRNAs. The best method is directional sequencing of infected cell RNA that has physically been depleted of ribosomal and mitochondrial RNA followed by bioinformatic steps to differentiate data originating from genomes from viral mRNAs and antigenomes. This approach has the advantage that the abundance of viral mRNA (and antigenomes) and genomes can be analyzed and quantified from the same data. We investigated the kinetics of viral transcription and replication during infection of A549 cells with parainfluenza virus type 2 (PIV2), PIV3, PIV5, or mumps virus and determined the abundances of individual viral mRNAs and readthrough mRNAs. We found that the mRNA abundance gradients differed significantly between all four viruses but that for each virus the pattern remained relatively stable throughout infection. We suggest that rapid degradation of non-poly(A) mRNAs may be primarily responsible for the shape of the mRNA abundance gradient in parainfluenza virus 3, whereas a combination of this factor and disengagement of RNA polymerase at intergenic sequences, particularly those at the NP:P and P:M gene boundaries, may be responsible in the other viruses.IMPORTANCE High-throughput sequencing (HTS) of virus-infected cells can be used to study in great detail the patterns of virus transcription and replication. For paramyxoviruses, and by analogy for all other negative-strand RNA viruses, we show that directional sequencing must be used to distinguish between genomic RNA and mRNA/antigenomic RNA because significant amounts of genomic RNA copurify with poly(A)-selected mRNA. We found that the best method is directional sequencing of total cell RNA, after the physical removal of rRNA (and mitochondrial RNA), because quantitative information on the abundance of both genomic RNA and mRNA/antigenomes can be simultaneously derived. Using this approach, we revealed new details of the kinetics of virus transcription and replication for parainfluenza virus (PIV) type 2, PIV3, PIV5, and mumps virus, as well as on the relative abundance of the individual viral mRNAs.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Infecciones por Paramyxoviridae/virología , Paramyxovirinae/fisiología , ARN Mensajero/genética , Secuenciación Completa del Genoma/métodos , Células A549 , Regulación Viral de la Expresión Génica , Tamaño del Genoma , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Paramyxovirinae/clasificación , Paramyxovirinae/patogenicidad , ARN Viral/genética , Especificidad de la Especie , Replicación ViralRESUMEN
Paramyxoviruses can establish persistent infections both in vitro and in vivo, some of which lead to chronic disease. However, little is known about the molecular events that contribute to the establishment of persistent infections by RNA viruses. Using parainfluenza virus type 5 (PIV5) as a model we show that phosphorylation of the P protein, which is a key component of the viral RNA polymerase complex, determines whether or not viral transcription and replication becomes repressed at late times after infection. If the virus becomes repressed, persistence is established, but if not, the infected cells die. We found that single amino acid changes at various positions within the P protein switched the infection phenotype from lytic to persistent. Lytic variants replicated to higher titres in mice than persistent variants and caused greater infiltration of immune cells into infected lungs but were cleared more rapidly. We propose that during the acute phases of viral infection in vivo, lytic variants of PIV5 will be selected but, as the adaptive immune response develops, variants in which viral replication can be repressed will be selected, leading to the establishment of prolonged, persistent infections. We suggest that similar selection processes may operate for other RNA viruses.
Asunto(s)
Infecciones por Paramyxoviridae/genética , Paramyxoviridae/genética , Fosfoproteínas/genética , Proteínas Virales/genética , Células A549 , Sustitución de Aminoácidos/genética , Animales , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Femenino , Células HEK293 , Humanos , Ratones , Ratones Endogámicos BALB C , Virus de la Parainfluenza 5/genética , Virus de la Parainfluenza 5/patogenicidad , Paramyxoviridae/patogenicidad , Infecciones por Paramyxoviridae/metabolismo , Fosfoproteínas/metabolismo , Fosfoproteínas/fisiología , Fosforilación , ARN Viral , Proteínas Virales/metabolismo , Proteínas Virales/fisiología , Replicación ViralRESUMEN
The molecular processes that determine the outcome of influenza virus infection in humans are multifactorial and involve a complex interplay between host, viral and bacterial factors1. However, it is generally accepted that a strong innate immune dysregulation known as 'cytokine storm' contributes to the pathology of infections with the 1918 H1N1 pandemic or the highly pathogenic avian influenza viruses of the H5N1 subtype2-4. The RNA sensor retinoic acid-inducible gene I (RIG-I) plays an important role in sensing viral infection and initiating a signalling cascade that leads to interferon expression5. Here, we show that short aberrant RNAs (mini viral RNAs (mvRNAs)), produced by the viral RNA polymerase during the replication of the viral RNA genome, bind to and activate RIG-I and lead to the expression of interferon-ß. We find that erroneous polymerase activity, dysregulation of viral RNA replication or the presence of avian-specific amino acids underlie mvRNA generation and cytokine expression in mammalian cells. By deep sequencing RNA samples from the lungs of ferrets infected with influenza viruses, we show that mvRNAs are generated during infection in vivo. We propose that mvRNAs act as the main agonists of RIG-I during influenza virus infection.
Asunto(s)
Proteína 58 DEAD Box/metabolismo , Inmunidad Innata/inmunología , Virus de la Influenza A/fisiología , Infecciones por Orthomyxoviridae/inmunología , ARN Viral/metabolismo , Animales , Línea Celular , Citocinas/metabolismo , Proteína 58 DEAD Box/genética , Femenino , Hurones , Virus de la Influenza A/genética , Interferón beta/genética , Interferón beta/metabolismo , Masculino , Mutación , Proteínas Virales/genética , Replicación ViralRESUMEN
Viral interferon (IFN) antagonists are a diverse class of viral proteins that counteract the host IFN response, which is important for controlling viral infections. Viral IFN antagonists are often multifunctional proteins that perform vital roles in virus replication beyond IFN antagonism. The critical importance of viral IFN antagonists is highlighted by the fact that almost all viruses encode one of these proteins. Inhibition of viral IFN antagonists has the potential to exert pleiotropic antiviral effects and thus this important protein class represents a diverse plethora of novel therapeutic targets. To exploit this, we have successfully developed and executed a novel modular cell-based platform that facilitates the safe and rapid screening for inhibitors of a viral IFN antagonist of choice. The platform is based on two reporter cell-lines that provide a simple method to detect activation of IFN induction or signaling via an eGFP gene placed under the control of the IFNß or an ISRE-containing promoter, respectively. Expression of a target IFN antagonist in the appropriate reporter cell-line will block the IFN response and hence eGFP expression. We hypothesized that addition of a compound that inhibits IFN antagonist function will release the block imposed on the IFN response and hence restore eGFP expression, providing a measurable parameter for high throughput screening (HTS). We demonstrate assay proof-of-concept by (i) exploiting hepatitis C virus (HCV) protease inhibitors to inhibit NS3-4A's capacity to block IFN induction and (ii) successfully executing two HTS targeting viral IFN antagonists that block IFN signaling; NS2 and IE1 from human respiratory syncytial virus (RSV) and cytomegalovirus (CMV) respectively, two clinically important viruses for which vaccine development has thus far been unsuccessful and new antivirals are required. Both screens performed robustly and Z' Factor scores of >0.6 were achieved. We identified (i) four hit compounds that specifically inhibit RSV NS2's ability to block IFN signaling by mediating STAT2 degradation and exhibit modest antiviral activity and (ii) two hit compounds that interfere with IE1 transcription and significantly impair CMV replication. Overall, we demonstrate assay proof-of-concept as we target viral IFN antagonists from unrelated viruses and demonstrate its suitability for HTS.
Asunto(s)
Antivirales/farmacología , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Ensayos Analíticos de Alto Rendimiento , Interferones/antagonistas & inhibidores , Interferones/farmacología , Proteínas Virales/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación Viral de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Unión Proteica , Virus Sincitial Respiratorio Humano/efectos de los fármacos , Virus Sincitial Respiratorio Humano/fisiología , Transducción de Señal , Replicación Viral/efectos de los fármacosRESUMEN
NS1 proteins of influenza A and B viruses share limited sequence homology, yet both are potent manipulators of host cell processes, particularly interferon (IFN) induction. Although many cellular partners are reported for A/NS1, only a few (e.g. PKR and ISG15) have been identified for B/NS1. Here, affinity-purification and mass spectrometry were used to expand the known host interactome of B/NS1. We identified 22 human proteins as new putative targets for B/NS1, validating several, including DHX9, ILF3, YBX1 and HNRNPC. Consistent with two RNA-binding domains in B/NS1, many of the identified factors bind RNA and some interact with B/NS1 in an RNA-dependent manner. Functional characterization of several B/NS1 interactors identified SNRNP200 as a potential positive regulator of host IFN responses, while ILF3 exhibited dual roles in both IFN induction and influenza B virus replication. These data provide a resource for future investigations into the mechanisms underpinning host cell modulation by influenza B virus NS1.
Asunto(s)
Virus de la Influenza B/aislamiento & purificación , Gripe Humana/metabolismo , Proteínas no Estructurales Virales/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Humanos , Virus de la Influenza B/genética , Virus de la Influenza B/metabolismo , Gripe Humana/genética , Gripe Humana/virología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas del Factor Nuclear 90/genética , Proteínas del Factor Nuclear 90/metabolismo , Unión Proteica , Proteínas no Estructurales Virales/genética , Proteína 1 de Unión a la Caja Y/genética , Proteína 1 de Unión a la Caja Y/metabolismoRESUMEN
Modern vaccinology has increasingly focused on non-living vaccines, which are more stable than live-attenuated vaccines but often show limited immunogenicity. Immunostimulatory substances, known as adjuvants, are traditionally used to increase the magnitude of protective adaptive immunity in response to a pathogen-associated antigen. Recently developed adjuvants often include substances that stimulate pattern recognition receptors (PRRs), essential components of innate immunity required for the activation of antigen-presenting cells (APCs), which serve as a bridge between innate and adaptive immunity. Nearly all PRRs are potential targets for adjuvants. Given the recent success of toll-like receptor (TLR) agonists in vaccine development, molecules with similar, but additional, immunostimulatory activity, such as defective interfering particles (DIPs) of viruses, represent attractive candidates for vaccine adjuvants. This review outlines some of the recent advances in vaccine development related to the use of TLR agonists, summarizes the current knowledge regarding DIP immunogenicity, and discusses the potential applications of DIPs in vaccine adjuvantation.
Asunto(s)
Adyuvantes Inmunológicos , Virus Defectuosos/inmunología , Receptores de Reconocimiento de Patrones/agonistas , Receptores de Reconocimiento de Patrones/inmunología , Vacunas/química , Inmunidad Adaptativa , Animales , Células Presentadoras de Antígenos/inmunología , Genoma Viral , Humanos , Inmunidad Innata , Inmunogenicidad Vacunal , Ratones , Receptores Toll-Like/agonistas , Vacunas/administración & dosificaciónRESUMEN
In a prototypical response to an acute viral infection it would be expected that the adaptive immune response would eliminate all virally infected cells within a few weeks of infection. However many (non-retrovirus) RNA viruses can establish 'within host' persistent infections that occasionally lead to chronic or reactivated disease. Despite the importance of 'within host' persistent RNA virus infections, much has still to be learnt about the molecular mechanisms by which RNA viruses establish persistent infections, why innate and adaptive immune responses fail to rapidly clear these infections, and the epidemiological and potential disease consequences of such infections.
Asunto(s)
Enfermedad Crónica , Interacciones Huésped-Patógeno , Infecciones por Virus ARN/inmunología , Infecciones por Virus ARN/virología , Latencia del Virus , Animales , HumanosRESUMEN
We have sequenced the parainfluenza virus 5 strain that persistently infects the commonly used AGS human cell line without causing cytopathology. This virus is most closely related to human strains, indicating that it may have originated from biopsy material or from laboratory contamination during generation of the cell line.
RESUMEN
Production of type I interferon (IFN) is an essential component of the innate immune response against invading pathogens. However, its production must be tightly regulated to avoid harmful effects. Compounds that modulate the IFN response are potentially valuable for a variety of applications due to IFN's beneficial and detrimental roles. We developed and executed a cell-based high-throughput screen (HTS) targeting components that participate in and/or regulate the IRF3 and nuclear factor (NF)-κB branches of the IFN induction pathway. The assay detects activation of the IFN induction pathway via an enhanced green fluorescent protein (eGFP) reporter gene under the control of the IFNß promoter and was optimized, miniaturized, and demonstrated suitable for HTS as robust Z' factor scores of >0.6 were consistently achieved. A diversity screening set of 15,667 small molecules was assayed and two novel hit compounds validated that specifically inhibit the IFN induction pathway. We demonstrate that one of these compounds acts at or upstream of IRF3 phosphorylation. A second cell-based assay to detect activation of the IFN signaling (Jak-Stat) pathway via an eGFP reporter gene under the control of an IFN-stimulated response element (ISRE) containing MxA promoter also performed well (robust Z' factor >0.7) and may therefore be similarly used to identify small molecules that modulate the IFN signaling pathway.