RESUMEN
Movement of macromolecules between the cytoplasm and the nucleus occurs through the nuclear pore complex (NPC). Karyopherins comprise a family of soluble transport factors facilitating the nucleocytoplasmic translocation of proteins through the NPC. In this study, we found that karyopherin α6 (KPNA6; also known as importin α7) was required for the optimal replication of porcine reproductive and respiratory syndrome virus (PRRSV) and Zika virus (ZIKV), which are positive-sense, single-stranded RNA viruses replicating in the cytoplasm. The KPNA6 protein level in virus-infected cells was much higher than that in mock-infected controls, whereas the KPNA6 transcript remains stable. Viral infection blocked the ubiquitin-proteasomal degradation of KPNA6, which led to an extension of the KPNA6 half-life and the elevation of the KPNA6 level in comparison to mock-infected cells. PRRSV nsp12 protein induced KPNA6 stabilization. KPNA6 silencing was detrimental to the replication of PRRSV, and KPNA6 knockout impaired ZIKV replication. Moreover, KPNA6 knockout blocked the nuclear translocation of PRRSV nsp1ß but had a minimal effect on two other PRRSV proteins with nuclear localization. Exogenous restitution of KPNA6 expression in the KPNA6-knockout cells results in restoration of the nuclear translocation of PRRSV nsp1ß and the replication of ZIKV. These results indicate that KPNA6 is an important cellular factor for the replication of PRRSV and ZIKV.IMPORTANCE Positive-sense, single-stranded RNA (+ssRNA) viruses replicate in the cytoplasm of infected cells. The roles of transport factors in the nucleocytoplasmic trafficking system for the replication of +ssRNA viruses are not known. In this study, we discovered that PRRSV and ZIKV viruses needed karyopherin α6 (KPNA6), one of the transport factors, to enhance the virus replication. Our data showed that viral infection induced an elevation of the KPNA6 protein level due to an extension of the KPNA6 half-life via viral interference of the ubiquitin-proteasomal degradation of KPNA6. Notably, KPNA6 silencing or knockout dramatically reduced the replication of PRRSV and ZIKV. PRRSV nsp1ß depended on KPNA6 to translocate into the nucleus. In addition, exogenous restitution of KPNA6 expression in KPNA6-knockout cells led to the restoration of nsp1ß nuclear translocation and ZIKV replication. These results reveal a new aspect in the virus-cell interaction and may facilitate the development of novel antiviral therapeutics.
Asunto(s)
Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Transporte de Proteínas/genética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/genética , Virus Zika/fisiología , alfa Carioferinas/genética , Aedes , Animales , Línea Celular Tumoral , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Poro Nuclear/genética , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Transducción de Señal/genética , Porcinos , Células Vero , Replicación Viral/fisiología , Virus Zika/genéticaRESUMEN
Signal transducer and activator of transcription 3 (STAT3) is a pleiotropic signaling mediator of many cytokines, including interleukin-6 (IL-6) and IL-10. STAT3 is known to play critical roles in cell growth, proliferation, differentiation, immunity and inflammatory responses. The objective of this study was to determine the effect of porcine reproductive and respiratory syndrome virus (PRRSV) infection on the STAT3 signaling since PRRSV induces a weak protective immune response in host animals. We report here that PRRSV infection of MARC-145 cells and primary porcine pulmonary alveolar macrophages led to significant reduction of STAT3 protein level. Several strains of both PRRSV type 1 and type 2 led to a similar reduction of STAT3 protein level but had a minimal effect on its transcripts. The PRRSV-mediated STAT3 reduction was in a dose-dependent manner as the STAT3 level decreased, along with incremental amounts of PRRSV inocula. Further study showed that nonstructural protein 5 (nsp5) of PRRSV induced the STAT3 degradation by increasing its polyubiquitination level and shortening its half-life from 24 h to â¼3.5 h. The C-terminal domain of nsp5 was shown to be required for the STAT3 degradation. Moreover, the STAT3 signaling in the cells transfected with nsp5 plasmid was significantly inhibited. These results indicate that PRRSV antagonizes the STAT3 signaling by accelerating STAT3 degradation via the ubiquitin-proteasomal pathway. This study provides insight into the PRRSV interference with the JAK/STAT3 signaling, leading to perturbation of the host innate and adaptive immune responses. IMPORTANCE: The typical features of immune responses in PRRSV-infected pigs are delayed onset and low levels of virus neutralizing antibodies, as well as weak cell-mediated immunity. Lymphocyte development and differentiation rely on cytokines, many of which signal through the JAK/STAT signaling pathway to exert their biological effects. Here, we discovered that PRRSV antagonizes the JAK/STAT3 signaling by inducing degradation of STAT3, a master transcription activator involved in multiple cellular processes and the host immune responses. The nsp5 protein of PRRSV is responsible for the accelerated STAT3 degradation. The PRRSV-mediated antagonizing STAT3 could lead to suppression of a broad spectrum of cytokines and growth factors to allow virus replication and spread in host animals. This may be one of the reasons for the PRRSV interference with the innate immunity and its poor elicitation of protective immunity. This finding provides insight into PRRSV pathogenesis and its interference with the host immune responses.
Asunto(s)
Quinasas Janus/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Proteínas no Estructurales Virales/metabolismo , Animales , Línea Celular , Humanos , Inmunidad Innata , Oncostatina M/farmacología , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteolisis , Transducción de Señal/efectos de los fármacos , Porcinos , Transcripción Genética , Ubiquitina/metabolismo , Ubiquitinación , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Replicación ViralRESUMEN
Porcine reproductive and respiratory syndrome virus (PRRSV) is known to antagonize the innate immune response. An atypical PRRSV strain A2MC2 is capable of inducing synthesis of type I interferons (IFNs) in cultured cells. Here, we show that the middle half of the A2MC2 genome is needed for triggering the IFN synthesis. First, a cDNA infectious clone of this atypical strain was constructed as a DNA-launched version. Virus recovery was achieved from the infectious clone and the recovered virus, rA2MC2, was characterized. The rA2MC2 retained the feature of IFN induction in cultured cells. Infection of pigs with the rA2MC2 virus caused viremia similar to that of the wild-type virus. Chimeric infectious clones were constructed by swapping genomic fragments with a cDNA clone of a moderately virulent strain VR-2385 that antagonizes IFN induction. Analysis of the rescued chimeric viruses demonstrated that the middle two fragments, ranging from nt4545 to nt12709 of the A2MC2 genome, were needed for the IFN induction, whereas the chimeric viruses containing any one of the two A2MC2 fragments failed to do so. The results and the cDNA infectious clone of the IFN-inducing A2MC2 will facilitate further study of its biology, ultimately leading towards the development of an improved vaccine against PRRS.
Asunto(s)
Genoma Viral , Interferón Tipo I/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Animales , Interferón Tipo I/genética , Síndrome Respiratorio y de la Reproducción Porcina/genética , Síndrome Respiratorio y de la Reproducción Porcina/metabolismo , Virus del Síndrome Respiratorio y Reproductivo Porcino/metabolismo , Porcinos , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
UNLABELLED: Hepatitis E virus (HEV) causes both the endemic and epidemic spread of acute hepatitis in many parts of the world. HEV open reading frame 3 (ORF3) encodes a 13-kDa multifunctional protein (vp13) that is essential for HEV infection of animals. The exact role of vp13 in HEV infection remains unclear. In this study, vp13 was found to enhance interferon (IFN) production induced by poly(I · C), a synthetic analog of double-stranded RNA. Poly(I · C) treatment induced a higher level of IFN-ß mRNA in HeLa cells stably expressing vp13 than in control cells. Using a luciferase reporter construct driven by the IFN-ß promoter, we demonstrated that vp13 enhanced retinoic acid-inducible gene I (RIG-I)-dependent luciferase expression. This enhancement was found to be due to both an increased level of RIG-I protein and its activation. The levels of both endogenous and exogenous RIG-I were increased by vp13 by extension of the half-life of RIG-I. Additionally, vp13 interacts with the RIG-I N-terminal domain and enhances its K63-linked ubiquitination, which is essential for RIG-I activation. Analysis of vp13 deletion constructs suggested that the C-terminal domain of vp13 was essential for the enhancement of RIG-I signaling. In HEV-infected hepatoma cells, wild-type HEV led to a higher level of RIG-I and more poly(I · C)-induced IFN-ß expression than did ORF3-null mutants. Analysis of vp13 from four HEV genotypes showed that vp13 from genotype I and III strains boosted RIG-I signaling, while vp13 from genotype II and IV strains had a minimal effect. These results indicate that vp13 enhances RIG-I signaling, which may play a role in HEV invasion. IMPORTANCE: Hepatitis E virus (HEV) is a significant pathogen causing hepatitis in many parts of the world, yet it is understudied compared with other viral hepatitis pathogens. Here we found that the HEV open reading frame 3 product, vp13, enhances interferon induction stimulated by a synthetic analog of double-stranded RNA. This enhancement may play a role in HEV invasion, as vp13 is essential for HEV infection in vivo. The results of this study provide insights into virus-cell interactions during HEV infection. In addition to revealing its possible roles in HEV interference with cellular signaling, these results suggest that the second half of the vp13 sequence can be ligated into the genomes of attenuated live viruses to induce an innate immune response for better protective immunity, as well as a marker for differentiation of vaccinated animals from those infected with the corresponding wild-type viruses.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Virus de la Hepatitis E/inmunología , Interferón beta/biosíntesis , Proteínas Virales/inmunología , Proteína 58 DEAD Box , Células HeLa , Humanos , Poli I-C/inmunología , Receptores InmunológicosRESUMEN
Hepatitis E virus (HEV) causes both endemic and epidemic human hepatitis by fecal-oral transmission in many parts of the world. Zoonotic transmission of HEV from animals to humans has been reported. Due to the lack of an efficient cell culture system, the molecular mechanisms of HEV infection remain largely unknown. In this study, we found that HEV replication in hepatoma cells inhibited poly(I·C)-induced beta interferon (IFN-ß) expression and that the HEV open reading frame 1 (ORF1) product was responsible for this inhibition. Two domains, X and the papain-like cysteine protease domain (PCP), of HEV ORF1 were identified as the putative IFN antagonists. When overexpressed in HEK293T cells, the X domain (or macro domain) inhibited poly(I·C)-induced phosphorylation of interferon regulatory factor 3 (IRF-3), which is the key transcription factor for IFN induction. The PCP domain was shown to have deubiquitinase activity for both RIG-I and TBK-1, whose ubiquitination is a key step in their activation in poly(I·C)-induced IFN induction. Furthermore, replication of a HEV replicon containing green fluorescent protein (GFP) (E2-GFP) in hepatoma cells led to impaired phosphorylation of IRF-3 and reduced ubiquitination of RIG-I and TBK-1, which confirmed our observations of X and PCP inhibitory effects in HEK293T cells. Altogether, our study identified the IFN antagonists within the HEV ORF1 polyprotein and expanded our understanding of the functions of several of the HEV ORF1 products, as well as the mechanisms of HEV pathogenesis. Importance: Type I interferons (IFNs) are important components of innate immunity and play a crucial role against viral infection. They also serve as key regulators to evoke an adaptive immune response. Virus infection can induce the synthesis of interferons; however, viruses have evolved many strategies to antagonize the induction of interferons. There is little knowledge about how hepatitis E virus (HEV) inhibits induction of host IFNs, though the viral genome was sequenced more than 2 decades ago. This is the first report of identification of the potential IFN antagonists encoded by HEV. By screening all the domains in the open reading frame 1 (ORF1) polyprotein, we identified two IFN antagonists and performed further research to determine how and at which step in the IFN induction pathway they antagonize host IFN induction. Our work provides valuable information about HEV-cell interaction and pathogenesis.
Asunto(s)
Virus de la Hepatitis E/fisiología , Interferón Tipo I/antagonistas & inhibidores , Sistemas de Lectura Abierta , Secuencia de Bases , Western Blotting , Cartilla de ADN , Células HEK293 , Humanos , Interferón Tipo I/biosíntesis , Fosforilación , Ubiquitinación , Proteínas Virales/fisiologíaRESUMEN
Porcine reproductive and respiratory syndrome virus (PRRSV) inhibits the interferon-mediated antiviral response. Type I interferons (IFNs) induce the expression of IFN-stimulated genes by activating phosphorylation of both signal transducer and activator of transcription 1 (STAT1) and STAT2, which form heterotrimers (interferon-stimulated gene factor 3 [ISGF3]) with interferon regulatory factor 9 (IRF9) and translocate to the nucleus. PRRSV Nsp1ß blocks the nuclear translocation of the ISGF3 complex by an unknown mechanism. In this study, we discovered that Nsp1ß induced the degradation of karyopherin-α1 (KPNA1, also called importin-α5), which is known to mediate the nuclear import of ISGF3. Overexpression of Nsp1ß resulted in a reduction of KPNA1 levels in a dose-dependent manner, and treatment of the cells with the proteasome inhibitor MG132 restored KPNA1 levels. Furthermore, the presence of Nsp1ß induced an elevation of KPNA1 ubiquitination and a shortening of its half-life. Our analysis of Nsp1ß deletion constructs showed that the N-terminal domain of Nsp1ß was involved in the ubiquitin-proteasomal degradation of KPNA1. A nucleotide substitution resulting in an amino acid change from valine to isoleucine at residue 19 of Nsp1ß diminished its ability to induce KPNA1 degradation and to inhibit IFN-mediated signaling. Interestingly, infection of MARC-145 cells by PRRSV strains VR-2332 and VR-2385 also resulted in KPNA1 reduction, whereas infection by an avirulent strain, Ingelvac PRRS modified live virus (MLV), did not. MLV Nsp1ß had no effect on KPNA1; however, a mutant with an amino acid change at residue 19 from isoleucine to valine induced KPNA1 degradation. These results indicate that Nsp1ß blocks ISGF3 nuclear translocation by inducing KPNA1 degradation and that valine-19 in Nsp1ß correlates with the inhibition.
Asunto(s)
Interferón-alfa/metabolismo , Quinasas Janus/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/metabolismo , Virus del Síndrome Respiratorio y Reproductivo Porcino/metabolismo , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT2/metabolismo , Proteínas no Estructurales Virales/metabolismo , alfa Carioferinas/metabolismo , Animales , Línea Celular , Regulación hacia Abajo , Interferón-alfa/genética , Quinasas Janus/genética , Síndrome Respiratorio y de la Reproducción Porcina/genética , Síndrome Respiratorio y de la Reproducción Porcina/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Proteolisis , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT2/genética , Transducción de Señal , Porcinos , Proteínas no Estructurales Virales/genética , alfa Carioferinas/genéticaRESUMEN
Hepatitis E virus (HEV) predominantly causes acute liver disease in humans and is transmitted via the fecal-oral route. HEV infection in pregnant women can result in grave consequences, with up to 30% fatality. The HEV strains infecting humans mainly belong to four genotypes. Genotypes 1 and 2 are restricted to human infection, while genotypes 3 and 4 are zoonotic. HEV genotype 3 (HEV-3) can cause both acute and chronic liver disease. Several cell lines (mainly hepatocytes) have been developed for HEV propagation and biological study. However, HEV production in these cell lines is suboptimal and inefficient. Here, we present methods for the isolation, propagation, and quantification of HEV. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation and propagation of hepatitis E virus in cultured cells from clinical HEV specimens Support Protocol 1: Quantification of HEV RNA by RT-qPCR Basic Protocol 2: Recovery of HEV from infectious cDNA clones and purification of the virus Support Protocol 2: Quantification of HEV live particles by infectivity assay.
Asunto(s)
Virus de la Hepatitis E , Hepatitis E , Embarazo , Humanos , Femenino , Virus de la Hepatitis E/genética , Hepatocitos , Línea CelularRESUMEN
Type I interferons (IFNs) IFN-α/ß play an important role in innate immunity against viral infections by inducing antiviral responses. Porcine reproductive and respiratory syndrome virus (PRRSV) inhibits the synthesis of type I IFNs. However, whether PRRSV can inhibit IFN signaling is less well understood. In the present study, we found that PRRSV interferes with the IFN signaling pathway. The transcript levels of IFN-stimulated genes ISG15 and ISG56 and protein level of signal transducer and activator of transcription 2 (STAT2) in PRRSV VR2385-infected MARC-145 cells were significantly lower than those in mock-infected cells after IFN-α treatment. IFN-induced phosphorylation of both STAT1 and STAT2 and their heterodimer formation in the PRRSV-infected cells were not affected. However, the majority of the STAT1/STAT2/IRF9 (IFN regulatory factor 9) heterotrimers remained in the cytoplasm of PRRSV-infected cells, which indicates that the nuclear translocation of the heterotrimers was blocked. Overexpression of NSP1ß of PRRSV VR2385 inhibited expression of ISG15 and ISG56 and blocked nuclear translocation of STAT1, which suggests that NSP1ß might be the viral protein responsible for the inhibition of IFN signaling. PRRSV infection in primary porcine pulmonary alveolar macrophages (PAMs) also inhibited IFN-α-stimulated expression of the ISGs and the STAT2 protein. In contrast, a licensed low-virulence vaccine strain, Ingelvac PRRS modified live virus (MLV), activated expression of IFN-inducible genes, including those of chemokines and antiviral proteins, in PAMs without the addition of external IFN and had no detectable effect on IFN signaling. These findings suggest that PRRSV interferes with the activation and signaling pathway of type I IFNs by blocking ISG factor 3 (ISGF3) nuclear translocation.
Asunto(s)
Transporte Activo de Núcleo Celular , Interferón Tipo I/metabolismo , Virus del Síndrome Respiratorio y Reproductivo Porcino/patogenicidad , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT2/metabolismo , Animales , Células HeLa , Humanos , Factor 3 de Genes Estimulados por el Interferón/metabolismo , Macrófagos Alveolares/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Transducción de Señal , PorcinosRESUMEN
Hepatitis E virus (HEV) is one of the causative agents for liver inflammation across the world. HEV is a positive-sense single-stranded RNA virus. Human HEV strains mainly belong to four major genotypes in the genus Orthohepevirus A, family Hepeviridae. Among the four genotypes, genotype 1 and 2 are obligate human pathogens, and genotype 3 and 4 cause zoonotic infections. HEV infection with genotype 1 and 2 mainly presents as acute and self-limiting hepatitis in young adults. However, HEV infection of pregnant women with genotype 1 strains can be exacerbated to fulminant hepatitis, resulting in a high rate of case fatality. As pregnant women maintain the balance of maternal-fetal tolerance and effective immunity against invading pathogens, HEV infection with genotype 1 might dysregulate the balance and cause the adverse outcome. Furthermore, HEV infection with genotype 3 can be chronic in immunocompromised patients, with rapid progression, which has been a challenge since it was reported years ago. The virus has a complex interaction with the host cells in downregulating antiviral factors and recruiting elements to generate a conducive environment of replication. The virus-cell interactions at an early stage might determine the consequence of the infection. In this review, advances in HEV virology, viral life cycle, viral interference with the immune response, and the pathogenesis in pregnant women are discussed, and perspectives on these aspects are presented.
Asunto(s)
Virus de la Hepatitis E/genética , Virus de la Hepatitis E/fisiología , Hepatitis E/patología , Interacciones Huésped-Patógeno/fisiología , Complicaciones Infecciosas del Embarazo/virología , Femenino , Genoma Viral/genética , Genotipo , Humanos , Evasión Inmune/inmunología , Hígado/patología , Hígado/virología , Sistemas de Lectura Abierta/genética , Embarazo , ARN Viral/genética , Replicación Viral/fisiologíaRESUMEN
Hepatitis E virus (HEV) is the causative agent of hepatitis E, a major form of viral hepatitis in developing countries. The open reading frame 3 (ORF3) of HEV encodes a phosphoprotein with a molecular mass of approximately 13 kDa (hereinafter called vp13). vp13 is essential for establishing HEV infections in animals, yet its exact functions are still obscure. Our current study found evidence showing interaction between vp13 and microtubules. Live-cell confocal fluorescence microscopy revealed both filamentous and punctate distribution patterns of vp13 in cells transfected with recombinant ORF3 reporter plasmids. The filamentous pattern of vp13 was altered by a microtubule-destabilizing drug. The vp13 expression led to elevation of acetylated alpha-tubulin, indicating increased microtubule stability. Its association with microtubules was further supported by its presence in microtubule-containing pellets in microtubule isolation assays. Exposure of these pellets to a high-salt buffer caused release of the vp13 to the supernatant, suggesting an electrostatic interaction. Inclusion of ATP and GTP in the lysis buffer during microtubule isolation also disrupted the interaction, indicating its sensitivity to the nucleotides. Further assays showed that motor proteins are needed for the vp13 association with the microtubules because disruption of dynein function abolished the vp13 filamentous pattern. Analysis of ORF3 deletion constructs found that both of the N-terminal hydrophobic domains of vp13 are needed for the interaction. Thus, our findings suggest that the vp13 interaction with microtubules might be needed for establishment of an HEV infection.
Asunto(s)
Virus de la Hepatitis E/genética , Microtúbulos/metabolismo , Sistemas de Lectura Abierta , Proteínas Virales/metabolismo , Dineínas/metabolismo , Células HeLa , Virus de la Hepatitis E/metabolismo , Humanos , Microscopía Confocal , Unión Proteica , Eliminación de Secuencia , Tubulina (Proteína)/metabolismo , Proteínas Virales/genéticaRESUMEN
KPNA2/importin-alpha1 (karyopherin subunit alpha 2) is the primary nucleocytoplasmic transporter for some transcription factors to activate cellular proliferation and differentiation. Aberrant increase of KPNA2 level is identified as a prognostic marker in a variety of cancers. Yet, the turnover mechanism of KPNA2 remains unknown. Here, we demonstrate that KPNA2 is degraded via the chaperone-mediated autophagy (CMA) and that Zika virus (ZIKV) enhances the KPNA2 degradation. KPNA2 contains a CMA motif, which possesses an indispensable residue Gln109 for the CMA-mediated degradation. RNAi-mediated knockdown of LAMP2A, a vital component of the CMA pathway, led to a higher level of KPNA2. Moreover, ZIKV reduced KPNA2 via the viral NS2A protein, which contains an essential residue Thr100 for inducing the CMA-mediated KPNA2 degradation. Notably, mutant ZIKV with T100A alteration in NS2A replicates much weaker than the wild-type virus. Also, knockdown of KPNA2 led to a higher ZIKV viral yield, which indicates that KPNA2 mediates certain antiviral effects. These data provide insights into the KPNA2 turnover and the ZIKV-cell interactions.
Asunto(s)
Autofagia Mediada por Chaperones , Proteolisis , Proteínas no Estructurales Virales/metabolismo , Virus Zika/metabolismo , alfa Carioferinas/metabolismo , Secuencias de Aminoácidos , Animales , Secuencia de Bases , Línea Celular Tumoral , Chlorocebus aethiops , Glutamina/genética , Células HEK293 , Semivida , Humanos , Lisosomas/metabolismo , Mutación/genética , Relación Estructura-Actividad , Treonina/metabolismo , Células Vero , Proteínas no Estructurales Virales/química , Replicación Viral , Virus Zika/fisiología , Infección por el Virus Zika/virología , alfa Carioferinas/químicaRESUMEN
BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) causes extensive economic losses in the swine industry. Current strategies and vaccines to control the disease are inadequate. We previously demonstrated that peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) could potently inhibit PRRS virus (PRRSV) replication in cell cultures. PPMOs are single-stranded DNA analogues containing a modified backbone and cell-penetrating peptide. PPMOs are nuclease-resistant, water-soluble, can enter cells readily and exhibit highly specific binding to complementary RNA. In this study, we examined PPMO-mediated inhibition of PRRSV replication in a primary culture of porcine pulmonary alveolar macrophages (PAMs). METHODS: PAMs were collected from piglets, pre-incubated in culture and infected with PRRSV. Viability, cytopathic effects, virus yield and apoptosis of PAMs in the presence or absence of a PPMO (5UP2) were examined. The 5UP2 PPMO is complementary to a conserved sequence in the 5'-terminal region of the PRRSV genome. The level of several interferon-associated gene products and activity of caspases were monitored. RESULTS: PRRSV infection induced the activity of caspases-3/7, -8 and -9 significantly. Treatment of PAMs with 5UP2 resulted in protection of the cells from PRRSV-induced cell death for at least 7 days and avoided the activation of the caspases evaluated. 5UP2 treatment of PRRSV-infected PAMs also prevented the vigorous induction of interferon-beta and chemokines observed in infected and mock-treated PAMs. CONCLUSIONS: PPMO-mediated suppression of PRRSV replication in PAMs was associated with a reduction of apoptotic and inflammatory responses. These results provide further rationale for the development of PPMO 5UP2 as an antiviral to control PRRSV infection.
Asunto(s)
Macrófagos Alveolares/efectos de los fármacos , Morfolinas/farmacología , Síndrome Respiratorio y de la Reproducción Porcina/tratamiento farmacológico , Virus del Síndrome Respiratorio y Reproductivo Porcino/efectos de los fármacos , Animales , Caspasas/biosíntesis , Técnicas de Cultivo de Célula , Muerte Celular/efectos de los fármacos , Quimiocinas/biosíntesis , Quimiocinas/genética , Inducción Enzimática/efectos de los fármacos , Regulación Viral de la Expresión Génica/efectos de los fármacos , Interacciones Huésped-Patógeno/efectos de los fármacos , Interferón beta/biosíntesis , Interferón beta/genética , Macrófagos Alveolares/fisiología , Macrófagos Alveolares/virología , Morfolinos , Síndrome Respiratorio y de la Reproducción Porcina/metabolismo , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Porcinos , Replicación Viral/efectos de los fármacosRESUMEN
Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8, is associated with several malignant disorders, including Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease. An early lytic gene of KSHV encodes viral interleukin-6 (vIL-6), a viral homologue of the proinflammatory cytokine and an autocrine/paracrine growth factor human IL-6. In this study, we examined the effects of suppressing vIL-6 expression in PEL cells with antisense peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO). PPMO are ssDNA-analogues that have a modified backbone and enter cells readily. Treatment of PEL cells with a PPMO designed against vIL-6 mRNA led to a marked reduction in the proportion of vIL-6-positive cells detected by immunofluorescence assay. Analysis by Western blot confirmed a specific reduction in the vIL-6 protein level and showed that the reduction was dependent on the dose of vIL-6 PPMO. PEL cells treated with the vIL-6 PPMO exhibited reduced levels of cellular growth, IL-6 expression and KSHV DNA, and an elevated level of p21 protein. Treatment of PEL cells with a combination of two vIL-6 PPMO compounds targeting different sequences in the vIL-6 mRNA led to an inhibitory effect that was greater than that achieved with either PPMO alone. These results show that PPMO targeting vIL-6 mRNA can potently reduce vIL-6 protein translation and indicate that further exploration of these compounds in an animal model for potential clinical application is warranted.
Asunto(s)
Herpesvirus Humano 8/fisiología , Interleucina-6/antagonistas & inhibidores , Polímeros/farmacología , Western Blotting , Línea Celular Tumoral , Técnica del Anticuerpo Fluorescente , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , ARN Mensajero/genética , Replicación ViralRESUMEN
Hepatitis E virus (HEV) causes predominantly acute and self-limiting hepatitis. However, in HEV-infected pregnant women, the case fatality rate because of fulminant hepatitis can be up to 30%. HEV infection is zoonotic for some genotypes. The HEV genome contains three open reading frames: ORF1 encodes the non-structural polyprotein involved in viral RNA replication; ORF2 encodes the capsid protein; ORF3 encodes a small multifunctional protein. Interferons (IFNs) play a significant role in the early stage of the host antiviral response. In this study, we discovered that the capsid protein antagonizes IFN induction. Mechanistically, the capsid protein blocked the phosphorylation of IFN regulatory factor 3 (IRF3) via interaction with the multiprotein complex consisting of mitochondrial antiviral-signaling protein (MAVS), TANK-binding kinase 1 (TBK1), and IRF3. The N-terminal domain of the capsid protein was found to be responsible for the inhibition of IRF3 activation. Further study showed that the arginine-rich-motif in the N-terminal domain is indispensable for the inhibition as mutations of any of the arginine residues abolished the blockage of IRF3 phosphorylation. These results provide further insight into HEV interference with the host innate immunity.
Asunto(s)
Proteínas de la Cápside/metabolismo , Virus de la Hepatitis E/fisiología , Hepatitis E/metabolismo , Hepatitis E/virología , Interacciones Huésped-Patógeno , Interferones/biosíntesis , Dominios y Motivos de Interacción de Proteínas , Proteínas de la Cápside/genética , Genotipo , Humanos , Factor 3 Regulador del Interferón/metabolismo , Interferones/química , Modelos Biológicos , Factores de Crecimiento Nervioso , Fosforilación , Poli I-C/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Zika virus (ZIKV) is a mosquito-borne positive-sense single-stranded RNA virus in the family of Flaviviridae. Unlike other flaviviruses, ZIKV infection of pregnant women may result in birth defects in their newborns, such as microcephaly or vision problem. ZIKV is known to antagonize the interferon (IFN) production in infected cells. However, the exact mechanism of this interference is not fully understood. Here, we demonstrate that NS5 protein of ZIKV MR766 strain antagonizes IFN production through inhibiting the activation of TANK-binding kinase 1 (TBK1), which phosphorylates the transcription activator IFN regulatory factor 3 (IRF3). Mechanistically, NS5 interacts with the ubiquitin-like domain of TBK1 and results in less complex of TBK1 and TNF (tumor necrosis factor) receptor-associated factor 6 (TRAF6), leading to dampened TBK1 activation and IRF3 phosphorylation. Our study provides insights into the mechanism of ZIKV evasion of IFN-mediated innate immunity.
Asunto(s)
Interferón beta/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas no Estructurales Virales/metabolismo , Infección por el Virus Zika/metabolismo , Virus Zika/fisiología , Dominio Catalítico , Línea Celular , Humanos , Inmunidad Innata , Factor 3 Regulador del Interferón/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/química , Transducción de Señal , Factor 6 Asociado a Receptor de TNF/metabolismo , Proteínas no Estructurales Virales/química , Infección por el Virus Zika/virologíaRESUMEN
Porcine reproductive and respiratory syndrome (PRRS) has been devastating the global swine industry for more than a decade, and current strategies to control PRRS are inadequate. In this study we characterized the inhibition of PRRS virus (PRRSV) replication by antisense phosphorodiamidate morpholino oligomers (PMO). Of 12 peptide-conjugated PMO (PPMO), four were found to be highly effective at inhibiting PRRSV replication in cell culture in a dose-dependant and sequence-specific manner. PPMO 5UP2 and 5HP are complementary to sequence in the 5' end of the PRRSV genome, and 6P1 and 7P1 to sequence in the translation initiation regions of ORF6 and ORF7, respectively. Treatment of cells with 5UP2 or 5HP caused a 4.5log(10) reduction in PRRSV yield, compared to a control PPMO. Combination of 6P1 and 7P1 led to higher level reduction than 6P1 or 7P1 alone. 5UP2, 5HP, and a combination of 6P1 and 7P1 inhibited PRRSV replication in porcine alveolar macrophages and protected the cells from PRRSV-induced cytopathic effect. Northern blot and real-time RT-PCR results demonstrated that the effective PPMO led to a reduction of PRRSV RNA level. 5UP2 and 5HP inhibited virus replication of 10 other strains of PRRSV. Results from this study suggest potential applications of PPMO for PRRS control.
Asunto(s)
Antivirales/farmacología , Morfolinas/farmacología , Péptidos/farmacología , Virus del Síndrome Respiratorio y Reproductivo Porcino/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Animales , Secuencia de Bases , Línea Celular , Supervivencia Celular/efectos de los fármacos , Sistema Libre de Células , Técnica del Anticuerpo Fluorescente Indirecta , Genes Reporteros , Humanos , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/virología , Ratones , Morfolinas/síntesis química , Morfolinos , Oligonucleótidos Antisentido/farmacología , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas/efectos de los fármacos , ARN Viral/biosíntesis , Alineación de Secuencia , PorcinosRESUMEN
Interferons (IFNs) are a group of secreted proteins that play critical roles in antiviral immunity, antitumor activity, activation of cytotoxic T cells, and modulation of host immune responses. IFNs are cytokines, and bind receptors on cell surfaces to trigger signal transduction. The major signaling pathway activated by IFNs is the JAK/STAT (Janus kinase/signal transducer and activator of transcription) pathway, a complex pathway involved in both viral and host survival strategies. On the one hand, viruses have evolved strategies to escape from antiviral host defenses evoked by IFN-activated JAK/STAT signaling. On the other hand, viruses have also evolved to exploit the JAK/STAT pathway to evoke activation of certain STATs that somehow promote viral pathogenesis. In this review, recent progress in our understanding of the virus-induced IFN-independent STAT signaling and its potential roles in viral induced inflammation and pathogenesis are summarized in detail, and perspectives are provided.
Asunto(s)
Interacciones Huésped-Patógeno , Inflamación/patología , Inflamación/virología , Factores de Transcripción STAT/biosíntesis , Transducción de Señal , Virosis/patología , Virosis/virología , Animales , HumanosRESUMEN
Phosphorodiamidate morpholino oligomers (PMO) are short single-stranded DNA analogs that are built upon a backbone of morpholine rings connected by phosphorodiamidate linkages. As uncharged nucleic acid analogs, PMO bind to complementary sequences of target mRNA by Watson-Crick base pairing to block protein translation through steric blockade. PMO interference of viral protein translation operates independently of RNase H. Meanwhile, PMO are resistant to a variety of enzymes present in biologic fluids, a characteristic that makes them highly suitable for in vivo applications. Notably, PMO-based therapy for Duchenne muscular dystrophy (DMD) has been approved by the United States Food and Drug Administration which is now a hallmark for PMO-based antisense therapy. In this review, the development history of PMO, delivery methods for improving cellular uptake of neutrally charged PMO molecules, past studies of PMO antagonism against RNA and DNA viruses, PMO target selection, and remaining questions of PMO antiviral strategies are discussed in detail and new insights are provided.
RESUMEN
Porcine reproductive and respiratory syndrome virus (PRRSV) is a member of the family Arteriviridae, order Nidovirale. PRRSV is an enveloped, single-stranded, positive-sense RNA virus with a genome around 15 kb in length. For propagation of PRRSV in vitro, the MARC-145 cell line is the most often used in a laboratory setting. Infectious cDNA clones of many PRRSV strains have been established, from which these viruses can be recovered. PRRSV titration is generally done in MARC-145 cells. PRRSV RNA copy numbers can be assessed by reverse transcription and real-time PCR. Here, protocols for PRRSV propagation, virus recovery from infectious cDNA clones, and quantification are presented. © 2018 by John Wiley & Sons, Inc.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Reacción en Cadena de la Polimerasa/métodos , Síndrome Respiratorio y de la Reproducción Porcina/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/crecimiento & desarrollo , Cultivo de Virus/métodos , Animales , Línea Celular , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Porcinos , Replicación ViralRESUMEN
Hepatitis E virus (HEV) predominantly causes acute liver disease in humans and is transmitted via the fecal-oral route. HEV infection in pregnant women can result in grave consequences, with up to 30% fatality. The HEV strains infecting humans mainly belong to four genotypes. Genotypes 1 and 2 are restricted to human infection, while genotypes 3 and 4 are zoonotic. HEV genotype 3 (HEV-3) can cause both acute and chronic liver diseases. Several cell lines (mainly hepatocytes) have been developed for HEV propagation and biological study. However, HEV production in these cell lines is suboptimal and inefficient. Here, we present methods for the isolation, propagation, and quantification of HEV. © 2018 by John Wiley & Sons, Inc.