RESUMEN
Coronavirus (CoV) replication requires efficient cleavage of viral polyproteins into an array of non-structural proteins involved in viral replication, organelle formation, viral RNA synthesis, and host shutoff. Human CoVs (HCoVs) encode two viral cysteine proteases, main protease (Mpro) and papain-like protease (PLpro), that mediate polyprotein cleavage. Using a structure-guided approach, a phenothiazine urea derivative that inhibits both SARS-CoV-2 Mpro and PLpro protease activity was identified. In silico docking studies also predicted the binding of the phenothiazine urea to the active sites of structurally similar Mpro and PLpro proteases from distantly related alphacoronavirus, HCoV-229 E (229 E), and the betacoronavirus, HCoV-OC43 (OC43). The lead phenothiazine urea derivative displayed broad antiviral activity against all three HCoVs tested in cellulo. It was further demonstrated that the compound inhibited 229 E and OC43 at an early stage of viral replication, with diminished formation of viral replication organelles, and the RNAs that are made within them, as expected following viral protease inhibition. These observations suggest that the phenothiazine urea derivative readily inhibits viral replication and may broadly inhibit proteases of diverse coronaviruses.
Asunto(s)
Péptido Hidrolasas , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Papaína/química , Proteasas Virales , Fenotiazinas/farmacología , Inhibidores de Proteasas/química , Antivirales/farmacología , Antivirales/químicaRESUMEN
There is an outstanding need for broadly acting antiviral drugs to combat emerging viral diseases. Here, we report that thiopurines inhibit the replication of the betacoronaviruses HCoV-OC43 and SARS-CoV-2. 6-Thioguanine (6-TG) disrupted early stages of infection, limiting accumulation of full-length viral genomes, subgenomic RNAs and structural proteins. In ectopic expression models, we observed that 6-TG increased the electrophoretic mobility of Spike from diverse betacoronaviruses, matching the effects of enzymatic removal of N-linked oligosaccharides from Spike in vitro. SARS-CoV-2 virus-like particles (VLPs) harvested from 6-TG-treated cells were deficient in Spike. 6-TG treatment had a similar effect on production of lentiviruses pseudotyped with SARS-CoV-2 Spike, yielding pseudoviruses deficient in Spike and unable to infect ACE2-expressing cells. Together, these findings from complementary ectopic expression and infection models strongly indicate that defective Spike trafficking and processing is an outcome of 6-TG treatment. Using biochemical and genetic approaches we demonstrated that 6-TG is a pro-drug that must be converted to the nucleotide form by hypoxanthine phosphoribosyltransferase 1 (HPRT1) to achieve antiviral activity. This nucleotide form has been shown to inhibit small GTPases Rac1, RhoA, and CDC42; however, we observed that selective chemical inhibitors of these GTPases had no effect on Spike processing or accumulation. By contrast, the broad GTPase agonist ML099 countered the effects of 6-TG, suggesting that the antiviral activity of 6-TG requires the targeting of an unknown GTPase. Overall, these findings suggest that small GTPases are promising targets for host-targeted antivirals.
Asunto(s)
COVID-19 , Proteínas de Unión al GTP Monoméricas , Profármacos , Enzima Convertidora de Angiotensina 2 , Antivirales/química , Antivirales/farmacología , Humanos , Hipoxantina Fosforribosiltransferasa/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Nucleótidos/metabolismo , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/metabolismo , Tioguanina , Virión/metabolismoRESUMEN
Photodynamic inactivation (PDI) employs a photosensitizer, light, and oxygen to create a local burst of reactive oxygen species (ROS) that can inactivate microorganisms. The botanical extract PhytoQuinTM is a powerful photosensitizer with antimicrobial properties. We previously demonstrated that photoactivated PhytoQuin also has antiviral properties against herpes simplex viruses and adenoviruses in a dose-dependent manner across a broad range of sub-cytotoxic concentrations. Here, we report that human coronaviruses (HCoVs) are also susceptible to photodynamic inactivation. Photoactivated-PhytoQuin inhibited the replication of the alphacoronavirus HCoV-229E and the betacoronavirus HCoV-OC43 in cultured cells across a range of sub-cytotoxic doses. This antiviral effect was light-dependent, as we observed minimal antiviral effect of PhytoQuin in the absence of photoactivation. Using RNase protection assays, we observed that PDI disrupted HCoV particle integrity allowing for the digestion of viral RNA by exogenous ribonucleases. Using lentiviruses pseudotyped with the SARS-CoV-2 Spike (S) protein, we once again observed a strong, light-dependent antiviral effect of PhytoQuin, which prevented S-mediated entry into human cells. We also observed that PhytoQuin PDI altered S protein electrophoretic mobility. The PhytoQuin constituent emodin displayed equivalent light-dependent antiviral activity to PhytoQuin in matched-dose experiments, indicating that it plays a central role in PhytoQuin PDI against CoVs. Together, these findings demonstrate that HCoV lipid envelopes and proteins are damaged by PhytoQuin PDI and expands the list of susceptible viruses.
Asunto(s)
Antivirales/farmacología , Coronavirus/efectos de los fármacos , Fármacos Fotosensibilizantes/farmacología , Inactivación de Virus/efectos de los fármacos , Animales , Antivirales/efectos de la radiación , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cricetinae , Emodina/farmacología , Emodina/efectos de la radiación , Humanos , Luz , Fármacos Fotosensibilizantes/efectos de la radiación , Extractos Vegetales/farmacología , Extractos Vegetales/efectos de la radiación , SARS-CoV-2/efectos de los fármacos , Glicoproteína de la Espiga del Coronavirus/efectos de los fármacos , Virión/efectos de los fármacosRESUMEN
Influenza A viruses (IAVs) utilize host shutoff mechanisms to limit antiviral gene expression and redirect translation machinery to the synthesis of viral proteins. Previously, we showed that IAV replication is sensitive to protein synthesis inhibitors that block translation initiation and induce formation of cytoplasmic condensates of untranslated messenger ribonucleoprotein complexes called stress granules (SGs). In this study, using an image-based high-content screen, we identified two thiopurines, 6-thioguanine (6-TG) and 6-thioguanosine (6-TGo), that triggered SG formation in IAV-infected cells and blocked IAV replication in a dose-dependent manner without eliciting SG formation in uninfected cells. 6-TG and 6-TGo selectively disrupted the synthesis and maturation of IAV glycoproteins hemagglutinin (HA) and neuraminidase (NA) without affecting the levels of the viral RNAs that encode them. By contrast, these thiopurines had minimal effect on other IAV proteins or the global host protein synthesis. Disruption of IAV glycoprotein accumulation by 6-TG and 6-TGo correlated with activation of unfolded protein response (UPR) sensors activating transcription factor-6 (ATF6), inositol requiring enzyme-1 (IRE1) and PKR-like endoplasmic reticulum kinase (PERK), leading to downstream UPR gene expression. Treatment of infected cells with the chemical chaperone 4-phenylbutyric acid diminished thiopurine-induced UPR activation and partially restored the processing and accumulation of HA and NA. By contrast, chemical inhibition of the integrated stress response downstream of PERK restored accumulation of NA monomers but did not restore processing of viral glycoproteins. Genetic deletion of PERK enhanced the antiviral effect of 6-TG without causing overt cytotoxicity, suggesting that while UPR activation correlates with diminished viral glycoprotein accumulation, PERK could limit the antiviral effects of drug-induced ER stress. Taken together, these data indicate that 6-TG and 6-TGo are effective host-targeted antivirals that trigger the UPR and selectively disrupt accumulation of viral glycoproteins.IMPORTANCESecreted and transmembrane proteins are synthesized in the endoplasmic reticulum (ER), where they are folded and modified prior to transport. Many viruses rely on the ER for the synthesis and processing of viral glycoproteins that will ultimately be incorporated into viral envelopes. Viral burden on the ER can trigger the unfolded protein response (UPR). Much remains to be learned about how viruses co-opt the UPR to ensure efficient synthesis of viral glycoproteins. Here, we show that two FDA-approved thiopurine drugs, 6-TG and 6-TGo, induce the UPR, which represents a previously unrecognized effect of these drugs on cell physiology. This thiopurine-mediated UPR activation blocks influenza virus replication by impeding viral glycoprotein accumulation. Our findings suggest that 6-TG and 6-TGo may have broad antiviral effect against enveloped viruses that require precise tuning of the UPR to support viral glycoprotein synthesis.
RESUMEN
Glioblastoma (GBM) is the most common primary malignant brain tumor, and it has a uniformly poor prognosis. Hypoxia is a feature of the GBM microenvironment, and previous work has shown that cancer cells residing in hypoxic regions resist treatment. Hypoxia can trigger the formation of stress granules (SGs), sites of mRNA triage that promote cell survival. A screen of 1120 FDA-approved drugs identified 129 candidates that delayed the dissolution of hypoxia-induced SGs following a return to normoxia. Amongst these candidates, the selective estrogen receptor modulator (SERM) raloxifene delayed SG dissolution in a dose-dependent manner. SG dissolution typically occurs by 15 min post-hypoxia, however pre-treatment of immortalized U251 and U3024 primary GBM cells with raloxifene prevented SG dissolution for up to 2 h. During this raloxifene-induced delay in SG dissolution, translational silencing was sustained, eIF2α remained phosphorylated and mTOR remained inactive. Despite its well-described role as a SERM, raloxifene-mediated delay in SG dissolution was unaffected by co-administration of ß-estradiol, nor did ß-estradiol alone have any effect on SGs. Importantly, the combination of raloxifene and hypoxia resulted in increased numbers of late apoptotic/necrotic cells. Raloxifene and hypoxia also demonstrated a block in late autophagy similar to the known autophagy inhibitor chloroquine (CQ). Genetic disruption of the SG-nucleating proteins G3BP1 and G3BP2 revealed that G3BP1 is required to sustain the raloxifene-mediated delay in SG dissolution. Together, these findings indicate that modulating the stress response can be used to exploit the hypoxic niche of GBM tumors, causing cell death by disrupting pro-survival stress responses and control of protein synthesis.
Asunto(s)
Antagonistas de Estrógenos/uso terapéutico , Glioblastoma/tratamiento farmacológico , Clorhidrato de Raloxifeno/uso terapéutico , Muerte Celular , Antagonistas de Estrógenos/farmacología , Humanos , Clorhidrato de Raloxifeno/farmacologíaRESUMEN
PURPOSE: Management of soft tissue and bone sarcoma presents many challenges, both diagnostically and therapeutically, and requires multidisciplinary collaboration; however, such collaboration is often challenging to establish, especially in the community setting. We share our experiences of a virtual multidisciplinary sarcoma case conference (VMSCC). METHODS: We conducted retrospective review of the VMSCC data-initially via Webex, now Microsoft Teams-and the surveys of referring physicians to understand the feasibility and value of the VMSCC. RESULTS: The VMSCC was established in March 2013 in Kaiser Permanente Northern California with consistent participation of the Departments of Musculoskeletal Oncology (orthopedic oncology), Musculoskeletal Radiology, Pathology, Medical Oncology, Radiation Oncology, Nuclear Medicine, Surgical Oncology, and Genetics. Pediatric Oncology participated ad hoc when pediatric sarcoma cases were presented. Referrals were from multiple specialties and regions, including the Kaiser Permanente Mid-Atlantic and Hawaii regions. From March 2013 to December 2019, 1,585 cases were reviewed encompassing 36 histologic types. More than 300 cases were reviewed per year from 2017 to 2019. Survey results of referring physicians demonstrate that the VMSCC enhanced the confidence of treating physicians, and its recommendations frequently led to treatment changes. CONCLUSION: Establishing a valuable community-based VMSCC is feasible. VMSCC recommendations frequently led to treatment changes and improved the confidence of treating physicians.
Asunto(s)
Oncología Médica/organización & administración , Sarcoma , Comunicación por Videoconferencia/organización & administración , Niño , Estudios de Factibilidad , Hawaii , Humanos , Estudios Retrospectivos , Sarcoma/diagnóstico , Sarcoma/terapiaRESUMEN
Basic leucine zipper (bZIP) transcription factors (TFs) govern diverse cellular processes and cell fate decisions. The hallmark of the leucine zipper domain is the heptad repeat, with leucine residues at every seventh position in the domain. These leucine residues enable homo- and heterodimerization between ZIP domain α-helices, generating coiled-coil structures that stabilize interactions between adjacent DNA-binding domains and target DNA substrates. Several cancer-causing viruses encode viral bZIP TFs, including human T-cell leukemia virus (HTLV), hepatitis C virus (HCV) and the herpesviruses Marek's disease virus (MDV), Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV). Here, we provide a comprehensive review of these viral bZIP TFs and their impact on viral replication, host cell responses and cell fate.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/fisiología , Virus Oncogénicos/metabolismo , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Deltaretrovirus/genética , Deltaretrovirus/metabolismo , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/metabolismo , Humanos , Mardivirus/genética , Mardivirus/metabolismo , Filogenia , Infecciones Tumorales por Virus/metabolismo , Infecciones Tumorales por Virus/virología , Respuesta de Proteína DesplegadaRESUMEN
PURPOSE: Management of soft tissue and bone sarcoma presents many challenges, both diagnostically and therapeutically, and requires multidisciplinary collaboration; however, such collaboration is often challenging to establish, especially in the community setting. We share our experiences of a virtual multidisciplinary sarcoma case conference (VMSCC). METHODS: We conducted retrospective review of the VMSCC data-initially via Webex, now Microsoft Teams-and the surveys of referring physicians to understand the feasibility and value of the VMSCC. RESULTS: The VMSCC was established in March 2013 in Kaiser Permanente Northern California with consistent participation of the Departments of Musculoskeletal Oncology (orthopedic oncology), Musculoskeletal Radiology, Pathology, Medical Oncology, Radiation Oncology, Nuclear Medicine, Surgical Oncology, and Genetics. Pediatric Oncology participated ad hoc when pediatric sarcoma cases were presented. Referrals were from multiple specialties and regions, including the Kaiser Permanente Mid-Atlantic and Hawaii regions. From March 2013 to December 2019, 1,585 cases were reviewed encompassing 36 histologic types. More than 300 cases were reviewed per year from 2017 to 2019. Survey results of referring physicians demonstrate that the VMSCC enhanced the confidence of treating physicians, and its recommendations frequently led to treatment changes. CONCLUSION: Establishing a valuable community-based VMSCC is feasible. VMSCC recommendations frequently led to treatment changes and improved the confidence of treating physicians.
RESUMEN
Influenza A virus (IAV) increases the presentation of class I human leukocyte antigen (HLA) proteins that limit antiviral responses mediated by natural killer (NK) cells, but molecular mechanisms for these processes have not yet been fully elucidated. We observed that infection with A/Fort Monmouth/1/1947(H1N1) IAV significantly increased the presentation of HLA-B, -C, and -E on lung epithelial cells. Virus entry was not sufficient to induce HLA upregulation because UV-inactivated virus had no effect. Aberrant internally deleted viral RNAs (vRNAs) known as mini viral RNAs (mvRNAs) and defective interfering RNAs (DI RNAs) expressed from an IAV minireplicon were sufficient for inducing HLA upregulation. These defective RNAs bind to retinoic acid-inducible gene I (RIG-I) and initiate mitochondrial antiviral signaling (MAVS) protein-dependent antiviral interferon (IFN) responses. Indeed, MAVS was required for HLA upregulation in response to IAV infection or ectopic mvRNA/DI RNA expression. The effect was partially due to paracrine signaling, as we observed that IAV infection or mvRNA/DI RNA-expression stimulated production of IFN-ß and IFN-λ1 and conditioned media from these cells elicited a modest increase in HLA surface levels in naive epithelial cells. HLA upregulation in response to aberrant viral RNAs could be prevented by the Janus kinase (JAK) inhibitor ruxolitinib. While HLA upregulation would seem to be advantageous to the virus, it is kept in check by the viral nonstructural 1 (NS1) protein; we determined that NS1 limits cell-intrinsic and paracrine mechanisms of HLA upregulation. Taken together, our findings indicate that aberrant IAV RNAs stimulate HLA presentation, which may aid viral evasion of innate immunity.IMPORTANCE Human leukocyte antigens (HLAs) are cell surface proteins that regulate innate and adaptive immune responses to viral infection by engaging with receptors on immune cells. Many viruses have evolved ways to evade host immune responses by modulating HLA expression and/or processing. Here, we provide evidence that aberrant RNA products of influenza virus genome replication can trigger retinoic acid-inducible gene I (RIG-I)/mitochondrial antiviral signaling (MAVS)-dependent remodeling of the cell surface, increasing surface presentation of HLA proteins known to inhibit the activation of an immune cell known as a natural killer (NK) cell. While this HLA upregulation would seem to be advantageous to the virus, it is kept in check by the viral nonstructural 1 (NS1) protein, which limits RIG-I activation and interferon production by the infected cell.
Asunto(s)
Genes MHC Clase I/genética , Antígenos HLA/metabolismo , Subtipo H1N1 del Virus de la Influenza A/genética , Células A549 , Proteínas Adaptadoras Transductoras de Señales/genética , Proteína 58 DEAD Box/genética , Bases de Datos Genéticas , Células Epiteliales/virología , Interacciones Huésped-Patógeno/genética , Humanos , Inmunidad Innata , Virus de la Influenza A/genética , Gripe Humana/genética , Células Asesinas Naturales/metabolismo , Pulmón/virología , ARN Viral/genética , Transducción de Señal , Activación Transcripcional , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/genéticaRESUMEN
Herpesviruses usurp cellular stress responses to promote viral replication and avoid immune surveillance. The unfolded protein response (UPR) is a conserved stress response that is activated when the protein load in the ER exceeds folding capacity and misfolded proteins accumulate. The UPR aims to restore protein homeostasis through translational and transcriptional reprogramming; if homeostasis cannot be restored, the UPR switches from "helper" to "executioner", triggering apoptosis. It is thought that the burst of herpesvirus glycoprotein synthesis during lytic replication causes ER stress, and that these viruses may have evolved mechanisms to manage UPR signaling to create an optimal niche for replication. The past decade has seen considerable progress in understanding how herpesviruses reprogram the UPR. Here we provide an overview of the molecular events of UPR activation, signaling and transcriptional outputs, and highlight key evidence that herpesviruses hijack the UPR to aid infection.
Asunto(s)
Infecciones por Herpesviridae/metabolismo , Infecciones por Herpesviridae/virología , Herpesviridae/fisiología , Interacciones Huésped-Patógeno , Respuesta de Proteína Desplegada , Factor de Transcripción Activador 6/genética , Factor de Transcripción Activador 6/metabolismo , Animales , Susceptibilidad a Enfermedades , Herpesviridae/clasificación , Humanos , Membranas Intracelulares/metabolismo , ProteolisisRESUMEN
Kaposi's sarcoma associated-herpesvirus (KSHV, also known as human herpesvirus-8) is a gammaherpesvirus that establishes life-long infection in human B lymphocytes. KSHV infection is typically asymptomatic, but immunosuppression can predispose KSHV-infected individuals to primary effusion lymphoma (PEL); a malignancy driven by aberrant proliferation of latently infected B lymphocytes, and supported by pro-inflammatory cytokines and angiogenic factors produced by cells that succumb to lytic viral replication. Here, we report the development of the first in vivo model for a virally induced lymphoma in zebrafish, whereby KSHV-infected PEL tumor cells engraft and proliferate in the yolk sac of zebrafish larvae. Using a PEL cell line engineered to produce the viral lytic switch protein RTA in the presence of doxycycline, we demonstrate drug-inducible reactivation from KSHV latency in vivo, which enabled real-time observation and evaluation of latent and lytic phases of KSHV infection. In addition, we developed a sensitive droplet digital PCR method to monitor latent and lytic viral gene expression and host cell gene expression in xenografts. The zebrafish yolk sac is not well vascularized, and by using fluorogenic assays, we confirmed that this site provides a hypoxic environment that may mimic the microenvironment of some human tumors. We found that PEL cell proliferation in xenografts was dependent on the host hypoxia-dependent translation initiation factor, eukaryotic initiation factor 4E2 (eIF4E2). This demonstrates that the zebrafish yolk sac is a functionally hypoxic environment, and xenografted cells must switch to dedicated hypoxic gene expression machinery to survive and proliferate. The establishment of the PEL xenograft model enables future studies that exploit the innate advantages of the zebrafish as a model for genetic and pharmacologic screens.
Asunto(s)
Susceptibilidad a Enfermedades , Infecciones por Herpesviridae/complicaciones , Infecciones por Herpesviridae/virología , Herpesvirus Humano 8/fisiología , Sarcoma de Kaposi/virología , Animales , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Xenoinjertos , Humanos , Pez CebraRESUMEN
Herpesviruses usurp host cell protein synthesis machinery to convert viral mRNAs into proteins, and the endoplasmic reticulum (ER) to ensure proper folding, post-translational modification and trafficking of secreted and transmembrane viral proteins. Overloading ER folding capacity activates the unfolded protein response (UPR), whereby sensor proteins ATF6, PERK and IRE1 initiate a stress-mitigating transcription program that accelerates catabolism of misfolded proteins while increasing ER folding capacity. Kaposi's sarcoma-associated herpesvirus (KSHV) can be reactivated from latency by chemical induction of ER stress, which causes accumulation of the XBP1s transcription factor that transactivates the viral RTA lytic switch gene. The presence of XBP1s-responsive elements in the RTA promoter suggests that KSHV evolved a mechanism to respond to ER stress. Here, we report that ATF6, PERK and IRE1 were activated upon reactivation from latency and required for efficient KSHV lytic replication; genetic or pharmacologic inhibition of each UPR sensor diminished virion production. Despite UPR sensor activation during KSHV lytic replication, downstream UPR transcriptional responses were restricted; 1) ATF6 was cleaved to activate the ATF6(N) transcription factor but ATF6(N)-responsive genes were not transcribed; 2) PERK phosphorylated eIF2α but ATF4 did not accumulate; 3) IRE1 caused XBP1 mRNA splicing, but XBP1s protein did not accumulate and XBP1s-responsive genes were not transcribed. Ectopic expression of the KSHV host shutoff protein SOX did not affect UPR gene expression, suggesting that alternative viral mechanisms likely mediate UPR suppression during lytic replication. Complementation of XBP1s deficiency during KSHV lytic replication inhibited virion production in a dose-dependent manner in iSLK.219 cells but not in TREx-BCBL1-RTA cells. However, genetically distinct KSHV virions harvested from these two cell lines were equally susceptible to XBP1s restriction following infection of naïve iSLK cells. This suggests that cell-intrinsic properties of BCBL1 cells may circumvent the antiviral effect of ectopic XBP1s expression. Taken together, these findings indicate that while XBP1s plays an important role in reactivation from latency, it can inhibit virus replication at a later step, which the virus overcomes by preventing its synthesis. These findings suggest that KSHV hijacks UPR sensors to promote efficient viral replication while sustaining ER stress.
Asunto(s)
Herpesvirus Humano 8/metabolismo , Respuesta de Proteína Desplegada/fisiología , Activación Viral/fisiología , Latencia del Virus/fisiología , Replicación Viral/fisiología , Línea Celular , Estrés del Retículo Endoplásmico/fisiología , Infecciones por Herpesviridae/virología , HumanosRESUMEN
Pseudomonas aeruginosa is an opportunistic pathogen that is a common cause of nosocomial infections. The molecular mechanisms governing immune responses to P. aeruginosa infection remain incompletely defined. Early growth response 1 (Egr-1) is a zinc-finger transcription factor that controls inflammatory responses. Here, we characterized the role of Egr-1 in host defense against P. aeruginosa infection in a mouse model of acute bacterial pneumonia. Egr-1 expression was rapidly and transiently induced in response to P. aeruginosa infection. Egr-1-deficient mice displayed decreased mortality, reduced levels of proinflammatory cytokines (tumor necrosis factor [TNF], interleukin-1ß [IL-1ß], IL-6, IL-12, and IL-17), and enhanced bacterial clearance from the lung. Egr-1 deficiency caused diminished NF-κB activation in P. aeruginosa-infected macrophages independently of IκBα phosphorylation. A physical interaction between Egr-1 and NF-κB p65 was found in P. aeruginosa-infected macrophages, suggesting that Egr-1 could be required for assembly of heterodimeric transcription factors that direct synthesis of inflammatory mediators. Interestingly, Egr-1 deficiency had no impact on neutrophil recruitment in vivo due to its differential effects on chemokine production, which included diminished accumulation of KC (CXCL1), MIP2 (CXCL2), and IP-10 (CXCL10) and increased accumulation of LIX (CXCL5). Importantly, Egr-1-deficient macrophages and neutrophils displayed significant increases in nitric oxide production and bacterial killing ability that correlated with enhanced bacterial clearance in Egr-1-deficient mice. Together, these findings suggest that Egr-1 plays a detrimental role in host defense against P. aeruginosa acute lung infection by promoting systemic inflammation and negatively regulating the nitric oxide production that normally assists with bacterial clearance.
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Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Pulmón/microbiología , Pulmón/patología , Neumonía Bacteriana/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Animales , Citocinas/análisis , Modelos Animales de Enfermedad , Proteína 1 de la Respuesta de Crecimiento Precoz/deficiencia , Factores Inmunológicos/análisis , Ratones , Análisis de SupervivenciaRESUMEN
Mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cellular metabolism. In nutrient-rich environments, mTORC1 kinase activity stimulates protein synthesis to meet cellular anabolic demands. Under nutrient-poor conditions or under stress, mTORC1 is rapidly inhibited, global protein synthesis is arrested, and a cellular catabolic process known as autophagy is activated. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes multiple proteins that stimulate mTORC1 activity or subvert autophagy, but precise roles for mTORC1 in different stages of KSHV infection remain incompletely understood. Here, we report that during latent and lytic stages of KSHV infection, chemical inhibition of mTORC1 caused eukaryotic initiation factor 4F (eIF4F) disassembly and diminished global protein synthesis, which indicated that mTORC1-mediated control of translation initiation was largely intact. We observed that mTORC1 was required for synthesis of the replication and transcription activator (RTA) lytic switch protein and reactivation from latency, but once early lytic gene expression had begun, mTORC1 was not required for genome replication, late gene expression, or the release of infectious progeny. Moreover, mTORC1 control of autophagy was dysregulated during lytic replication, whereby chemical inhibition of mTORC1 prevented ULK1 phosphorylation but did not affect autophagosome formation or rates of autophagic flux. Together, these findings suggest that mTORC1 is dispensable for viral protein synthesis and viral control of autophagy during lytic infection and that KSHV undermines mTORC1-dependent cellular processes during the lytic cycle to ensure efficient viral replication.IMPORTANCE All viruses require host cell machinery to synthesize viral proteins. A host cell protein complex known as mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of protein synthesis. Under nutrient-rich conditions, mTORC1 is active and promotes protein synthesis to meet cellular anabolic demands. Under nutrient-poor conditions or under stress, mTORC1 is rapidly inhibited, global protein synthesis is arrested, and a cellular catabolic process known as autophagy is activated. Kaposi's sarcoma-associated herpesvirus (KSHV) stimulates mTORC1 activity and utilizes host machinery to synthesize viral proteins. However, we discovered that mTORC1 activity was largely dispensable for viral protein synthesis, genome replication, and the release of infectious progeny. Likewise, during lytic replication, mTORC1 was no longer able to control autophagy. These findings suggest that KSHV undermines mTORC1-dependent cellular processes during the lytic cycle to ensure efficient viral replication.
Asunto(s)
Autofagia , Herpesvirus Humano 8/fisiología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Sarcoma de Kaposi/virología , Activación Viral , Autofagia/efectos de los fármacos , Ácido Butírico/farmacología , Línea Celular , Factor 4F Eucariótico de Iniciación/metabolismo , Interacciones Huésped-Patógeno , Humanos , Proteínas Inmediatas-Precoces/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/patología , Sirolimus/farmacología , Transactivadores/metabolismo , Virión/metabolismo , Activación Viral/efectos de los fármacos , Latencia del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Many viruses shut off host gene expression to inhibit antiviral responses. Viral proteins and host proteins required for viral replication are typically spared in this process, but the mechanisms of target selectivity during host shutoff remain poorly understood. Using transcriptome-wide and targeted reporter experiments, we demonstrate that the influenza A virus endoribonuclease PA-X usurps RNA splicing to selectively target host RNAs for destruction. Proximity-labeling proteomics reveals that PA-X interacts with cellular RNA processing proteins, some of which are partially required for host shutoff. Thus, PA-X taps into host nuclear pre-mRNA processing mechanisms to destroy nascent mRNAs shortly after their synthesis. This mechanism sets PA-X apart from other viral host shutoff proteins that target actively translating mRNAs in the cytoplasm. Our study reveals a unique mechanism of host shutoff that helps us understand how influenza viruses suppress host gene expression.
Asunto(s)
Virus de la Influenza A/fisiología , Empalme del ARN , ARN Mensajero/metabolismo , Proteínas Represoras/metabolismo , Proteínas no Estructurales Virales/metabolismo , Células A549 , Factor de Especificidad de Desdoblamiento y Poliadenilación/antagonistas & inhibidores , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Regulación hacia Abajo , Endorribonucleasas/metabolismo , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Interferones/genética , Interferones/metabolismo , Mutagénesis Sitio-Dirigida , Interferencia de ARN , Precursores del ARN/metabolismo , Sitios de Empalme de ARN , ARN Interferente Pequeño/metabolismo , Proteínas Represoras/genética , Regulación hacia Arriba , Proteínas no Estructurales Virales/genética , Factores de Escisión y Poliadenilación de ARNm/antagonistas & inhibidores , Factores de Escisión y Poliadenilación de ARNm/genética , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
The 2nd Symposium of the Canadian Society for Virology (CSV2018) was held in June 2018 in Halifax, Nova Scotia, Canada, as a featured event marking the 200th anniversary of Dalhousie University. CSV2018 attracted 175 attendees from across Canada and around the world, more than double the number that attended the first CSV symposium two years earlier. CSV2018 provided a forum to discuss a wide range of topics in virology including human, veterinary, plant, and microbial pathogens. Invited keynote speakers included David Kelvin (Dalhousie University and Shantou University Medical College) who provided a historical perspective on influenza on the 100th anniversary of the 1918 pandemic; Sylvain Moineau (Université Laval) who described CRISPR-Cas systems and anti-CRISPR proteins in warfare between bacteriophages and their host microbes; and Kate O'Brien (then from Johns Hopkins University, now relocated to the World Health Organization where she is Director of Immunization, Vaccines and Biologicals), who discussed the underlying viral etiology for pneumonia in the developing world, and the evidence for respiratory syncytial virus (RSV) as a primary cause. Reflecting a strong commitment of Canadian virologists to science communication, CSV2018 featured the launch of Halifax's first annual Soapbox Science event to enable public engagement with female scientists, and the live-taping of the 499th episode of the This Week in Virology (TWIV) podcast, hosted by Vincent Racaniello (Columbia University) and science writer Alan Dove. TWIV featured interviews of CSV co-founders Nathalie Grandvaux (Université de Montréal) and Craig McCormick (Dalhousie University), who discussed the origins and objectives of the new society; Ryan Noyce (University of Alberta), who discussed technical and ethical considerations of synthetic virology; and Kate O'Brien, who discussed vaccines and global health. Finally, because CSV seeks to provide a better future for the next generation of Canadian virologists, the symposium featured a large number of oral and poster presentations from trainees and closed with the awarding of presentation prizes to trainees, followed by a tour of the Halifax Citadel National Historic Site and an evening of entertainment at the historic Alexander Keith's Brewery.
Asunto(s)
Congresos como Asunto , Virología , Distinciones y Premios , Canadá , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Vacunas contra la Influenza , Virus Sincitiales RespiratoriosRESUMEN
Plant cell walls are composed of cellulose, hemicellulose, and lignin, collectively known as lignocellulose. Microorganisms degrade lignocellulose to liberate sugars to meet metabolic demands. Using a metagenomic sequencing approach, we previously demonstrated that the microbiome of the North American porcupine (Erethizon dorsatum) is replete with genes that could encode lignocellulose-degrading enzymes. Here, we report the identification, synthesis and partial characterization of four novel genes from the porcupine microbiome encoding putative lignocellulose-degrading enzymes: ß-glucosidase, α-L-arabinofuranosidase, ß-xylosidase, and endo-1,4-ß-xylanase. These genes were identified via conserved catalytic domains associated with cellulose- and hemicellulose-degradation. Phylogenetic trees were created for each of these putative enzymes to depict genetic relatedness to known enzymes. Candidate genes were synthesized and cloned into plasmid expression vectors for inducible protein expression and secretion. The putative ß-glucosidase fusion protein was efficiently secreted but did not permit Escherichia coli (E. coli) to use cellobiose as a sole carbon source, nor did the affinity purified enzyme cleave p-Nitrophenyl ß-D-glucopyranoside (p-NPG) substrate in vitro over a range of physiological pH levels (pH 5-7). The putative hemicellulose-degrading ß-xylosidase and α-L-arabinofuranosidase enzymes also lacked in vitro enzyme activity, but the affinity purified endo-1,4-ß-xylanase protein cleaved a 6-chloro-4-methylumbelliferyl xylobioside substrate in acidic and neutral conditions, with maximal activity at pH 7. At this optimal pH, KM, Vmax, and kcat were determined to be 32.005 ± 4.72 µM, 1.16x10-5 ± 3.55x10-7 M/s, and 94.72 s-1, respectively. Thus, our pipeline enabled successful identification and characterization of a novel hemicellulose-degrading enzyme from the porcupine microbiome. Progress towards the goal of introducing a complete lignocellulose-degradation pathway into E. coli will be accelerated by combining synthetic metagenomic approaches with functional metagenomic library screening, which can identify novel enzymes unrelated to those found in available databases.
Asunto(s)
Lignina/metabolismo , Microbiota/genética , Microbiota/fisiología , Puercoespines/microbiología , Animales , Endo-1,4-beta Xilanasas/genética , Endo-1,4-beta Xilanasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentación , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Cinética , Metagenómica , Filogenia , Puercoespines/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Biología Sintética , Xilosidasas/genética , Xilosidasas/metabolismo , beta-Glucosidasa/genética , beta-Glucosidasa/metabolismoRESUMEN
Post-transcriptional regulation is an important aspect of the control of gene expression. mRNAs are translated with variable efficiencies, and these efficiencies can change rapidly during adaptation to diverse environmental factors, including cellular stresses and microbial infections. Polysome profiling analysis utilizes ultracentrifugation to isolate complexes of mRNAs in the process of translation and corresponding proteins on the basis of density. Here we describe polysome profiling analysis using a continuous ultraviolet spectrophotometer and a gradient fractionator. We provide protocols for processing sucrose gradient fractions for isolation of RNA for RT-qPCR analysis and isolation of protein for SDS-PAGE and immunoblot analysis. © 2018 by John Wiley & Sons, Inc.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Fraccionamiento Celular/métodos , Reacción en Cadena de la Polimerasa/métodos , Polirribosomas/genética , Biosíntesis de Proteínas , Proteínas/genética , ARN Mensajero/genética , Animales , Sistema Libre de Células/química , Humanos , Polirribosomas/metabolismo , Proteínas/aislamiento & purificación , Proteínas/metabolismo , ARN Mensajero/aislamiento & purificación , ARN Mensajero/metabolismoRESUMEN
Herpes simplex virus (HSV) infections can be treated with direct acting antivirals like acyclovir and foscarnet, but long-term use can lead to drug resistance, which motivates research into broadly-acting antivirals that can provide a greater genetic barrier to resistance. Photodynamic inactivation (PDI) employs a photosensitizer, light, and oxygen to create a local burst of reactive oxygen species that inactivate microorganisms. The botanical plant extract OrthoquinTM is a powerful photosensitizer with antimicrobial properties. Here we report that Orthoquin also has antiviral properties. Photoactivated Orthoquin inhibited herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) infection of target cells in a dose-dependent manner across a broad range of sub-cytotoxic concentrations. HSV inactivation required direct contact between Orthoquin and the inoculum, whereas pre-treatment of target cells had no effect. Orthoquin did not cause appreciable damage to viral capsids or premature release of viral genomes, as measured by qPCR for the HSV-1 genome. By contrast, immunoblotting for HSV-1 antigens in purified virion preparations suggested that higher doses of Orthoquin had a physical impact on certain HSV-1 proteins that altered protein mobility or antigen detection. Orthoquin PDI also inhibited the non-enveloped adenovirus (AdV) in a dose-dependent manner, whereas Orthoquin-mediated inhibition of the enveloped vesicular stomatitis virus (VSV) was light-independent. Together, these findings suggest that the broad antiviral effects of Orthoquin-mediated PDI may stem from damage to viral attachment proteins.
Asunto(s)
Antivirales/uso terapéutico , Herpes Simple/tratamiento farmacológico , Herpesvirus Humano 1/efectos de los fármacos , Herpesvirus Humano 2/efectos de los fármacos , Fotoquimioterapia , Fármacos Fotosensibilizantes/uso terapéutico , Animales , Antivirales/farmacología , Chlorocebus aethiops , Fallopia japonica/química , Células HEK293 , Células HeLa , Herpes Simple/virología , Humanos , Fármacos Fotosensibilizantes/farmacología , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Raíces de Plantas/química , Células VeroRESUMEN
Toll-like receptors (TLRs) recognize the conserved molecular patterns in microorganisms and trigger myeloid differentiation primary response 88 (MyD88) and/or TIR-domain-containing adapter-inducing interferon-ß (TRIF) pathways that are critical for host defense against microbial infection. However, the molecular mechanisms that govern TLR signaling remain incompletely understood. Regulator of calcineurin-1 (RCAN1), a small evolutionarily conserved protein that inhibits calcineurin phosphatase activity, suppresses inflammation during Pseudomonas aeruginosa infection. Here, we define the roles for RCAN1 in P. aeruginosa lipopolysaccharide (LPS)-activated TLR4 signaling. We compared the effects of P. aeruginosa LPS challenge on bone marrow-derived macrophages from both wild-type and RCAN1-deficient mice and found that RCAN1 deficiency increased the MyD88-NF-κB-mediated cytokine production (IL-6, TNF and MIP-2), whereas TRIF-interferon-stimulated response elements (ISRE)-mediated cytokine production (IFNß, RANTES and IP-10) was suppressed. RCAN1 deficiency caused increased IκBα phosphorylation and NF-κB activity in the MyD88-dependent pathway, but impaired ISRE activation and reduced IRF7 expression in the TRIF-dependent pathway. Complementary studies of a mouse model of P. aeruginosa LPS-induced acute pneumonia confirmed that RCAN1-deficient mice displayed greatly enhanced NF-κB activity and MyD88-NF-κB-mediated cytokine production, which correlated with enhanced pulmonary infiltration of neutrophils. By contrast, RCAN1 deficiency had little effect on the TRIF pathway in vivo. These findings demonstrate a novel regulatory role of RCAN1 in TLR signaling, which differentially regulates MyD88 and TRIF pathways.