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BACKGROUND & AIMS: Diarrhea occurs in up to 50% of cases of COVID-19. Nonetheless, the pathophysiologic mechanism(s) have not been determined. METHODS: This was examined using normal human enteroid monolayers exposed apically to live SARS-CoV-2 or non-replicating virus-like particles (VLPs) bearing the 4 SARS-CoV-2 structural proteins or irradiated virus, all of which bound and entered enterocytes. RESULTS: Live virus and VLPs increased secretion of multiple cytokines and reduced mRNAs of ACE2, NHE3, and DRA. Interleukin (IL)-6 plus IL-8 alone reduced NHE3 mRNA and protein and DRA mRNA. Neither VLPs nor IL-6 plus IL-8 alone altered Cl- secretion, but together they caused Cl- secretion, which was Ca2+-dependent, CFTR-independent, blocked partially by a specific TMEM16 A inhibitor, and entirely by a general TMEM16 family inhibitor. VLPs and irradiated virus, but not IL-6 plus IL-8, produced Ca2+ waves that began within minutes of VLP exposure, lasted for at least 60 minutes, and were prevented by pretreatment with apyrase, a P2Y1 receptor antagonist, and general TMEM16 family inhibitor but not by the specific TMEM16A inhibitor. CONCLUSIONS: The pathophysiology of COVID-19 diarrhea appears to be a unique example of a calcium-dependent inflammatory diarrhea that is caused by direct viral effects plus the virus-induced intestinal epithelial cytokine secretion.
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Orthohantaviruses are rodent-borne, negative-sense RNA viruses that are capable of causing severe vascular disease in humans. Over the course of viral evolution, these viruses have tailored their replication cycles in such a way as to avoid and/or antagonize host innate immune responses. In the rodent reservoir, this results in life long asymptomatic infections. However, in hosts other than its co-evolved reservoir, the mechanisms for subduing the innate immune response may be less efficient or absent, potentially leading to disease and/or viral clearance. In the case of human orthohantavirus infection, the interaction of the innate immune response with viral replication is thought to give rise to severe vascular disease. The orthohantavirus field has made significant advancements in understanding how these viruses replicate and interact with host innate immune responses since their identification by Dr. Ho Wang Lee and colleagues in 1976. Therefore, the purpose of this review, as part of this special issue dedicated to Dr. Lee, was to summarize the current knowledge of orthohantavirus replication, how viral replication activates innate immunity, and how the host antiviral response, in turn, impacts viral replication.
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Infecciones por Hantavirus , Orthohantavirus , Virus ARN , Humanos , Inmunidad Innata , Antivirales , Replicación ViralRESUMEN
This paper presents results of a study of a new cationic oligomer that contains end groups and a chromophore affording inactivation of SARS-CoV-2 by visible light irradiation in solution or as a solid coating on paper wipes and glass fiber filtration substrates. A key finding of this study is that the cationic oligomer with a central thiophene ring and imidazolium charged groups gives outstanding performance in both the killing of E. coli bacterial cells and inactivation of the virus at very short times. Our introduction of cationic N-methyl imidazolium groups enhances the light activation process for both E. coli and SARS-CoV-2 but dampens the killing of the bacteria and eliminates the inactivation of the virus in the dark. For the studies with this oligomer in solution at a concentration of 1 µg/mL and E. coli, we obtain 3 log killing of the bacteria with 10 min of irradiation with LuzChem cool white lights (mimicking indoor illumination). With the oligomer in solution at a concentration of 10 µg/mL, we observe 4 log inactivation (99.99%) in 5 min of irradiation and total inactivation after 10 min. The oligomer is quite active against E. coli on oligomer-coated paper wipes and glass fiber filter supports. The SARS-CoV-2 is also inactivated by oligomer-coated glass fiber filter papers. This study indicates that these oligomer-coated materials may be very useful as wipes and filtration materials.
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Antivirales/farmacología , COVID-19/terapia , SARS-CoV-2/efectos de la radiación , COVID-19/genética , COVID-19/virología , Cationes/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/efectos de la radiación , Humanos , Luz , Fototerapia , SARS-CoV-2/patogenicidad , Rayos Ultravioleta , Inactivación de Virus/efectos de los fármacos , Inactivación de Virus/efectos de la radiaciónRESUMEN
The genus Orthohantavirus (family Hantaviridae, order Bunyavirales) consists of numerous genetic and pathologically distinct viral species found within rodent and mammalian insectivore populations world-wide. Although reservoir hosts experience persistent asymptomatic infection, numerous rodent-borne orthohantaviruses cause severe disease when transmitted to humans, with case-fatality rates up to 40%. The first isolation of an orthohantavirus occurred in 1976 and, since then, the field has made significant progress in understanding the immune correlates of disease, viral interactions with the human innate immune response, and the immune kinetics of reservoir hosts. Much still remains elusive regarding the molecular mechanisms of orthohantavirus recognition by the innate immune response and viral antagonism within the reservoir host, however. This review provides a summary of the last 45 years of research into orthohantavirus interaction with the host innate immune response. This summary includes discussion of current knowledge involving human, non-reservoir rodent, and reservoir innate immune responses to viruses which cause hemorrhagic fever with renal syndrome and hantavirus cardio-pulmonary syndrome. Review of the literature concludes with a brief proposition for the development of novel tools needed to drive forward investigations into the molecular mechanisms of innate immune activation and consequences for disease outcomes in the various hosts for orthohantaviruses.
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Infecciones por Hantavirus , Orthohantavirus , Animales , Orthohantavirus/genética , Orthohantavirus/inmunología , Infecciones por Hantavirus/inmunología , Humanos , Inmunidad InnataRESUMEN
SARS-CoV-2 infection depends on binding its spike (S) protein to angiotensin-converting enzyme 2 (ACE2). The S protein expresses an RGD motif, suggesting that integrins may be co-receptors. Here, we UV-inactivated SARS-CoV-2 and fluorescently labeled the envelope membrane with octadecyl rhodamine B (R18) to explore the role of integrin activation in mediating cell entry and productive infection. We used flow cytometry and confocal microscopy to show that SARS-CoV-2R18 particles engage basal-state integrins. Furthermore, we demonstrate that Mn2+, which induces integrin extension, enhances cell entry of SARS-CoV-2R18. We also show that one class of integrin antagonist, which binds to the αI MIDAS site and stabilizes the inactive, closed conformation, selectively inhibits the engagement of SARS-CoV-2R18 with basal state integrins, but is ineffective against Mn2+-activated integrins. RGD-integrin antagonists inhibited SARS-CoV-2R18 binding regardless of integrin activation status. Integrins transmit signals bidirectionally: 'inside-out' signaling primes the ligand-binding function of integrins via a talin-dependent mechanism, and 'outside-in' signaling occurs downstream of integrin binding to macromolecular ligands. Outside-in signaling is mediated by Gα13. Using cell-permeable peptide inhibitors of talin and Gα13 binding to the cytoplasmic tail of an integrin's ß subunit, we demonstrate that talin-mediated signaling is essential for productive infection.
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COVID-19/metabolismo , Integrinas/metabolismo , SARS-CoV-2/fisiología , Internalización del Virus , Animales , Chlorocebus aethiops , Interacciones Huésped-Patógeno , Humanos , Transducción de Señal , Células VeroRESUMEN
Many host RNA sensors are positioned in the cytosol to detect viral RNA during infection. However, most positive-strand RNA viruses replicate within a modified organelle co-opted from intracellular membranes of the endomembrane system, which shields viral products from cellular innate immune sensors. Targeting innate RNA sensors to the endomembrane system may enhance their ability to sense RNA generated by viruses that use these compartments for replication. Here, we reveal that an isoform of oligoadenylate synthetase 1, OAS1 p46, is prenylated and targeted to the endomembrane system. Membrane localization of OAS1 p46 confers enhanced access to viral replication sites and results in increased antiviral activity against a subset of RNA viruses including flaviviruses, picornaviruses, and SARS-CoV-2. Finally, our human genetic analysis shows that the OAS1 splice-site SNP responsible for production of the OAS1 p46 isoform correlates with protection from severe COVID-19. This study highlights the importance of endomembrane targeting for the antiviral specificity of OAS1 and suggests that early control of SARS-CoV-2 replication through OAS1 p46 is an important determinant of COVID-19 severity.
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2',5'-Oligoadenilato Sintetasa/metabolismo , COVID-19/virología , SARS-CoV-2/metabolismo , Animales , COVID-19/inmunología , Sistemas CRISPR-Cas , Línea Celular , Edición Génica , Humanos , Polimorfismo de Nucleótido Simple , SARS-CoV-2/aislamiento & purificaciónRESUMEN
Cellular entry of coronaviruses depends on binding of the viral spike (S) protein to a specific cellular receptor, the angiotensin-converting enzyme 2 (ACE2). Furthermore, the viral spike protein expresses an RGD motif, suggesting that cell surface integrins may be attachment co-receptors. However, using infectious SARS-CoV-2 requires a biosafety level 3 laboratory (BSL-3), which limits the techniques that can be used to study the mechanism of cell entry. Here, we UV-inactivated SARS-CoV-2 and fluorescently labeled the envelope membrane with octadecyl rhodamine B (R18) to explore the role of integrin activation in mediating both cell entry and productive infection. We used flow cytometry and confocal fluorescence microscopy to show that fluorescently labeled SARS-CoV-2 R18 particles engage basal-state integrins. Furthermore, we demonstrate that Mn 2+ , which activates integrins and induces integrin extension, enhances cell binding and entry of SARS-CoV-2 R18 in proportion to the fraction of integrins activated. We also show that one class of integrin antagonist, which binds to the αI MIDAS site and stabilizes the inactive, closed conformation, selectively inhibits the engagement of SARS-CoV-2 R18 with basal state integrins, but is ineffective against Mn 2+ -activated integrins. At the same time, RGD-integrin antagonists inhibited SARS-CoV-2 R18 binding regardless of integrin activity state. Integrins transmit signals bidirectionally: 'inside-out' signaling primes the ligand binding function of integrins via a talin dependent mechanism and 'outside-in' signaling occurs downstream of integrin binding to macromolecular ligands. Outside-in signaling is mediated by Gα 13 and induces cell spreading, retraction, migration, and proliferation. Using cell-permeable peptide inhibitors of talin, and Gα 13 binding to the cytoplasmic tail of an integrin's ß subunit, we further demonstrate that talin-mediated signaling is essential for productive infection by SARS-CoV-2.
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Diarrhea occurs in 2-50% of cases of COVID-19 (â¼8% is average across series). The diarrhea does not appear to account for the disease mortality and its contribution to the morbidity has not been defined, even though it is a component of Long Covid or post-infectious aspects of the disease. Even less is known about the pathophysiologic mechanism of the diarrhea. To begin to understand the pathophysiology of COVID-19 diarrhea, we exposed human enteroid monolayers obtained from five healthy subjects and made from duodenum, jejunum, and proximal colon to live SARS-CoV-2 and virus like particles (VLPs) made from exosomes expressing SARS-CoV-2 structural proteins (Spike, Nucleocapsid, Membrane and Envelope). Results: 1) Live virus was exposed apically for 90 min, then washed out and studied 2 and 5 days later. SARS-Cov-2 was taken up by enteroids and live virus was present in lysates and in the apical>>basolateral media of polarized enteroids 48 h after exposure. This is the first demonstration of basolateral appearance of live virus after apical exposure. High vRNA concentration was detected in cell lysates and in the apical and basolateral media up to 5 days after exposure. 2) Two days after viral exposure, cytokine measurements of media showed significantly increased levels of IL-6, IL-8 and MCP-1. 3) Two days after viral exposure, mRNA levels of ACE2, NHE3 and DRA were reduced but there was no change in mRNA of CFTR. NHE3 protein was also decreased. 4) Live viral studies were mimicked by some studies with VLP exposure for 48 h. VLPs with Spike-D614G bound to the enteroid apical surface and was taken up; this resulted in decreased mRNA levels of ACE2, NHE3, DRA and CFTR. 4) VLP effects were determined on active anion secretion measured with the Ussing chamber/voltage clamp technique. S-D614G acutely exposed to apical surface of human ileal enteroids did not alter the short-circuit current (Isc). However, VLPS-D614G exposure to enteroids that were pretreated for â¼24 h with IL-6 plus IL-8 induced a concentration dependent increase in Isc indicating stimulated anion secretion, that was delayed in onset by â¼8 min. The anion secretion was inhibited by apical exposure to a specific calcium activated Cl channel (CaCC) inhibitor (AO1) but not by a specific CFTR inhibitor (BP027); was inhibited by basolateral exposure to the K channel inhibit clortimazole; and was prevented by pretreatment with the calcium buffer BAPTA-AM. 5) The calcium dependence of the VLP-induced increase in Isc was studied in Caco-2/BBe cells stably expressing the genetically encoded Ca2+ sensor GCaMP6s. 24 h pretreatment with IL-6/IL-8 did not alter intracellular Ca2+. However, in IL-6/IL-8 pretreated cells, VLP S-D614G caused appearance of Ca 2+ waves and an overall increase in intracellular Ca 2+ with a delay of â¼10 min after VLP addition. We conclude that the diarrhea of COVID-19 appears to an example of a calcium dependent inflammatory diarrhea that involves both acutely stimulated Ca2+ dependent anion secretion (stimulated Isc) that involves CaCC and likely inhibition of neutral NaCl absorption (decreased NHE3 protein and mRNA and decreased DRA mRNA).
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The ongoing pandemic of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has placed a substantial strain on the supply of personal protective equipment, particularly the availability of N95 respirators for frontline healthcare personnel. These shortages have led to the creation of protocols to disinfect and reuse potentially contaminated personal protective equipment. A simple and inexpensive decontamination procedure that does not rely on the use of consumable supplies is dry heat incubation. Although reprocessing with this method has been shown to maintain the integrity of N95 respirators after multiple decontamination procedures, information on the ability of dry heat incubation to inactivate SARS-CoV-2 is largely unreported. Here, we show that dry heat incubation does not consistently inactivate SARS-CoV-2-contaminated N95 respirators, and that variation in experimental conditions can dramatically affect viability of the virus. Furthermore, we show that SARS-CoV-2 can survive on N95 respirators that remain at room temperature for at least five days. Collectively, our findings demonstrate that dry heat incubation procedures and ambient temperature for five days are not viable methods for inactivating SARS-CoV-2 on N95 respirators for potential reuse. We recommend that decontamination procedures being considered for the reuse of N95 respirators be validated at each individual site and that validation of the process must be thoroughly conducted using a defined protocol.
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COVID-19 , Calor , Máscaras , Pandemias , SARS-CoV-2/metabolismo , Inactivación de Virus , Animales , COVID-19/epidemiología , COVID-19/metabolismo , COVID-19/prevención & control , COVID-19/terapia , Chlorocebus aethiops , Humanos , Células VeroRESUMEN
In the present study, we examined the inactivation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by synthetic conjugated polymers and oligomers developed in our laboratories as antimicrobials for bacteria, fungi, and nonenveloped viruses. The results show highly effective light-induced inactivation with several of these oligomers and polymers including irradiation with near-UV and visible light. In the best case, one oligomer induced a 5-log reduction in pfu/mL within 10 min. In general, the oligomers are more active than the polymers; however, the polymers are active with longer wavelength visible irradiation. Although not studied quantitatively, the results show that in the presence of the agents at concentrations similar to those used in the light studies, there is essentially no dark inactivation of the virus. Because three of the five materials/compounds examined are quaternary ammonium derivatives, this study indicates that conventional quaternary ammonium antimicrobials may not be active against SARS-CoV-2. Our results suggest several applications involving the incorporation of these materials in wipes, sprays, masks, and clothing and other personal protection equipment that can be useful in preventing infections and the spreading of this deadly virus and future outbreaks from similar viruses.
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Tratamiento Farmacológico de COVID-19 , Polímeros/farmacología , SARS-CoV-2/efectos de los fármacos , Animales , COVID-19/virología , Chlorocebus aethiops , Humanos , Luz , Polímeros/efectos de la radiación , SARS-CoV-2/patogenicidad , SARS-CoV-2/efectos de la radiación , Rayos Ultravioleta , Células Vero , Inactivación de Virus/efectos de los fármacos , Inactivación de Virus/efectos de la radiaciónRESUMEN
The current Covid-19 Pandemic caused by the highly contagious SARS-CoV-2 virus has proven extremely difficult to prevent or control. Currently there are few treatment options and very few long-lasting disinfectants available to prevent the spread. While masks and protective clothing and social distancing may offer some protection, their use has not always halted or slowed the spread. Several vaccines are currently undergoing testing; however there is still a critical need to provide new methods for inactivating the virus before it can spread and infect humans. In the present study we examined the inactivation of SARS-CoV-2 by synthetic conjugated polymers and oligomers developed in our laboratories as antimicrobials for bacteria, fungi and non-enveloped viruses. Our results show that we can obtain highly effective light induced inactivation with several of these oligomers and polymers including irradiation with near-UV and visible light. With both the oligomers and polymers, we can reach several logs of inactivation with relatively short irradiation times. Our results suggest several applications involving the incorporation of these materials in wipes, sprays, masks and clothing and other Personal Protection Equipment (PPE) that can be useful in preventing infections and the spreading of this deadly virus and future outbreaks from similar viruses.
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TRIM5α is a key cross-species barrier to retroviral infection, with certain TRIM5 alleles conferring increased risk of HIV-1 infection in humans. TRIM5α is best known as a species-specific restriction factor that directly inhibits the viral life cycle. Additionally, it is also a pattern-recognition receptor (PRR) that activates inflammatory signaling. How TRIM5α carries out its multi-faceted actions in antiviral defense remains incompletely understood. Here, we show that proteins required for autophagy, a cellular self-digestion pathway, play an important role in TRIM5α's function as a PRR. Genetic depletion of proteins involved in all stages of the autophagy pathway prevented TRIM5α-driven expression of NF-κB and AP1 responsive genes. One of these genes is the preeminent antiviral cytokine interferon ß (IFN-ß), whose TRIM5-dependent expression was lost in cells lacking the autophagy proteins ATG7, BECN1, and ULK1. Moreover, we found that the ability of TRIM5α to stimulate IFN-ß expression in response to recognition of a TRIM5α-restricted HIV-1 capsid mutant (P90A) was abrogated in cells lacking autophagy factors. Stimulation of human macrophage-like cells with the P90A virus protected them against subsequent infection with an otherwise resistant wild type HIV-1 in a manner requiring TRIM5α, BECN1, and ULK1. Mechanistically, TRIM5α was attenuated in its ability to activate the kinase TAK1 in autophagy deficient cells, and both BECN1 and ATG7 contributed to the assembly of TRIM5α-TAK1 complexes. These data demonstrate a non-canonical role for the autophagy machinery in assembling antiviral signaling complexes and in establishing a TRIM5α-dependent antiviral state.
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Autofagia/fisiología , Receptores de Reconocimiento de Patrones/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Factores de Restricción Antivirales , Autofagia/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia , Beclina-1 , Cápside/metabolismo , Proteínas de la Cápside/metabolismo , Proteínas Portadoras/metabolismo , Línea Celular , Células HEK293 , Infecciones por VIH/virología , VIH-1/genética , Células HeLa , Humanos , Interferón beta/metabolismo , Péptidos y Proteínas de Señalización Intracelular , FN-kappa B/metabolismo , Péptidos/metabolismo , Receptores de Reconocimiento de Patrones/fisiología , Infecciones por Retroviridae/virología , Especificidad de la Especie , Células THP-1 , Proteínas de Motivos Tripartitos/fisiología , Ubiquitina-Proteína Ligasas/fisiologíaRESUMEN
Pathogenic hantaviruses, genus Orthohantaviridae, are maintained in rodent reservoirs with zoonotic transmission to humans occurring through inhalation of rodent excreta. Hantavirus disease in humans is characterized by localized vascular leakage and elevated levels of circulating proinflammatory cytokines. Despite the constant potential for deadly zoonotic transmission to humans, specific virus-host interactions of hantaviruses that lead to innate immune activation, and how these processes impart disease, remain unclear. In this study, we examined the mechanisms of viral recognition and innate immune activation of Hantaan orthohantavirus (HTNV) infection. We identified the RIG-I-like receptor (RLR) pathway as essential for innate immune activation, interferon (IFN) production, and interferon stimulated gene (ISG) expression in response to HTNV infection in human endothelial cells, and in murine cells representative of a non-reservoir host. Our results demonstrate that innate immune activation and signaling through the RLR pathway depends on viral replication wherein the host response can significantly restrict replication in target cells in a manner dependent on the type 1 interferon receptor (IFNAR). Importantly, following HTNV infection of a non-reservoir host murine model, IFNAR-deficient mice had higher viral loads, increased persistence, and greater viral dissemination to lung, spleen, and kidney compared to wild-type animals. Surprisingly, this response was MAVS independent in vivo. Innate immune profiling in these tissues demonstrates that HTNV infection triggers expression of IFN-regulated cytokines early during infection. We conclude that the RLR pathway is essential for recognition of HTNV infection to direct innate immune activation and control of viral replication in vitro, and that additional virus sensing and innate immune response pathways of IFN and cytokine regulation contribute to control of HTNV in vivo. These results reveal a critical role for innate immune regulation in driving divergent outcomes of HTNV infection, and serve to inform studies to identify therapeutic targets to alleviate human hantavirus disease.
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Proteína 58 DEAD Box/inmunología , Infecciones por Hantavirus/inmunología , Interferón Tipo I/inmunología , Orthohantavirus/fisiología , Replicación Viral/fisiología , Animales , Chlorocebus aethiops , Citocinas/inmunología , Citocinas/metabolismo , Proteína 58 DEAD Box/metabolismo , ARN Helicasas DEAD-box/metabolismo , Células Endoteliales/metabolismo , Orthohantavirus/inmunología , Orthohantavirus/metabolismo , Orthohantavirus/patogenicidad , Infecciones por Hantavirus/metabolismo , Infecciones por Hantavirus/virología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inmunidad Innata , Interferón Tipo I/metabolismo , Interferón beta/metabolismo , Ratones , Receptor de Interferón alfa y beta/metabolismo , Receptores Inmunológicos , Transducción de Señal/inmunología , Células VeroRESUMEN
The initial response to viral infection is anticipatory, with host antiviral restriction factors and pathogen sensors constantly surveying the cell to rapidly mount an antiviral response through the synthesis and downstream activity of interferons. After pathogen clearance, the host's ability to resolve this antiviral response and return to homeostasis is critical. Here, we found that isoforms of the RNA-binding protein ZAP functioned as both a direct antiviral restriction factor and an interferon-resolution factor. The short isoform of ZAP bound to and mediated the degradation of several host interferon messenger RNAs, and thus acted as a negative feedback regulator of the interferon response. In contrast, the long isoform of ZAP had antiviral functions and did not regulate interferon. The two isoforms contained identical RNA-targeting domains, but differences in their intracellular localization modulated specificity for host versus viral RNA, which resulted in disparate effects on viral replication during the innate immune response.
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Infecciones por Alphavirus/inmunología , Interferones/genética , Isoformas de Proteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Proteínas Represoras/metabolismo , Virus Sindbis/fisiología , Infecciones por Alphavirus/genética , Retroalimentación Fisiológica , Células HEK293 , Células Hep G2 , Homeostasis , Humanos , Inmunidad Innata , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Unión Proteica , Isoformas de Proteínas/genética , ARN/genética , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/genética , Proteínas Represoras/genética , Replicación ViralRESUMEN
Hepatitis C virus (HCV) infects 200 million people globally, and 60-80% of cases persist as a chronic infection that will progress to cirrhosis and liver cancer in 2-10% of patients. We recently demonstrated that HCV induces aberrant expression of two host microRNAs (miRNAs), miR-208b and miR-499a-5p, encoded by myosin genes in infected hepatocytes. These miRNAs, along with AU-rich-element-mediated decay, suppress IFNL2 and IFNL3, members of the type III interferon (IFN) gene family, to support viral persistence. In this study, we show that miR-208b and miR-499a-5p also dampen type I IFN signaling in HCV-infected hepatocytes by directly down-regulating expression of the type I IFN receptor chain, IFNAR1. Inhibition of these miRNAs by using miRNA inhibitors during HCV infection increased expression of IFNAR1. Additionally, inhibition rescued the antiviral response to exogenous type I IFN, as measured by a marked increase in IFN-stimulated genes and a decrease in HCV load. Treatment of HCV-infected hepatocytes with type I IFN increased expression of myosins over HCV infection alone. Since these miRNAs can suppress type III IFN family members, these data collectively define a novel cross-regulation between type I and III IFNs during HCV infection.
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Regulación de la Expresión Génica/inmunología , Hepacivirus/inmunología , Hepatitis C Crónica/inmunología , Hepatocitos/inmunología , Interferón Tipo I/inmunología , MicroARNs/inmunología , Sistemas CRISPR-Cas , Regulación hacia Abajo , Técnicas de Inactivación de Genes , Células Hep G2 , Hepatitis C/inmunología , Humanos , Interferones , Interleucinas/inmunología , Miosinas/metabolismo , Receptor de Interferón alfa y beta/genéticaRESUMEN
Interferon (IFN) lambdas are critical antiviral effectors in hepatic and mucosal infections. Although IFNλ1, IFNλ2, and IFNλ3 act antiviral, genetic association studies have shown that expression of the recently discovered IFNL4 is detrimental to hepatitis C virus (HCV) infection through a yet unknown mechanism. Intriguingly, human IFNL4 harbors a genetic variant that introduces a premature stop codon. We performed a molecular and biochemical characterization of IFNλ4 to determine its role and regulation of expression. We found that IFNλ4 exhibits similar antiviral activity to IFNλ3 without negatively affecting antiviral IFN activity or cell survival. We show that humans deploy several mechanisms to limit expression of functional IFNλ4 through noncoding splice variants and nonfunctional protein isoforms. Furthermore, protein-coding IFNL4 mRNA are not loaded onto polyribosomes and lack a strong polyadenylation signal, resulting in poor translation efficiency. This study provides mechanistic evidence that humans suppress IFNλ4 expression, suggesting that immune function is dependent on other IFNL family members.
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Interacciones Huésped-Patógeno , Interleucinas/metabolismo , Virosis/metabolismo , Empalme Alternativo/genética , Animales , Antivirales/farmacología , Secuencia de Bases , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Espacio Extracelular/metabolismo , Mutación del Sistema de Lectura/genética , Hepacivirus/efectos de los fármacos , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Interferones , Interleucinas/farmacología , Espacio Intracelular/metabolismo , Modelos Biológicos , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Isoformas de Proteínas/metabolismo , Receptores de Citocinas/metabolismo , Receptores de Interferón , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
UNLABELLED: Stimulation of the antiviral response depends on the sensing of viral pathogen-associated molecular patterns (PAMPs) by specialized cellular proteins. During infection with RNA viruses, 5'-di- or -triphosphates accompanying specific single or double-stranded RNA motifs trigger signaling of intracellular RIG-I-like receptors (RLRs) and initiate the antiviral response. Although these molecular signatures are present during the replication of many viruses, it is unknown whether they are sufficient for strong activation of RLRs during infection. Immunostimulatory defective viral genomes (iDVGs) from Sendai virus (SeV) are among the most potent natural viral triggers of antiviral immunity. Here we describe an RNA motif (DVG(70-114)) that is essential for the potent immunostimulatory activity of 5'-triphosphate-containing SeV iDVGs. DVG(70-114) enhances viral sensing by the host cell independently of the long stretches of complementary RNA flanking the iDVGs, and it retains its stimulatory potential when transferred to otherwise inert viral RNA. In vitro analysis showed that DVG(70-114) augments the binding of RIG-I to viral RNA and promotes enhanced RIG-I polymerization, thereby facilitating the onset of the antiviral response. Together, our results define a new natural viral PAMP enhancer motif that promotes viral recognition by RLRs and confers potent immunostimulatory activity to viral RNA. IMPORTANCE: A discrete group of molecular motifs, including 5'-triphosphates associated with double-stranded RNA, have been identified as essential for the triggering of antiviral immunity. Most RNA viruses expose these motifs during their replication; however, successful viruses normally evade immune recognition and replicate to high levels before detection, indicating that unknown factors drive antiviral immunity. DVGs from SeV are among the most potent natural viral stimuli of the antiviral response known to date. These studies define a new natural viral motif present in DVGs that maximizes viral recognition by the intracellular sensor RIG-I, allowing fast and strong antiviral responses even in the presence of viral-encoded immune antagonists. This motif can be harnessed to increase the immunostimulatory potential of otherwise inert viral RNAs and represents a novel immunostimulatory enhancer that could be used in the development of vaccine adjuvants and antivirals.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Inmunidad Innata , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismo , ARN Viral/metabolismo , Virus Sendai/inmunología , Animales , Línea Celular , Proteína 58 DEAD Box , Humanos , Macaca mulatta , Unión Proteica , Receptores InmunológicosRESUMEN
UNLABELLED: Despite the introduction of direct-acting antiviral (DAA) drugs against hepatitis C virus (HCV), infection remains a major public health concern because DAA therapeutics do not prevent reinfection and patients can still progress to chronic liver disease. Chronic HCV infection is supported by a variety of viral immune evasion strategies, but, remarkably, 20% to 30% of acute infections spontaneously clear prior to development of adaptive immune responses, thus implicating innate immunity in resolving acute HCV infection. However, the virus-host interactions regulating acute infection are unknown. Transmission of HCV involves one or a few transmitted/founder (T/F) variants. In infected hepatocytes, the retinoic acid-inducible gene I (RIG-I) protein recognizes 5' triphosphate (5'ppp) of the HCV RNA and a pathogen-associated molecular pattern (PAMP) motif located within the 3' untranslated region consisting of poly-U/UC. PAMP binding activates RIG-I to induce innate immune signaling and type 1 interferon antiviral defenses. HCV poly-U/UC sequences can differ in length and complexity, suggesting that PAMP diversity in T/F genomes could regulate innate immune control of acute HCV infection. Using 14 unique poly-U/UC sequences from HCV T/F genomes recovered from acute-infection patients, we tested whether RIG-I recognition and innate immune activation correlate with PAMP sequence characteristics. We show that T/F variants are recognized by RIG-I in a manner dependent on length of the U-core motif of the poly-U/UC PAMP and are recognized by RIG-I to induce innate immune responses that restrict acute infection. PAMP recognition of T/F HCV variants by RIG-I may therefore impart innate immune signaling and HCV restriction to impact acute-phase-to-chronic-phase transition. IMPORTANCE: Recognition of nonself molecular patterns such as those seen with viral nucleic acids is an essential step in triggering the immune response to virus infection. Innate immunity is induced by hepatitis C virus infection through the recognition of viral RNA by the cellular RIG-I protein, where RIG-I recognizes a poly-uridine/cytosine motif in the viral genome. Variation within this motif may provide an immune evasion strategy for transmitted/founder viruses during acute infection. Using 14 unique poly-U/UC sequences from HCV T/F genomes recovered from acutely infected HCV patients, we demonstrate that RIG-I binding and activation of innate immunity depend primarily on the length of the uridine core within this motif. T/F variants found in acute infection contained longer U cores within the motif and could activate RIG-I and induce innate immune signaling sufficient to restrict viral infection. Thus, recognition of T/F variants by RIG-I could significantly impact the transition from acute to chronic infection.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Variación Genética , Hepacivirus/genética , Hepatitis C/inmunología , Inmunidad Innata/inmunología , Poli U/metabolismo , Línea Celular , Proteína 58 DEAD Box , Ensayo de Cambio de Movilidad Electroforética , Hepacivirus/metabolismo , Hepatocitos/metabolismo , Hepatocitos/virología , Interacciones Huésped-Patógeno , Humanos , Immunoblotting , Plásmidos/genética , Poli U/genética , Reacción en Cadena de la Polimerasa , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores InmunológicosRESUMEN
Antiviral immunity is initiated upon host recognition of viral products via non-self molecular patterns known as pathogen-associated molecular patterns (PAMPs). Such recognition initiates signaling cascades that induce intracellular innate immune defenses and an inflammatory response that facilitates development of the acquired immune response. The retinoic acid-inducible gene I (RIG-I) and the RIG-I-like receptor (RLR) protein family are key cytoplasmic pathogen recognition receptors that are implicated in the recognition of viruses across genera and virus families, including functioning as major sensors of RNA viruses, and promoting recognition of some DNA viruses. RIG-I, the charter member of the RLR family, is activated upon binding to PAMP RNA. Activated RIG-I signals by interacting with the adapter protein MAVS leading to a signaling cascade that activates the transcription factors IRF3 and NF-κB. These actions induce the expression of antiviral gene products and the production of type I and III interferons that lead to an antiviral state in the infected cell and surrounding tissue. RIG-I signaling is essential for the control of infection by many RNA viruses. Recently, RIG-I crosstalk with other pathogen recognition receptors and components of the inflammasome has been described. In this review, we discuss the current knowledge regarding the role of RIG-I in recognition of a variety of virus families and its role in programming the adaptive immune response through cross-talk with parallel arms of the innate immune system, including how RIG-I can be leveraged for antiviral therapy.
Asunto(s)
Citoplasma/virología , ARN Helicasas DEAD-box/metabolismo , Interferones/metabolismo , Virus ARN/inmunología , ARN Viral/metabolismo , Receptores de Reconocimiento de Patrones/metabolismo , Transducción de Señal , Inmunidad Adaptativa , Citoplasma/inmunología , ARN Helicasas DEAD-box/inmunología , Virus ADN/inmunología , Inmunidad Innata , Factor 3 Regulador del Interferón/metabolismo , FN-kappa B/metabolismo , Receptores de Reconocimiento de Patrones/inmunologíaRESUMEN
Viral genotype displacement events are characterized by the replacement of a previously dominant virus genotype by a novel genotype of the same virus species in a given geographic region. We examine here the fitness of three pairs of infectious hematopoietic necrosis virus (IHNV) genotypes involved in three major genotype displacement events in Washington state over the last 30 years to determine whether increased virus fitness correlates with displacement. Fitness was assessed using in vivo assays to measure viral replication in single infection, simultaneous co-infection, and sequential superinfection in the natural host, steelhead trout. In addition, virion stability of each genotype was measured in freshwater and seawater environments at various temperatures. By these methods, we found no correlation between increased viral fitness and displacement in the field. These results suggest that other pressures likely exist in the field with important consequences for IHNV evolution.