RESUMEN
Macrophage activation is essential for effective immunity to infection but can also contribute to disease through incompletely understood mechanisms. In this issue of Immunity, Simpson et al. reveal that death of activated macrophages integrates extrinsic and intrinsic pathways of apoptosis that contribute to damaging host responses.
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Interferón gamma , Activación de Macrófagos , Apoptosis , Caspasa 8/metabolismo , Muerte Celular , Interferón gamma/metabolismo , Ligandos , Macrófagos/inmunologíaRESUMEN
Outbreaks of Ebola virus disease are caused by multiple virus species although current therapeutic monoclonal antibodies (mAb) are essentially limited to treating one species, Zaire ebolavirus. In this issue of Immunity, Gilchuk et al. (2018) identify new mAbs with potent cross-neutralizing activity against three ebolavirus species pathogenic to humans.
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Ebolavirus/inmunología , Fiebre Hemorrágica Ebola , Animales , Anticuerpos Monoclonales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Glicoproteínas , HumanosRESUMEN
Zika virus (ZIKV) belongs to the family Flaviviridae, and is related to other viruses that cause human diseases. Unlike other flaviviruses, ZIKV infection can cause congenital neurological disorders and replicates efficiently in reproductive tissues1-3. Here we show that the envelope protein (E) of ZIKV is polyubiquitinated by the E3 ubiquitin ligase TRIM7 through Lys63 (K63)-linked polyubiquitination. Accordingly, ZIKV replicates less efficiently in the brain and reproductive tissues of Trim7-/- mice. Ubiquitinated E is present on infectious virions of ZIKV when they are released from specific cell types, and enhances virus attachment and entry into cells. Specifically, K63-linked polyubiquitin chains directly interact with the TIM1 (also known as HAVCR1) receptor of host cells, which enhances virus entry in cells as well as in brain tissue in vivo. Recombinant ZIKV mutants that lack ubiquitination are attenuated in human cells and in wild-type mice, but not in live mosquitoes. Monoclonal antibodies against K63-linked polyubiquitin specifically neutralize ZIKV and reduce viraemia in mice. Our results demonstrate that the ubiquitination of ZIKV E is an important determinant of virus entry, tropism and pathogenesis.
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Ubiquitinación , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus , Virus Zika/metabolismo , Virus Zika/patogenicidad , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Encéfalo/metabolismo , Línea Celular , Culicidae/citología , Culicidae/virología , Endosomas/metabolismo , Femenino , Receptor Celular 1 del Virus de la Hepatitis A/metabolismo , Humanos , Masculino , Fusión de Membrana , Ratones , Especificidad de Órganos , Poliubiquitina/inmunología , Poliubiquitina/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Tropismo Viral , Viremia/inmunología , Viremia/prevención & control , Viremia/virología , Replicación Viral , Virus Zika/química , Virus Zika/genética , Infección por el Virus Zika/prevención & control , Infección por el Virus Zika/virologíaRESUMEN
The pediatric live-attenuated bovine/human parainfluenza virus type 3 (B/HPIV3)-vectored vaccine expressing the prefusion-stabilized SARS-CoV-2 spike (S) protein (B/HPIV3/S-2P) was previously evaluated in vitro and in hamsters. To improve its immunogenicity, we generated B/HPIV3/S-6P, expressing S further stabilized with 6 proline mutations (S-6P). Intranasal immunization of hamsters with B/HPIV3/S-6P reproducibly elicited significantly higher serum anti-S IgA/IgG titers than B/HPIV3/S-2P; hamster sera efficiently neutralized variants of concern (VoCs), including Omicron variants. B/HPIV3/S-2P and B/HPIV3/S-6P immunization protected hamsters against weight loss and lung inflammation following SARS-CoV-2 challenge with the vaccine-matched strain WA1/2020 or VoCs B.1.1.7/Alpha or B.1.351/Beta and induced near-sterilizing immunity. Three weeks post-challenge, B/HPIV3/S-2P- and B/HPIV3/S-6P-immunized hamsters exhibited a robust anamnestic serum antibody response with increased neutralizing potency to VoCs, including Omicron sublineages. B/HPIV3/S-6P primed for stronger anamnestic antibody responses after challenge with WA1/2020 than B/HPIV3/S-2P. B/HPIV3/S-6P will be evaluated as an intranasal vaccine to protect infants against both HPIV3 and SARS-CoV-2.
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COVID-19 , Infecciones por Paramyxoviridae , Cricetinae , Humanos , Animales , Bovinos , Niño , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/genética , Anticuerpos Antivirales , Proteínas Virales de Fusión , Vacunas Atenuadas , COVID-19/prevención & control , Virus de la Parainfluenza 3 Humana , Anticuerpos NeutralizantesRESUMEN
The immunological surveillance factors controlling vulnerability of the female reproductive tract (FRT) to sexually transmitted viral infections are not well understood. Interferon-epsilon (IFNÉ) is a distinct, immunoregulatory type-I IFN that is constitutively expressed by FRT epithelium and is not induced by pathogens like other antiviral IFNs α, ß and λ. We show the necessity of IFNÉ for Zika Virus (ZIKV) protection by: increased susceptibility of IFNÉ-/- mice; their "rescue" by intravaginal recombinant IFNÉ treatment and blockade of protective endogenous IFNÉ by neutralising antibody. Complementary studies in human FRT cell lines showed IFNÉ had potent anti-ZIKV activity, associated with transcriptome responses similar to IFNλ but lacking the proinflammatory gene signature of IFNα. IFNÉ activated STAT1/2 pathways similar to IFNα and λ that were inhibited by ZIKV-encoded non-structural (NS) proteins, but not if IFNε exposure preceded infection. This scenario is provided by the constitutive expression of endogenous IFNε. However, the IFNÉ expression was not inhibited by ZIKV NS proteins despite their ability to antagonise the expression of IFNß or λ. Thus, the constitutive expression of IFNÉ provides cellular resistance to viral strategies of antagonism and maximises the antiviral activity of the FRT. These results show that the unique spatiotemporal properties of IFNε provides an innate immune surveillance network in the FRT that is a significant barrier to viral infection with important implications for prevention and therapy.
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Infección por el Virus Zika , Virus Zika , Animales , Femenino , Humanos , Ratones , Antivirales/farmacología , Genitales Femeninos , Factores Inmunológicos , Interferón-alfa/farmacología , Virus Zika/genéticaRESUMEN
The tick-borne encephalitis virus (TBEV) serocomplex includes several medically important flavivirus members endemic to Europe, Asia, and North America, which can induce severe neuroinvasive or viscerotropic diseases with unclear mechanisms of pathogenesis. Langat virus (LGTV) shares a high sequence identity with TBEV but exhibits lower pathogenic potential in humans and serves as a model for virus-host interactions. In this study, we demonstrated that LGTV infection inhibits the activation of gp130/JAK/STAT (Janus kinases (JAK) and signal transducer and activator of transcription (STAT)) signaling, which plays a pivotal role in numerous biological processes. Our data show that the LGTV-infected cells had significantly lower phosphorylated STAT3 (pSTAT3) protein upon oncostatin M (OSM) stimulation than the mock-infected control. LGTV infection blocked the nuclear translocation of STAT3 without a significant effect on total STAT3 protein level. LGTV inhibited JAK1 activation and reduced gp130 protein expression in infected cells, with the viral NS5 protein mediating this effect. TBEV infection also reduces gp130 level. On the other hand, pretreatment of Vero cells with OSM significantly reduces LGTV replication, and STAT1/STAT2 knockdown had little effect on OSM-mediated antiviral effect, which suggests it is independent of STAT1/STAT2 and, instead, it is potentially mediated by STAT3 signlaing. These findings shed light on the LGTV and TBEV-cell interactions, offering insights for the future development of antiviral therapeutics and improved vaccines.
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Fenómenos Biológicos , Virus de la Encefalitis Transmitidos por Garrapatas , Animales , Chlorocebus aethiops , Humanos , Quinasas Janus/metabolismo , Células Vero , Receptor gp130 de Citocinas/metabolismo , Antivirales/metabolismoRESUMEN
Our recent data demonstrate a critical role of the RIG-I-like receptor family in regulating antifungal immunity against Aspergillus fumigatus in a murine model. However, the importance of this pathway in humans and the cell types that use this innate immune receptor family to detect A. fumigatus remain unresolved. In this study, using patients who underwent hematopoietic stem cell transplantation, we demonstrate that a polymorphism in human MAVS present in the donor genome was associated with the incidence of invasive pulmonary aspergillosis. Moreover, in a separate cohort of confirmed invasive pulmonary aspergillosis patients, polymorphisms in the IFIH1 gene alter the inflammatory response, including IFN-responsive chemokines. Returning to our murine model, we now demonstrate that CD11c+ Siglec F+ alveolar macrophages require Mavs expression to maintain host resistance against A. fumigatus. Our data support the role of MAVS signaling in mediating antifungal immunity in both mice and humans at least in part through the role of MAVS-dependent signaling in alveolar macrophages.
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Aspergillus fumigatus , Aspergilosis Pulmonar Invasiva , Animales , Antifúngicos , Modelos Animales de Enfermedad , Humanos , Macrófagos Alveolares , RatonesRESUMEN
Single-dose vaccines with the ability to restrict SARS-CoV-2 replication in the respiratory tract are needed for all age groups, aiding efforts toward control of COVID-19. We developed a live intranasal vector vaccine for infants and children against COVID-19 based on replication-competent chimeric bovine/human parainfluenza virus type 3 (B/HPIV3) that express the native (S) or prefusion-stabilized (S-2P) SARS-CoV-2 S spike protein, the major protective and neutralization antigen of SARS-CoV-2. B/HPIV3/S and B/HPIV3/S-2P replicated as efficiently as B/HPIV3 in vitro and stably expressed SARS-CoV-2 S. Prefusion stabilization increased S expression by B/HPIV3 in vitro. In hamsters, a single intranasal dose of B/HPIV3/S-2P induced significantly higher titers compared to B/HPIV3/S of serum SARS-CoV-2-neutralizing antibodies (12-fold higher), serum IgA and IgG to SARS-CoV-2 S protein (5-fold and 13-fold), and IgG to the receptor binding domain (10-fold). Antibodies exhibited broad neutralizing activity against SARS-CoV-2 of lineages A, B.1.1.7, and B.1.351. Four weeks after immunization, hamsters were challenged intranasally with 104.5 50% tissue-culture infectious-dose (TCID50) of SARS-CoV-2. In B/HPIV3 empty vector-immunized hamsters, SARS-CoV-2 replicated to mean titers of 106.6 TCID50/g in lungs and 107 TCID50/g in nasal tissues and induced moderate weight loss. In B/HPIV3/S-immunized hamsters, SARS-CoV-2 challenge virus was reduced 20-fold in nasal tissues and undetectable in lungs. In B/HPIV3/S-2P-immunized hamsters, infectious challenge virus was undetectable in nasal tissues and lungs; B/HPIV3/S and B/HPIV3/S-2P completely protected against weight loss after SARS-CoV-2 challenge. B/HPIV3/S-2P is a promising vaccine candidate to protect infants and young children against HPIV3 and SARS-CoV-2.
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Vacunas contra la COVID-19/administración & dosificación , COVID-19/prevención & control , SARS-CoV-2/inmunología , Administración Intranasal , Animales , Anticuerpos Antivirales/sangre , Vacunas contra la COVID-19/genética , Vacunas contra la COVID-19/inmunología , Cricetinae , Vectores Genéticos , Inmunización , Virus de la Parainfluenza 3 Bovina/genética , Virus de la Parainfluenza 3 Humana/genética , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
Kyasanur Forest disease virus (KFDV) and the closely related Alkhurma hemorrhagic disease virus (AHFV) are emerging flaviviruses that cause severe viral hemorrhagic fevers in humans. Increasing geographical expansion and case numbers, particularly of KFDV in southwest India, class these viruses as a public health threat. Viral pathogenesis is not well understood and additional vaccines and antivirals are needed to effectively counter the impact of these viruses. However, current animal models of KFDV pathogenesis do not accurately reproduce viral tissue tropism or clinical outcomes observed in humans. Here, we show that pigtailed macaques (Macaca nemestrina) infected with KFDV or AHFV develop viremia that peaks 2 to 4 days following inoculation. Over the course of infection, animals developed lymphocytopenia, thrombocytopenia, and elevated liver enzymes. Infected animals exhibited hallmark signs of human disease characterized by a flushed appearance, piloerection, dehydration, loss of appetite, weakness, and hemorrhagic signs including epistaxis. Virus was commonly present in the gastrointestinal tract, consistent with human disease caused by KFDV and AHFV where gastrointestinal symptoms (hemorrhage, vomiting, diarrhea) are common. Importantly, RNAseq of whole blood revealed that KFDV downregulated gene expression of key clotting factors that was not observed during AHFV infection, consistent with increased severity of KFDV disease observed in this model. This work characterizes a nonhuman primate model for KFDV and AHFV that closely resembles human disease for further utilization in understanding host immunity and development of antiviral countermeasures.
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Modelos Animales de Enfermedad , Virus de la Encefalitis Transmitidos por Garrapatas/patogenicidad , Encefalitis Transmitida por Garrapatas/virología , Fiebres Hemorrágicas Virales/virología , Macaca nemestrina , Animales , Chlorocebus aethiops , Citocinas/sangre , Virus de la Encefalitis Transmitidos por Garrapatas/genética , Virus de la Encefalitis Transmitidos por Garrapatas/inmunología , Encefalitis Transmitida por Garrapatas/inmunología , Encefalitis Transmitida por Garrapatas/patología , Femenino , Células HEK293 , Fiebres Hemorrágicas Virales/inmunología , Fiebres Hemorrágicas Virales/patología , Humanos , Ganglios Linfáticos/virología , Células Vero , ViremiaRESUMEN
SARS-CoV-2 emerged in late 2019 and resulted in the ongoing COVID-19 pandemic. Several animal models have been rapidly developed that recapitulate the asymptomatic to moderate disease spectrum. Now, there is a direct need for additional small animal models to study the pathogenesis of severe COVID-19 and for fast-tracked medical countermeasure development. Here, we show that transgenic mice expressing the human SARS-CoV-2 receptor (angiotensin-converting enzyme 2 [hACE2]) under a cytokeratin 18 promoter (K18) are susceptible to SARS-CoV-2 and that infection resulted in a dose-dependent lethal disease course. After inoculation with either 104 TCID50 or 105 TCID50, the SARS-CoV-2 infection resulted in rapid weight loss in both groups and uniform lethality in the 105 TCID50 group. High levels of viral RNA shedding were observed from the upper and lower respiratory tract and intermittent shedding was observed from the intestinal tract. Inoculation with SARS-CoV-2 resulted in upper and lower respiratory tract infection with high infectious virus titers in nasal turbinates, trachea and lungs. The observed interstitial pneumonia and pulmonary pathology, with SARS-CoV-2 replication evident in pneumocytes, were similar to that reported in severe cases of COVID-19. SARS-CoV-2 infection resulted in macrophage and lymphocyte infiltration in the lungs and upregulation of Th1 and proinflammatory cytokines/chemokines. Extrapulmonary replication of SARS-CoV-2 was observed in the cerebral cortex and hippocampus of several animals at 7 DPI but not at 3 DPI. The rapid inflammatory response and observed pathology bears resemblance to COVID-19. Additionally, we demonstrate that a mild disease course can be simulated by low dose infection with 102 TCID50 SARS-CoV-2, resulting in minimal clinical manifestation and near uniform survival. Taken together, these data support future application of this model to studies of pathogenesis and medical countermeasure development.
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Enzima Convertidora de Angiotensina 2/genética , COVID-19/genética , COVID-19/patología , Queratina-18/genética , Enzima Convertidora de Angiotensina 2/inmunología , Animales , COVID-19/inmunología , COVID-19/virología , Modelos Animales de Enfermedad , Femenino , Humanos , Queratina-18/inmunología , Pulmón/inmunología , Pulmón/patología , Linfocitos/inmunología , Macrófagos/inmunología , Masculino , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas , SARS-CoV-2/fisiología , Tráquea/inmunología , Tráquea/virologíaRESUMEN
Malaria infection induces complex and diverse immune responses. To elucidate the mechanisms underlying host-parasite interaction, we performed a genetic screen during early (24 h) Plasmodium yoelii infection in mice and identified a large number of interacting host and parasite genes/loci after transspecies expression quantitative trait locus (Ts-eQTL) analysis. We next investigated a host E3 ubiquitin ligase gene (March1) that was clustered with interferon (IFN)-stimulated genes (ISGs) based on the similarity of the genome-wide pattern of logarithm of the odds (LOD) scores (GPLS). March1 inhibits MAVS/STING/TRIF-induced type I IFN (IFN-I) signaling in vitro and in vivo. However, in malaria-infected hosts, deficiency of March1 reduces IFN-I production by activating inhibitors such as SOCS1, USP18, and TRIM24 and by altering immune cell populations. March1 deficiency increases CD86+DC (dendritic cell) populations and levels of IFN-γ and interleukin 10 (IL-10) at day 4 post infection, leading to improved host survival. T cell depletion reduces IFN-γ level and reverse the protective effects of March1 deficiency, which can also be achieved by antibody neutralization of IFN-γ. This study reveals functions of MARCH1 (membrane-associated ring-CH-type finger 1) in innate immune responses and provides potential avenues for activating antimalaria immunity and enhancing vaccine efficacy.
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Malaria/inmunología , Plasmodium yoelii/fisiología , Linfocitos T/inmunología , Ubiquitina-Proteína Ligasas/inmunología , Animales , Modelos Animales de Enfermedad , Femenino , Interacciones Huésped-Parásitos , Humanos , Inmunidad Innata , Interferón Tipo I/genética , Interferón Tipo I/inmunología , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-10/genética , Interleucina-10/inmunología , Malaria/enzimología , Malaria/genética , Malaria/parasitología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Plasmodium yoelii/inmunología , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Cellular factors have important roles in all facets of the flavivirus replication cycle. Deciphering viral-host protein interactions is essential for understanding the flavivirus life cycle as well as development of effective antiviral strategies. To uncover novel host factors that are co-opted by multiple flaviviruses, a CRISPR/Cas9 genome wide knockout (KO) screen was employed to identify genes required for replication of Zika virus (ZIKV). Receptor for Activated Protein C Kinase 1 (RACK1) was identified as a novel host factor required for ZIKV replication, which was confirmed via complementary experiments. Depletion of RACK1 via siRNA demonstrated that RACK1 is important for replication of a wide range of mosquito- and tick-borne flaviviruses, including West Nile Virus (WNV), Dengue Virus (DENV), Powassan Virus (POWV) and Langat Virus (LGTV) as well as the coronavirus SARS-CoV-2, but not for YFV, EBOV, VSV or HSV. Notably, flavivirus replication was only abrogated when RACK1 expression was dampened prior to infection. Utilising a non-replicative flavivirus model, we show altered morphology of viral replication factories and reduced formation of vesicle packets (VPs) in cells lacking RACK1 expression. In addition, RACK1 interacted with NS1 protein from multiple flaviviruses; a key protein for replication complex formation. Overall, these findings reveal RACK1's crucial role to the biogenesis of pan-flavivirus replication organelles. IMPORTANCE Cellular factors are critical in all facets of viral lifecycles, where overlapping interactions between the virus and host can be exploited as possible avenues for the development of antiviral therapeutics. Using a genome-wide CRISPR knockout screening approach to identify novel cellular factors important for flavivirus replication we identified RACK1 as a pro-viral host factor for both mosquito- and tick-borne flaviviruses in addition to SARS-CoV-2. Using an innovative flavivirus protein expression system, we demonstrate for the first time the impact of the loss of RACK1 on the formation of viral replication factories known as 'vesicle packets' (VPs). In addition, we show that RACK1 can interact with numerous flavivirus NS1 proteins as a potential mechanism by which VP formation can be induced by the former.
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Sistemas CRISPR-Cas , Flavivirus/genética , Proteínas de Neoplasias/genética , Receptores de Cinasa C Activada/genética , Replicación Viral , Células A549 , Aedes , Animales , COVID-19 , Chlorocebus aethiops , Culicidae , Virus del Dengue/genética , Estudio de Asociación del Genoma Completo , Células HEK293 , Interacciones Huésped-Patógeno/genética , Humanos , ARN Interferente Pequeño/metabolismo , ARN Viral/metabolismo , SARS-CoV-2 , Células Vero , Virus del Nilo Occidental/genética , Virus Zika/genética , Infección por el Virus Zika/virologíaRESUMEN
BACKGROUND: SARS-CoV-2 RNA prevalence in blood donors from large geographic areas of high community transmission is limited. We tested residual donor plasma minipools (MPs) to determine SARS-CoV-2 RNAemia prevalence in six United States areas. STUDY DESIGN/METHODS: Blood donations collected from 7 March 2020 to 25 September 2020 were tested for SARS-CoV-2 RNA (vRNA) in MP of 6 or 16 donations using the Grifols Procleix SARS-CoV-2 research-use only (RUO) transcription-mediated amplification (TMA) assay. Reactive results were confirmed using an alternate target region TMA assay. Reactive MPs were tested by TMA after serial dilution to estimate viral load. Testing for anti-SARS-CoV-2 antibodies and infectivity was performed. RESULTS: A total of 17,995 MPs corresponding to approximately 258,000 donations were tested for vRNA. Three confirmed reactive MP16 were identified. The estimated prevalence of vRNA reactive donations was 1.16/100,000 (95% CI 0.40, 3.42). The vRNA-reactive samples were non-reactive for antibody, and the estimated viral loads of the (presumed single) positive donations within each MP ranged from <1000 to <4000 copies/ml. When tested, no infectivity was observed in inoculated permissive cell cultures. DISCUSSION: Blood donation MP-nucleic acid testing (NAT) indicated that SARS-CoV-2 RNAemia is infrequent and, when detected, the vRNA was at low concentrations. Only one RNA-reactive MP could be tested for infectivity for operational reasons and was not infectious in cell culture. These findings support current recommendations from international and national regulatory agencies to not screen donors by NAT.
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Donantes de Sangre , Seguridad de la Sangre , Prueba de Ácido Nucleico para COVID-19 , COVID-19/diagnóstico , ARN Viral/análisis , SARS-CoV-2/aislamiento & purificación , Humanos , Estados UnidosRESUMEN
Inflammatory monocyte (iMO) recruitment to the brain is a hallmark of many neurologic diseases. Prior to entering the brain, iMOs must egress into the blood from the bone marrow through a mechanism, which for known encephalitic viruses, is CCR2 dependent. In this article, we show that during La Crosse Virus-induced encephalitis, egress of iMOs was surprisingly independent of CCR2, with similar percentages of iMOs in the blood and brain of heterozygous and CCR2-/- mice following infection. Interestingly, CCR2 was required for iMO trafficking from perivascular areas to sites of virus infection within the brain. Thus, CCR2 was not essential for iMO trafficking to the blood or the brain but was essential for trafficking within the brain parenchyma. Analysis of other orthobunyaviruses showed that Jamestown Canyon virus also induced CCR2-independent iMO egress to the blood. These studies demonstrate that the CCR2 requirement for iMO egress to the blood is not universal for all viruses.
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Antígenos Ly/metabolismo , Encefalitis de California/inmunología , Encefalitis de California/metabolismo , Virus La Crosse , Monocitos/inmunología , Monocitos/metabolismo , Receptores CCR2/metabolismo , Animales , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Encéfalo/inmunología , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/virología , Quimiotaxis de Leucocito/inmunología , Modelos Animales de Enfermedad , Encefalitis de California/virología , Femenino , Masculino , Ratones , Ratones Transgénicos , Monocitos/patologíaAsunto(s)
COVID-19 , Modelos Animales de Enfermedad , SARS-CoV-2 , COVID-19/virología , COVID-19/genética , Animales , Ratones , HumanosRESUMEN
BACKGROUND: La Crosse virus (LACV) is the leading cause of pediatric arboviral encephalitis in the USA. LACV encephalitis can result in learning and memory deficits, which may be due to infection and apoptosis of neurons in the brain. Despite neurons being the primary cell infected in the brain by LACV, little is known about neuronal responses to infection. METHODS: Human cerebral organoids (COs), which contain a spectrum of developing neurons, were used to examine neuronal responses to LACV. Plaque assay and quantitative reverse transcription (qRT) PCR were used to determine the susceptibility of COs to LACV infection. Immunohistochemistry, flow cytometry, and single-cell transcriptomics were used to determine specific neuronal subpopulation responses to the virus. RESULTS: Overall, LACV readily infected COs causing reduced cell viability and increased apoptosis. However, it was determined that neurons at different stages of development had distinct responses to LACV. Both neural progenitors and committed neurons were infected with LACV, however, committed neurons underwent apoptosis at a higher rate. Transcriptomic analysis showed that committed neurons expressed fewer interferon (IFN)-stimulated genes (ISGs) and genes involved IFN signaling in response to infection compared to neural progenitors. Furthermore, induction of interferon signaling in LACV-infected COs by application of recombinant IFN enhanced cell viability. CONCLUSIONS: These findings indicate that neuronal maturation increases the susceptibility of neurons to LACV-induced apoptosis. This susceptibility is likely due, at least in part, to mature neurons being less responsive to virus-induced IFN as evidenced by their poor ISG response to LACV. Furthermore, exogenous administration of recombinant IFN to LACV COs rescued cellular viability suggesting that increased IFN signaling is overall protective in this complex neural tissue. Together these findings indicate that induction of IFN signaling in developing neurons is an important deciding factor in virus-induced cell death.
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Encefalitis de California/inmunología , Interferón Tipo I/inmunología , Células-Madre Neurales/virología , Neuronas/virología , Apoptosis/fisiología , Células Cultivadas , Encefalitis de California/patología , Humanos , Células Madre Pluripotentes Inducidas , Células-Madre Neurales/patología , Neuronas/citología , Neuronas/patología , OrganoidesRESUMEN
Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that has evolved effective mechanisms to counteract the type I interferon (IFN) response. Upon recognition of the virus, cells secrete IFNs, which signal through transmembrane receptors (IFNAR) to phosphorylate STAT proteins (pSTAT). pSTAT dimers are transported into the nucleus by importin-α5 and activate the transcription of IFN-stimulated genes (ISGs), increasing cellular resistance to infection. Subsequently, STAT proteins are shuttled back into the cytoplasm by the exportin CRM1. CHIKV nonstructural protein 2 (nsP2) reduces ISG expression by inhibiting general host cell transcription and by specifically reducing the levels of nuclear pSTAT1 via an unknown mechanism. To systematically examine where nsP2 acts within the JAK/STAT signaling cascade, we used two well-characterized mutants of nsP2, P718S and KR649AA. Both mutations abrogate nsP2's ability to shut off host transcription, but only the KR649AA mutant localizes exclusively to the cytoplasm and no longer specifically inhibits JAK/STAT signaling. These mutant nsP2 proteins did not differentially affect IFNAR expression levels or STAT1 phosphorylation in response to IFNs. Coimmunoprecipitation experiments showed that in the presence of nsP2, STAT1 still effectively bound importin-α5. Chemically blocking CRM1-mediated nuclear export in the presence of nsP2 additionally showed that nuclear translocation of STAT1 is not affected by nsP2. nsP2 putatively has five domains. Redirecting the nsP2 KR649AA mutant or just nsP2's C-terminal methyltransferase-like domain into the nucleus strongly reduced nuclear pSTAT in response to IFN stimulation. This demonstrates that the C-terminal domain of nuclear nsP2 specifically inhibits the IFN response by promoting the nuclear export of STAT1.IMPORTANCE Chikungunya virus is an emerging pathogen associated with large outbreaks on the African, Asian, European, and both American continents. In most patients, infection results in high fever, rash, and incapacitating (chronic) arthralgia. CHIKV effectively inhibits the first line of defense, the innate immune response. As a result, stimulation of the innate immune response with interferons (IFNs) is ineffective as a treatment for CHIKV disease. The IFN response requires an intact downstream signaling cascade called the JAK/STAT signaling pathway, which is effectively inhibited by CHIKV nonstructural protein 2 (nsP2) via an unknown mechanism. The research described here specifies where in the JAK/STAT signaling cascade the IFN response is inhibited and which protein domain of nsP2 is responsible for IFN inhibition. The results illuminate new aspects of antiviral defense and CHIKV counterdefense strategies and will direct the search for novel antiviral compounds.
Asunto(s)
Virus Chikungunya/enzimología , Cisteína Endopeptidasas/genética , Inmunidad Innata , Interferón Tipo I/antagonistas & inhibidores , Factor de Transcripción STAT1/genética , Línea Celular , Virus Chikungunya/genética , Virus Chikungunya/fisiología , Simulación por Computador , Cisteína Endopeptidasas/metabolismo , Células HEK293 , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunoprecipitación , Interferón Tipo I/genética , Interferón Tipo I/inmunología , Metiltransferasas/metabolismo , Mutación , Transducción de Señal/genética , Transducción de Señal/inmunología , Replicación ViralRESUMEN
The recent association between Zika virus (ZIKV) and neurologic complications, including Guillain-Barré syndrome in adults and CNS abnormalities in fetuses, highlights the importance in understanding the immunological mechanisms controlling this emerging infection. Studies have indicated that ZIKV evades the human type I IFN response, suggesting a role for the adaptive immune response in resolving infection. However, the inability of ZIKV to antagonize the mouse IFN response renders the virus highly susceptible to circulating IFN in murine models. Thus, as we show in this article, although wild-type C57BL/6 mice mount cell-mediated and humoral adaptive immune responses to ZIKV, these responses were not required to prevent disease. However, when the type I IFN response of mice was suppressed, then the adaptive immune responses became critical. For example, when type I IFN signaling was blocked by Abs in Rag1-/- mice, the mice showed dramatic weight loss and ZIKV infection in the brain and testes. This phenotype was not observed in Ig-treated Rag1-/- mice or wild-type mice treated with anti-type I IFNR alone. Furthermore, we found that the CD8+ T cell responses of pregnant mice to ZIKV infection were diminished compared with nonpregnant mice. It is possible that diminished cell-mediated immunity during pregnancy could increase virus spread to the fetus. These results demonstrate an important role for the adaptive immune response in the control of ZIKV infection and imply that vaccination may prevent ZIKV-related disease, particularly when the type I IFN response is suppressed as it is in humans.
Asunto(s)
Inmunidad Adaptativa , Encéfalo/virología , Linfocitos T CD8-positivos/virología , Complicaciones Infecciosas del Embarazo/inmunología , Testículo/virología , Infección por el Virus Zika/inmunología , Virus Zika/inmunología , Animales , Anticuerpos Bloqueadores/administración & dosificación , Encéfalo/inmunología , Linfocitos T CD8-positivos/inmunología , Modelos Animales de Enfermedad , Femenino , Proteínas de Homeodominio/genética , Humanos , Evasión Inmune , Interferón Tipo I/inmunología , Interferón Tipo I/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Embarazo/inmunología , Testículo/inmunología , Infección por el Virus Zika/epidemiologíaRESUMEN
The vector-borne flaviviruses cause severe disease in humans on every inhabited continent on earth. Their transmission by arthropods, particularly mosquitoes, facilitates large emergence events such as witnessed with Zika virus (ZIKV) or West Nile virus in the Americas. Every vector-borne flavivirus examined thus far that causes disease in humans, from dengue virus to ZIKV, antagonizes the host type I interferon (IFN-I) response by preventing JAK-STAT signaling, suggesting that suppression of this pathway is an important determinant of infection. The most direct and potent viral inhibitor of this pathway is the nonstructural protein NS5. However, the mechanisms utilized by NS5 from different flaviviruses are often quite different, sometimes despite close evolutionary relationships between viruses. The varied mechanisms of NS5 as an IFN-I antagonist are also surprising given that the evolution of NS5 is restrained by the requirement to maintain function of two enzymatic activities critical for virus replication, the methyltransferase and RNA-dependent RNA polymerase. This review discusses the different strategies used by flavivirus NS5 to evade the antiviral effects of IFN-I and how this information can be used to better model disease and develop antiviral countermeasures.
Asunto(s)
Infecciones por Flavivirus/metabolismo , Infecciones por Flavivirus/virología , Flavivirus/fisiología , Interferón Tipo I/metabolismo , Transducción de Señal , Proteínas no Estructurales Virales/metabolismo , Animales , Vectores Artrópodos/virología , Flavivirus/clasificación , Infecciones por Flavivirus/transmisión , Interacciones Huésped-Patógeno , Humanos , Interferón Tipo I/antagonistas & inhibidores , Quinasas Janus/metabolismo , Unión Proteica , Factores de Transcripción STAT/metabolismo , Relación Estructura-Actividad , Proteínas no Estructurales Virales/química , Proteínas Virales/metabolismoRESUMEN
Selective autophagy of the endoplasmic reticulum (termed ER-phagy) is controlled by members of the FAM134 reticulon protein family. Here we used mouse embryonic fibroblasts from mice deficient in FAM134B to examine the role of the ER in replication of historic (Mayinga) or contemporary (Makona GCO7) strains of Ebola virus (EBOV). Loss of FAM134B resulted in 1-2 log10 higher production of infectious EBOV, which was associated with increased production of viral proteins GP and VP40 and greater accumulation of nucleocaspid lattices. In addition, only 10% of wild-type cells contained detectable nucleoprotein, whereas knockout of FAM134B resulted in 80% of cells positive for nucleoprotein. Together, these data suggest that FAM134B-dependent ER-phagy is an important limiting event in EBOV replication in mouse cells and may have implications for further development of antiviral therapeutics and murine models of infection.