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Tick-borne encephalitis (TBE) virus is the most prevalent tick-transmitted orthoflavivirus in Europe. Due to the nonspecific nature of its symptoms, TBE is primarily diagnosed by ELISA-based detection of specific antibodies in the patient serum. However, cross-reactivity between orthoflaviviruses complicates the diagnosis. Specificity issues may be mitigated by serum neutralization assays (SNT), although the handling of clinically relevant orthoflaviviruses requires biosafety level (BSL) 3 conditions and they have highly divergent viral kinetics and cell tropisms. In the present study, we established a reporter virus particle (RVP)-based SNT in which the infectivity is measured by luminescence and that can be performed under BSL-2 conditions. The RVP-based SNT for TBEV exhibited a highly significant correlation with the traditional virus-based SNT (R2 = 0.8637, p < 0.0001). The RVP-based assay demonstrated a sensitivity of 92.3% (95% CI: 79.7%-97.4%) and specificity of 100% (95% CI: 81.6%-100%). We also tested the cross-reactivity of serum samples in RVP-based assays against other orthoflaviviruses (yellow fever virus, dengue virus type 2, Zika virus, West Nile virus and Japanese encephalitis virus). Interestingly, all serum samples which had tested TBEV-positive by ELISA but negative by RVP-based SNT were reactive for antibodies against other orthoflaviviruses. Thus, the RVP-based seroneutralization assay provides an added value in clinical diagnostics as well as in epidemiological studies.
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Anticuerpos Antivirales , Reacciones Cruzadas , Virus de la Encefalitis Transmitidos por Garrapatas , Encefalitis Transmitida por Garrapatas , Ensayo de Inmunoadsorción Enzimática , Pruebas de Neutralización , Sensibilidad y Especificidad , Virus de la Encefalitis Transmitidos por Garrapatas/inmunología , Humanos , Anticuerpos Antivirales/sangre , Pruebas de Neutralización/métodos , Encefalitis Transmitida por Garrapatas/diagnóstico , Encefalitis Transmitida por Garrapatas/virología , Ensayo de Inmunoadsorción Enzimática/métodos , Virión/inmunología , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , AnimalesRESUMEN
Flaviviruses such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV) are spread by mosquitoes and cause human disease and mortality in tropical areas. In contrast, Powassan virus (POWV), which causes severe neurologic illness, is a flavivirus transmitted by ticks in temperate regions of the Northern hemisphere. We find serologic neutralizing activity against POWV in individuals living in Mexico and Brazil. Monoclonal antibodies P002 and P003, which were derived from a resident of Mexico (where POWV is not reported), neutralize POWV lineage I by recognizing an epitope on the virus envelope domain III (EDIII) that is shared with a broad range of tick- and mosquito-borne flaviviruses. Our findings raise the possibility that POWV, or a flavivirus closely related to it, infects humans in the tropics.
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Anticuerpos Neutralizantes , Humanos , Brasil , Anticuerpos Neutralizantes/inmunología , México , Anticuerpos Antivirales/inmunología , Animales , Virus de la Encefalitis Transmitidos por Garrapatas/inmunología , Flavivirus/inmunología , Epítopos/inmunología , Anticuerpos Monoclonales/inmunología , Garrapatas/virología , Garrapatas/inmunología , Femenino , MasculinoRESUMEN
Introduction: Lyme disease (LD) affects â¼476,000 people each year in the United States. Symptoms are variable and include rash and flu-like symptoms. Reasons for the wide variation in disease outcomes are unknown. Powassan virus (POWV) is a tick-borne flavivirus that causes disease ranging from asymptomatic infection to encephalitis, neurologic damage, and death. POWV and LD geographic case distributions overlap, with Ixodes species ticks as the common vectors. Clinical ramifications of coinfection or sequential infection are unknown. Objectives: This study's primary objective was to determine the prevalence of POWV-reactive antibodies in sera samples collected from previously studied cohorts of individuals with self-reported LD history residing in the Northeastern United States. As a secondary objective, we studied clinical differences between people with self-reported LD history and low versus high POWV antibody levels. Methods: We used an enzyme-linked immunosorbent assay (ELISA) to quantify IgG directed at the POWV envelope (E) protein domain III in 538 samples from individuals with self-reported LD history and 16 community controls. The samples were also tested with an ELISA assay to quantify IgG directed at the POWV NS1 protein. Results: The percentage of individuals with LD history and possible evidence of POWV exposure varied depending on the assay utilized. We found no significant difference in clinical symptoms between those with low or high POWV IgG levels in the in-house assay. Congruence of the EDIII and NS1 assays was low with only 12% of those positive in the in-house EDIII ELISA testing positive in the POWV NS1 ELISA. Conclusions: The results highlight the difficulty in flavivirus diagnostic testing, particularly in the retrospective detection of flavivirus exposure. The findings suggest that a prospective study with symptomatic patients using approved clinical testing is necessary to address the incidence and clinical implications of LD and POWV co-infection or sequential infection.
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Virus de la Encefalitis Transmitidos por Garrapatas , Encefalitis Transmitida por Garrapatas , Ixodes , Enfermedad de Lyme , Animales , Humanos , Estados Unidos/epidemiología , Prevalencia , Estudios Retrospectivos , Estudios Prospectivos , Encefalitis Transmitida por Garrapatas/veterinaria , Enfermedad de Lyme/epidemiología , Enfermedad de Lyme/veterinaria , New England/epidemiología , Anticuerpos Antivirales , Inmunoglobulina GRESUMEN
Vaccination will likely be a key component of strategies to curtail or prevent future sarbecovirus pandemics and to reduce the prevalence of infection and disease by future SARS-CoV-2 variants. A "pan-sarbecovirus" vaccine, that provides maximum possible mitigation of human disease, should elicit neutralizing antibodies with maximum possible breadth. By positioning multiple different receptor binding domain (RBD) antigens in close proximity on a single immunogen, it is postulated that cross-reactive B cell receptors might be selectively engaged. Heteromultimeric vaccines could therefore elicit individual antibodies that neutralize a broad range of viral species. Here, we use model systems to investigate the ability of multimeric sarbecovirus RBD immunogens to expand cross-reactive B cells and elicit broadly reactive antibodies. Homomultimeric RBD immunogens generated higher serum neutralizing antibody titers than the equivalent monomeric immunogens, while heteromultimeric RBD immunogens generated neutralizing antibodies recognizing each RBD component. Moreover, RBD heterodimers elicited a greater fraction of cross-reactive germinal center B cells and cross-reactive RBD binding antibodies than did homodimers. However, when serum antibodies from RBD heterodimer-immunized mice were depleted using one RBD component, neutralization activity against the homologous viral pseudotype was removed, but neutralization activity against pseudotypes corresponding to the other RBD component was unaffected. Overall, simply combining divergent RBDs in a single immunogen generates largely separate sets of individual RBD-specific neutralizing serum antibodies that are mostly incapable of neutralizing viruses that diverge from the immunogen components.
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Anticuerpos Neutralizantes , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo , Animales , Ratones , Humanos , Anticuerpos Antivirales , Pruebas de Neutralización , Vacunación , Glicoproteína de la Espiga del Coronavirus/químicaRESUMEN
Although dengue virus (DENV) infection typically causes asymptomatic disease, DENV-infected patients can experience severe complications. A risk factor for symptomatic disease is pre-existing anti-DENV IgG antibodies. Cellular assays suggested that these antibodies can enhance viral infection of Fcγ receptor (FcγR)-expressing myeloid cells. Recent studies, however, revealed more complex interactions between anti-DENV antibodies and specific FcγRs by demonstrating that modulation of the IgG Fc glycan correlates with disease severity. To investigate the in vivo mechanisms of antibody-mediated dengue pathogenesis, we developed a mouse model for dengue disease that recapitulates the unique complexity of human FcγRs. In in vivo mouse models of dengue disease, we discovered that the pathogenic activity of anti-DENV antibodies is exclusively mediated through engagement of FcγRIIIa on splenic macrophages, resulting in inflammatory sequelae and mortality. These findings highlight the importance of IgG-FcγRIIIa interactions in dengue, with important implications for the design of safer vaccination approaches and effective therapeutic strategies.
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Virus del Dengue , Dengue , Humanos , Animales , Ratones , Receptores de IgG , Macrófagos , Inmunoglobulina GRESUMEN
Mosquito-borne West Nile virus (WNV) infection is benign in most individuals but can cause encephalitis in <1% of infected individuals. We show that â¼35% of patients hospitalized for WNV disease (WNVD) in six independent cohorts from the EU and USA carry auto-Abs neutralizing IFN-α and/or -ω. The prevalence of these antibodies is highest in patients with encephalitis (â¼40%), and that in individuals with silent WNV infection is as low as that in the general population. The odds ratios for WNVD in individuals with these auto-Abs relative to those without them in the general population range from 19.0 (95% CI 15.0-24.0, P value <10-15) for auto-Abs neutralizing only 100 pg/ml IFN-α and/or IFN-ω to 127.4 (CI 87.1-186.4, P value <10-15) for auto-Abs neutralizing both IFN-α and IFN-ω at a concentration of 10 ng/ml. These antibodies block the protective effect of IFN-α in Vero cells infected with WNV in vitro. Auto-Abs neutralizing IFN-α and/or IFN-ω underlie â¼40% of cases of WNV encephalitis.
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Interferón Tipo I , Fiebre del Nilo Occidental , Virus del Nilo Occidental , Animales , Chlorocebus aethiops , Humanos , Células Vero , Autoanticuerpos , Anticuerpos Antivirales , Interferón-alfaRESUMEN
Powassan virus (POWV) is an emerging tick-borne virus and cause of lethal encephalitis in humans. The lack of treatment or prevention strategies for POWV disease underscores the need for an effective POWV vaccine. Here, we took two independent approaches to develop vaccine candidates. First, we recoded the POWV genome to increase the dinucleotide frequencies of CpG and UpA to potentially attenuate the virus by raising its susceptibility to host innate immune factors, such as the zinc-finger antiviral protein (ZAP). Secondly, we took advantage of the live-attenuated yellow fever virus vaccine 17D strain (YFV-17D) as a vector to express the structural genes pre-membrane (prM) and envelope (E) of POWV. The chimeric YFV-17D-POWV vaccine candidate was further attenuated for in vivo application by removing an N-linked glycosylation site within the nonstructural protein (NS)1 of YFV-17D. This live-attenuated chimeric vaccine candidate significantly protected mice from POWV disease, conferring a 70% survival rate after lethal challenge when administered in a homologous two-dose regimen. Importantly, when given in a heterologous prime-boost vaccination scheme, in which vaccination with the initial chimeric virus was followed by a protein boost with the envelope protein domain III (EDIII), 100% of the mice were protected without showing any signs of morbidity. Combinations of this live-attenuated chimeric YFV-17D-POWV vaccine candidate with an EDIII protein boost warrant further studies for the development of an effective vaccine strategy for the prevention of POWV disease.
RESUMEN
ADP-ribosylation is a highly dynamic posttranslational modification frequently studied in stress response pathways with recent attention given to its role in response to viral infection. Notably, the alphaviruses encode catalytically active macrodomains capable of ADP-ribosylhydrolase (ARH) activities, implying a role in remodeling the cellular ADP-ribosylome. This report decouples mono- and poly-ARH contributions to macrodomain function using a newly engineered Sindbis virus (SINV) mutant with attenuated poly-ARH activity. Our findings indicate that viral poly-ARH activity is uniquely required for high titer replication in mammalian systems. Despite translating incoming genomic RNA as efficiently as WT virus, mutant viruses have a reduced capacity to establish productive infection, offering a more complete understanding of the kinetics and role of the alphavirus macrodomain with important implications for broader ADP-ribosyltransferase biology. IMPORTANCE Viral macrodomains have drawn attention in recent years due to their high degree of conservation in several virus families (e.g., coronaviruses and alphaviruses) and their potential druggability. These domains erase mono- or poly-ADP-ribose, posttranslational modifications written by host poly-ADP-ribose polymerase (PARP) proteins, from undetermined host or viral proteins to enhance replication. Prior work determined that efficient alphavirus replication requires catalytically active macrodomains; however, which form of the modification requires removal and from which protein(s) had not been determined. Here, we present evidence for the specific requirement of poly-ARH activity to ensure efficient productive infection and virus replication.
Asunto(s)
Coronavirus , Hidrolasas , ARN Viral , Virus Sindbis , Animales , Coronavirus/genética , Hidrolasas/metabolismo , Mamíferos/genética , Poli Adenosina Difosfato Ribosa/metabolismo , ARN Viral/genética , Virus Sindbis/enzimología , Virus Sindbis/genética , Replicación ViralRESUMEN
Zika virus (ZIKV) is a re-emerging flavivirus that has caused large-scale epidemics. Infection during pregnancy can lead to neurologic developmental abnormalities in children. There is no approved vaccine or therapy for ZIKV. To uncover cellular pathways required for ZIKV that can be therapeutically targeted, we transcriptionally upregulated all known human coding genes with an engineered CRISPR-Cas9 activation complex in human fibroblasts deficient in interferon (IFN) signaling. We identified Ras homolog family member V (RhoV) and WW domain-containing transcription regulator 1 (WWTR1) as proviral factors, and found them to play important roles during early ZIKV infection in A549 cells. We then focused on RhoV, a Rho GTPase with atypical terminal sequences and membrane association, and validated its proviral effects on ZIKV infection and virion production in SNB-19 cells. We found that RhoV promotes infection of some flaviviruses and acts at the step of viral entry. Furthermore, RhoV proviral effects depend on the complete GTPase cycle. By depleting Rho GTPases and related proteins, we identified RhoB and Pak1 as additional proviral factors. Taken together, these results highlight the positive role of RhoV in ZIKV infection and confirm CRISPR activation as a relevant method to identify novel host-pathogen interactions.
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Proteínas de Unión al GTP/metabolismo , Proteínas de Neoplasias/metabolismo , Infección por el Virus Zika/enzimología , Virus Zika/fisiología , Proteína de Unión al GTP rhoB/metabolismo , Células A549 , Sistemas CRISPR-Cas , Proteínas de Unión al GTP/genética , Humanos , Proteínas de Neoplasias/genética , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ/genética , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ/metabolismo , Internalización del Virus , Replicación Viral , Virus Zika/genética , Infección por el Virus Zika/genética , Infección por el Virus Zika/virología , Quinasas p21 Activadas/genética , Quinasas p21 Activadas/metabolismo , Proteína de Unión al GTP rhoB/genéticaRESUMEN
Molecular virology tools are critical for basic studies of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and for developing new therapeutics. Experimental systems that do not rely on viruses capable of spread are needed for potential use in lower-containment settings. In this work, we use a yeast-based reverse genetics system to develop spike-deleted SARS-CoV-2 self-replicating RNAs. These noninfectious self-replicating RNAs, or replicons, can be trans-complemented with viral glycoproteins to generate replicon delivery particles for single-cycle delivery into a range of cell types. This SARS-CoV-2 replicon system represents a convenient and versatile platform for antiviral drug screening, neutralization assays, host factor validation, and viral variant characterization.
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ARN Viral/genética , Replicón/fisiología , SARS-CoV-2/genética , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Antivirales/farmacología , Línea Celular , Humanos , Interferones/farmacología , Pruebas de Sensibilidad Microbiana , Mutación , Plásmidos , ARN Viral/metabolismo , Replicón/genética , Genética Inversa , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/fisiología , Saccharomyces cerevisiae/genética , Glicoproteína de la Espiga del Coronavirus/genética , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Pseudotipado Viral , Virión/genética , Virión/fisiología , Replicación ViralRESUMEN
Tick-borne encephalitis virus (TBEV) is an emerging human pathogen that causes potentially fatal disease with no specific treatment. Mouse monoclonal antibodies are protective against TBEV, but little is known about the human antibody response to infection. Here, we report on the human neutralizing antibody response to TBEV in a cohort of infected and vaccinated individuals. Expanded clones of memory B cells expressed closely related anti-envelope domain III (EDIII) antibodies in both groups of volunteers. However, the most potent neutralizing antibodies, with IC50s below 1 ng/ml, were found only in individuals who recovered from natural infection. These antibodies also neutralized other tick-borne flaviviruses, including Langat, louping ill, Omsk hemorrhagic fever, Kyasanur forest disease, and Powassan viruses. Structural analysis revealed a conserved epitope near the lateral ridge of EDIII adjoining the EDI-EDIII hinge region. Prophylactic or early therapeutic antibody administration was effective at low doses in mice that were lethally infected with TBEV.
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Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Virus de la Encefalitis Transmitidos por Garrapatas/inmunología , Encefalitis Transmitida por Garrapatas/inmunología , Inmunoglobulina G/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/genética , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Neutralizantes/genética , Anticuerpos Antivirales/administración & dosificación , Anticuerpos Antivirales/genética , Células Cultivadas , Estudios de Cohortes , Reacciones Cruzadas/inmunología , Virus de la Encefalitis Transmitidos por Garrapatas/efectos de los fármacos , Virus de la Encefalitis Transmitidos por Garrapatas/fisiología , Encefalitis Transmitida por Garrapatas/prevención & control , Encefalitis Transmitida por Garrapatas/virología , Epítopos/inmunología , Femenino , Humanos , Inmunoglobulina G/administración & dosificación , Ratones Endogámicos BALB C , Homología de Secuencia de Aminoácido , Análisis de Supervivencia , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Yellow fever virus (YFV) live attenuated vaccine can, in rare cases, cause life-threatening disease, typically in patients with no previous history of severe viral illness. Autosomal recessive (AR) complete IFNAR1 deficiency was reported in one 12-yr-old patient. Here, we studied seven other previously healthy patients aged 13 to 80 yr with unexplained life-threatening YFV vaccine-associated disease. One 13-yr-old patient had AR complete IFNAR2 deficiency. Three other patients vaccinated at the ages of 47, 57, and 64 yr had high titers of circulating auto-Abs against at least 14 of the 17 individual type I IFNs. These antibodies were recently shown to underlie at least 10% of cases of life-threatening COVID-19 pneumonia. The auto-Abs were neutralizing in vitro, blocking the protective effect of IFN-α2 against YFV vaccine strains. AR IFNAR1 or IFNAR2 deficiency and neutralizing auto-Abs against type I IFNs thus accounted for more than half the cases of life-threatening YFV vaccine-associated disease studied here. Previously healthy subjects could be tested for both predispositions before anti-YFV vaccination.
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Anticuerpos Neutralizantes/inmunología , Autoanticuerpos/inmunología , Enfermedades Autoinmunes , COVID-19 , Enfermedades Genéticas Congénitas , Interferón-alfa , Receptor de Interferón alfa y beta , SARS-CoV-2 , Vacuna contra la Fiebre Amarilla , Virus de la Fiebre Amarilla , Adolescente , Adulto , Anciano , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , COVID-19/genética , COVID-19/inmunología , Femenino , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/inmunología , Células HEK293 , Humanos , Interferón-alfa/genética , Interferón-alfa/inmunología , Masculino , Persona de Mediana Edad , Receptor de Interferón alfa y beta/deficiencia , Receptor de Interferón alfa y beta/inmunología , SARS-CoV-2/genética , SARS-CoV-2/inmunología , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vacuna contra la Fiebre Amarilla/efectos adversos , Vacuna contra la Fiebre Amarilla/genética , Vacuna contra la Fiebre Amarilla/inmunología , Virus de la Fiebre Amarilla/genética , Virus de la Fiebre Amarilla/inmunologíaRESUMEN
Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection, we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results, we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms present at nearly 20% in East Asian populations reduce flavivirus infection. Based on our mechanistic studies, we propose that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication.
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Infecciones por Flavivirus/genética , Flavivirus/fisiología , Proteínas de la Membrana/metabolismo , Animales , Pueblo Asiatico/genética , Autofagia , COVID-19/genética , COVID-19/metabolismo , COVID-19/virología , Sistemas CRISPR-Cas , Línea Celular , Infecciones por Flavivirus/inmunología , Infecciones por Flavivirus/metabolismo , Infecciones por Flavivirus/virología , Técnicas de Inactivación de Genes , Estudio de Asociación del Genoma Completo , Interacciones Huésped-Patógeno , Humanos , Inmunidad Innata , Proteínas de la Membrana/genética , Polimorfismo de Nucleótido Simple , SARS-CoV-2/fisiología , Replicación Viral , Virus de la Fiebre Amarilla/fisiología , Virus Zika/fisiologíaRESUMEN
The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has devastated the global economy and claimed more than 1.7 million lives, presenting an urgent global health crisis. To identify host factors required for infection by SARS-CoV-2 and seasonal coronaviruses, we designed a focused high-coverage CRISPR-Cas9 library targeting 332 members of a recently published SARS-CoV-2 protein interactome. We leveraged the compact nature of this library to systematically screen SARS-CoV-2 at two physiologically relevant temperatures along with three related coronaviruses (human coronavirus 229E [HCoV-229E], HCoV-NL63, and HCoV-OC43), allowing us to probe this interactome at a much higher resolution than genome-scale studies. This approach yielded several insights, including potential virus-specific differences in Rab GTPase requirements and glycosylphosphatidylinositol (GPI) anchor biosynthesis, as well as identification of multiple pan-coronavirus factors involved in cholesterol homeostasis. This coronavirus essentiality catalog could inform ongoing drug development efforts aimed at intercepting and treating coronavirus disease 2019 (COVID-19) and help prepare for future coronavirus outbreaks.
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COVID-19/virología , SARS-CoV-2/metabolismo , Sistemas CRISPR-Cas , Coronavirus Humano 229E/genética , Coronavirus Humano 229E/metabolismo , Coronavirus Humano NL63/genética , Coronavirus Humano NL63/metabolismo , Coronavirus Humano OC43 , Genes Virales , Interacciones Huésped-Patógeno , Humanos , SARS-CoV-2/genética , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The coronavirus disease 2019 (COVID-19) pandemic has claimed the lives of over one million people worldwide. The causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a member of the Coronaviridae family of viruses that can cause respiratory infections of varying severity. The cellular host factors and pathways co-opted during SARS-CoV-2 and related coronavirus life cycles remain ill defined. To address this gap, we performed genome-scale CRISPR knockout screens during infection by SARS-CoV-2 and three seasonal coronaviruses (HCoV-OC43, HCoV-NL63, and HCoV-229E). These screens uncovered host factors and pathways with pan-coronavirus and virus-specific functional roles, including major dependency on glycosaminoglycan biosynthesis, sterol regulatory element-binding protein (SREBP) signaling, bone morphogenetic protein (BMP) signaling, and glycosylphosphatidylinositol biosynthesis, as well as a requirement for several poorly characterized proteins. We identified an absolute requirement for the VMP1, TMEM41, and TMEM64 (VTT) domain-containing protein transmembrane protein 41B (TMEM41B) for infection by SARS-CoV-2 and three seasonal coronaviruses. This human coronavirus host factor compendium represents a rich resource to develop new therapeutic strategies for acute COVID-19 and potential future coronavirus pandemics.
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Infecciones por Coronavirus/genética , Estudio de Asociación del Genoma Completo , SARS-CoV-2/fisiología , Células A549 , Línea Celular , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Coronavirus Humano 229E/fisiología , Infecciones por Coronavirus/virología , Coronavirus Humano NL63/fisiología , Coronavirus Humano OC43/fisiología , Técnicas de Inactivación de Genes , Células HEK293 , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Proteínas de la Membrana/metabolismo , Redes y Vías Metabólicas/efectos de los fármacos , Mapeo de Interacción de ProteínasRESUMEN
The COVID-19 pandemic has claimed the lives of more than one million people worldwide. The causative agent, SARS-CoV-2, is a member of the Coronaviridae family, which are viruses that cause respiratory infections of varying severity. The cellular host factors and pathways co-opted by SARS-CoV-2 and other coronaviruses in the execution of their life cycles remain ill-defined. To develop an extensive compendium of host factors required for infection by SARS-CoV-2 and three seasonal coronaviruses (HCoV-OC43, HCoV-NL63, and HCoV-229E), we performed parallel genome-scale CRISPR knockout screens. These screens uncovered multiple host factors and pathways with pan-coronavirus and virus-specific functional roles, including major dependency on glycosaminoglycan biosynthesis, SREBP signaling, and glycosylphosphatidylinositol biosynthesis, as well as an unexpected requirement for several poorly characterized proteins. We identified an absolute requirement for the VTT-domain containing protein TMEM41B for infection by SARS-CoV-2 and all other coronaviruses. This human Coronaviridae host factor compendium represents a rich resource to develop new therapeutic strategies for acute COVID-19 and potential future coronavirus spillover events. HIGHLIGHTS: Genome-wide CRISPR screens for SARS-CoV-2, HCoV-OC43, HCoV-NL63, and HCoV-229E coronavirus host factors.Parallel genome-wide CRISPR screening uncovered host factors and pathways with pan-coronavirus and virus-specific functional roles.Coronaviruses co-opt multiple biological pathways, including glycosaminoglycan biosynthesis, SREBP signaling, and glycosylphosphatidylinositol biosynthesis and anchoring, among others.TMEM41B - a poorly understood factor with roles in autophagy and lipid mobilization - is a critical pan-coronavirus host factor.
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Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms (SNPs) present at nearly twenty percent in East Asian populations reduce flavivirus infection. Based on our mechanistic studies we hypothesize that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication. HIGHLIGHTS: TMEM41B and VMP1 are required for both autophagy and flavivirus infection, however, autophagy is not required for flavivirus infection.TMEM41B associates with viral proteins and likely facilitates membrane remodeling to establish viral RNA replication complexes.TMEM41B single nucleotide polymorphisms (SNPs) present at nearly twenty percent in East Asian populations reduce flavivirus infection.TMEM41B-deficient cells display an exaggerated innate immune response upon high multiplicity flavivirus infection.
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The ongoing SARS-CoV-2 pandemic has devastated the global economy and claimed nearly one million lives, presenting an urgent global health crisis. To identify host factors required for infection by SARS-CoV-2 and seasonal coronaviruses, we designed a focused high-coverage CRISPR-Cas9 library targeting 332 members of a recently published SARS-CoV-2 protein interactome. We leveraged the compact nature of this library to systematically screen four related coronaviruses (HCoV-229E, HCoV-NL63, HCoV-OC43 and SARS-CoV-2) at two physiologically relevant temperatures (33 °C and 37 °C), allowing us to probe this interactome at a much higher resolution relative to genome scale studies. This approach yielded several new insights, including unexpected virus and temperature specific differences in Rab GTPase requirements and GPI anchor biosynthesis, as well as identification of multiple pan-coronavirus factors involved in cholesterol homeostasis. This coronavirus essentiality catalog could inform ongoing drug development efforts aimed at intercepting and treating COVID-19, and help prepare for future coronavirus outbreaks. HIGHLIGHTS: Focused CRISPR screens targeting host factors in the SARS-CoV-2 interactome were performed for SARS-CoV-2, HCoV-229E, HCoV-NL63, and HCoV-OC43 coronaviruses.Focused interactome CRISPR screens achieve higher resolution compared to genome-wide screens, leading to the identification of critical factors missed by the latter.Parallel CRISPR screens against multiple coronaviruses uncover host factors and pathways with pan-coronavirus and virus-specific functional roles.The number of host proteins that interact with a viral bait protein is not proportional to the number of functional interactors.Novel SARS-CoV-2 host factors are expressed in relevant cell types in the human airway.
RESUMEN
OBJECTIVES: Screening for genes differentially expressed in placental tissues, aiming to identify transcriptional signatures that may be involved in ZIKV congenital pathogenesis. METHODS: Transcriptome data from placental tissues of pregnant women naturally infected with Zika virus during the third trimester were compared to those from women who tested negative for Zika infection. The findings were validated using both a cell culture model and an immunohistochemistry/morphological analysis of naturally infected placental tissues. RESULTS: Transcriptome analysis revealed that Zika virus infection induces downregulation of insulin-like growth factor II (IGF2) gene, an essential factor for fetal development. The Caco-2 cell culture model that constitutively expresses IGF2 was used for the transcriptome validation. Asiatic and African Zika virus strains infection caused downregulated IGF2 gene expression in Caco-2 cells, whereas other flaviviruses, such as dengue serotype 1, West Nile and wild-type yellow fever viruses, had no effect on this gene expression. Immunohistochemical assays on decidual tissues corroborated our transcriptome analysis, showing that IGF2 is reduced in the decidua of Zika virus-infected women. CONCLUSIONS: Our results draw attention to IGF2 modulation in uterine tissues, and this finding is expected to support future studies on strategies to ameliorate the harmful effects of Zika virus infection during pregnancy.
Asunto(s)
Infección por el Virus Zika , Virus Zika , Brasil , Células CACO-2 , Regulación hacia Abajo , Femenino , Humanos , Factor II del Crecimiento Similar a la Insulina/genética , Embarazo , Tercer Trimestre del Embarazo , Virus Zika/genéticaRESUMEN
Yellow fever virus (YFV), a member of the Flaviviridae family, is an arthropod-borne virus that can cause severe disease in humans with a lethality rate of up to 60%. Since 2017, increases in YFV activity in areas of South America and Africa have been described. Although a vaccine is available, named strain 17D (Theiler and Smith, 1937), it is contraindicated for use in the elderly, expectant mothers, immunocompromised people, among others. To this day there is no antiviral treatment against YFV to reduce the severity of viral infection. Here, we used a circular polymerase extension reaction (CPER)-based reverse genetics approach to generate a full-length reporter virus (YFVhb) by introducing a small HiBit tag in the NS1 protein. The reporter virus replicates at a similar rate to the parental YFV in HuH-7 cells. Using YFVhb, we designed a high throughput antiviral screening luciferase-based assay to identify inhibitors that target any step of the viral replication cycle. We validated our assay by using a range of inhibitors including drugs, immune sera and neutralizing single chain variable fragments (scFv). In light of the recent upsurge in YFV and a potential spread of the virus, this assay is a further tool in the development of antiviral therapy against YFV.