RESUMEN
IMPORTANCE: Mouse models of viral infection play an especially large role in virology. In 1960, a mouse virus, lactate dehydrogenase-elevating virus (LDV), was discovered and found to have the peculiar ability to evade clearance by the immune system, enabling it to persistently infect an individual mouse for its entire lifespan without causing overt disease. However, researchers were unable to grow LDV in culture, ultimately resulting in the demise of this system as a model of failed immunity. We solve this problem by identifying the cell-surface molecule CD163 as the critical missing component in cell-culture systems, enabling the growth of LDV in immortalized cell lines for the first time. This advance creates abundant opportunities for further characterizing LDV in order to study both failed immunity and the family of viruses to which LDV belongs, Arteriviridae (aka, arteriviruses).
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Antígenos CD , Antígenos de Diferenciación Mielomonocítica , Técnicas de Cultivo de Célula , Expresión Génica Ectópica , Virus Elevador de Lactato Deshidrogenasa , Receptores de Superficie Celular , Animales , Ratones , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/metabolismo , Línea Celular/virología , Virus Elevador de Lactato Deshidrogenasa/genética , Virus Elevador de Lactato Deshidrogenasa/crecimiento & desarrollo , Virus Elevador de Lactato Deshidrogenasa/inmunología , Virus Elevador de Lactato Deshidrogenasa/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Factores de TiempoRESUMEN
We previously identified a subset of interferon-stimulated genes (ISGs) upregulated by West Nile virus (WNV) infection in wild-type mouse embryo fibroblasts (MEFs) after viral proteins had inhibited type I interferon (IFN)-mediated JAK-STAT signaling and also in WNV-infected RIG-I-/-, MDA5-/-, STAT1-/-, STAT2-/-, IFNAR-/-, IRF3-/-, IRF7-/-, and IRF3/7-/- MEFs. In this study, ISG upregulation by WNV infection in IFNAR-/- MEFs was confirmed by transcriptome sequencing (RNA-seq). ISG upregulation by WNV infection was inhibited in RIG-I/MDA5-/- MEFs. ISGs were upregulated in IRF1-/- and IRF5-/- MEFs but only minimally upregulated in IRF3/5/7-/- MEFs, suggesting redundant IRF involvement. We previously showed that a single proximal interferon-stimulated response element (ISRE) in the Oas1a and Oas1b promoters bound the ISGF3 complex after type I IFN treatment. In this study, we used wild-type and mutant promoter luciferase reporter constructs to identify critical regions in the Oas1b and Ifit1 promoters for gene activation in infected IFNAR-/- MEFs. Two ISREs were required in both promoters. Mutation of these ISREs in an Ifit1 promoter DNA probe reduced in vitro complex formation with infected nuclear extracts. An NF-κB inhibitor decreased Ifit1 promoter activity in cells and in vitro complex formation. IRF3 and p50 promoter binding was detected by chromatin immunoprecipitation (ChIP) for upregulated ISGs with two proximal ISREs. The data indicate that ISREs function cooperatively to upregulate the expression of some ISGs when type I IFN signaling is absent, with the binding complex consisting of IRF3, IRF5, and/or IRF7 and an NF-κB component(s) as well as other, as-yet-unknown factors. IMPORTANCE Type I IFN signaling in mammalian cells induces formation of the ISGF3 transcription factor complex, which binds to interferon stimulated response elements (ISREs) in the promoters of interferon-stimulated genes (ISGs) in the cell nucleus. Flavivirus proteins counteract type I IFN signaling by preventing either the formation or nuclear localization of ISGF3. A subset of ISRE-regulated ISGs was still induced in West Nile virus (WNV)-infected mouse embryo fibroblasts (MEFs), indicating that cells have an alternative mechanism for activating these ISGs. In this study, cellular components involved in this ISG upregulation mechanism were identified using gene knockout MEFs and ChIP, and critical promoter regions for gene activation were mapped using reporter assays. The data indicate a cooperative function between two ISREs and required binding of IRF3, IRF5, and/or IRF7 and an NF-κB component(s). Moreover, type I IFN signaling-independent ISG activation requires different additional promoter activation regions than type I IFN-dependent activation.
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Fibroblastos , Regulación de la Expresión Génica/inmunología , Interferón Tipo I/inmunología , Fiebre del Nilo Occidental/inmunología , Virus del Nilo Occidental/inmunología , Animales , Fibroblastos/inmunología , Fibroblastos/virología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Elementos de Respuesta/inmunologíaRESUMEN
RNase L is an ankyrin repeat domain-containing dual endoribonuclease-pseudokinase that is activated by unusual 2,'5'-oligoadenylate (2-5A) second messengers and which impedes viral infections in higher vertebrates. Despite its importance in interferon-regulated antiviral innate immunity, relatively little is known about its precise mechanism of action. Here we present a functional characterization of 2.5 Å and 3.25 Å X-ray crystal and small-angle X-ray scattering structures of RNase L bound to a natural 2-5A activator with and without ADP or the nonhydrolysable ATP mimetic AMP-PNP. These studies reveal how recognition of 2-5A through interactions with the ankyrin repeat domain and the pseudokinase domain, together with nucleotide binding, imposes a rigid intertwined dimer configuration that is essential for RNase catalytic and antiviral functions. The involvement of the pseudokinase domain of RNase L in 2-5A sensing, nucleotide binding, dimerization, and ribonuclease functions highlights the evolutionary adaptability of the eukaryotic protein kinase fold.
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Nucleótidos de Adenina/química , Endorribonucleasas/química , Oligorribonucleótidos/química , Adenosina Difosfato/química , Adenilil Imidodifosfato/química , Animales , Repetición de Anquirina , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Virus de la Encefalomiocarditis , Endorribonucleasas/genética , Endorribonucleasas/fisiología , Células HeLa , Humanos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Picornaviridae , Estructura Terciaria de Proteína , Dispersión de Radiación , Relación Estructura-Actividad , Sus scrofaRESUMEN
The family Arteriviridae comprises enveloped RNA viruses with a linear, positive-sense genome of approximately 12.7 to 15.7 kb. The spherical, pleomorphic virions have a median diameter of 50-74 nm and include eight to eleven viral proteins. Arteriviruses infect non-human mammals in a vector-independent manner. Infections are often persistent and can either be asymptomatic or produce overt disease. Some arteriviruses are important veterinary pathogens while others infect particular species of wild rodents or African non-human primates. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Arteriviridae, which is available at ictv.global/report/arteriviridae.
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Arteriviridae/clasificación , Arteriviridae/genética , Filogenia , Animales , Arteriviridae/ultraestructura , Arterivirus/clasificación , Arterivirus/genética , Endocitosis , Genoma Viral , Primates , Infecciones por Virus ARN , Proteínas Virales/genética , Virión/clasificación , Virión/genética , Virión/ultraestructura , Acoplamiento Viral , Replicación ViralRESUMEN
Mammalian ATP-binding cassette (ABC) subfamily F member 3 (ABCF3) is a class 2 ABC protein that has previously been identified as a partner of the mouse flavivirus resistance protein 2',5'-oligoadenylate synthetase 1B (OAS1B). The functions and natural substrates of ABCF3 are not known. In this study, analysis of purified ABCF3 showed that it is an active ATPase, and binding analyses with a fluorescent ATP analog suggested unequal contributions by the two nucleotide-binding domains. We further showed that ABCF3 activity is increased by lipids, including sphingosine, sphingomyelin, platelet-activating factor, and lysophosphatidylcholine. However, cholesterol inhibited ABCF3 activity, whereas alkyl ether lipids either inhibited or resulted in a biphasic response, suggesting small changes in lipid structure differentially affect ABCF3 activity. Point mutations in the two nucleotide-binding domains of ABCF3 affected sphingosine-stimulated ATPase activity differently, further supporting different roles for the two catalytic pockets. We propose a model in which pocket 1 is the site of basal catalysis, whereas pocket 2 engages in ligand-stimulated ATP hydrolysis. Co-localization of the ABCF3-OAS1B complex to the virus-remodeled endoplasmic reticulum membrane has been shown before. We also noted that co-expression of ABCF3 and OAS1B in bacteria alleviated growth inhibition caused by expression of OAS1B alone, and ABCF3 significantly enhanced OAS1B levels, indirectly showing interaction between these two proteins in bacterial cells. As viral RNA synthesis requires large amounts of ATP, we conclude that lipid-stimulated ATP hydrolysis may contribute to the reduction in viral RNA production characteristic of the flavivirus resistance phenotype.
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2',5'-Oligoadenilato Sintetasa/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Flavivirus/fisiología , Adenosina Trifosfato/metabolismo , Animales , Hidrólisis , Espacio Intracelular/metabolismo , Ligandos , Ratones , Unión ProteicaRESUMEN
Zika virus (ZIKV) infection attenuates the growth of human neural progenitor cells (hNPCs). As these hNPCs generate the cortical neurons during early brain development, the ZIKV-mediated growth retardation potentially contributes to the neurodevelopmental defects of the congenital Zika syndrome. Here, we investigate the mechanism by which ZIKV manipulates the cell cycle in hNPCs and the functional consequence of cell cycle perturbation on the replication of ZIKV and related flaviviruses. We demonstrate that ZIKV, but not dengue virus (DENV), induces DNA double-strand breaks (DSBs), triggering the DNA damage response through the ATM/Chk2 signaling pathway while suppressing the ATR/Chk1 signaling pathway. Furthermore, ZIKV infection impedes the progression of cells through S phase, thereby preventing the completion of host DNA replication. Recapitulation of the S-phase arrest state with inhibitors led to an increase in ZIKV replication, but not of West Nile virus or DENV. Our data identify ZIKV's ability to induce DSBs and suppress host DNA replication, which results in a cellular environment favorable for its replication.IMPORTANCE Clinically, Zika virus (ZIKV) infection can lead to developmental defects in the cortex of the fetal brain. How ZIKV triggers this event in developing neural cells is not well understood at a molecular level and likely requires many contributing factors. ZIKV efficiently infects human neural progenitor cells (hNPCs) and leads to growth arrest of these cells, which are critical for brain development. Here, we demonstrate that infection with ZIKV, but not dengue virus, disrupts the cell cycle of hNPCs by halting DNA replication during S phase and inducing DNA damage. We further show that ZIKV infection activates the ATM/Chk2 checkpoint but prevents the activation of another checkpoint, the ATR/Chk1 pathway. These results unravel an intriguing mechanism by which an RNA virus interrupts host DNA replication. Finally, by mimicking virus-induced S-phase arrest, we show that ZIKV manipulates the cell cycle to benefit viral replication.
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Daño del ADN , Células-Madre Neurales/metabolismo , Células-Madre Neurales/virología , Replicación Viral , Infección por el Virus Zika/genética , Infección por el Virus Zika/virología , Virus Zika/fisiología , Biomarcadores , Ciclo Celular , Línea Celular , Interacciones Huésped-Patógeno/genética , Humanos , Modelos BiológicosRESUMEN
Members of the order Nidovirales express their structural protein ORFs from a nested set of 3' subgenomic mRNAs (sg mRNAs), and for most of these ORFs, a single genomic transcription regulatory sequence (TRS) was identified. Nine TRSs were previously reported for the arterivirus Simian hemorrhagic fever virus (SHFV). In the present study, which was facilitated by next-generation sequencing, 96 SHFV body TRSs were identified that were functional in both infected MA104 cells and macaque macrophages. The abundance of sg mRNAs produced from individual TRSs was consistent over time in the two different cell types. Most of the TRSs are located in the genomic 3' region, but some are in the 5' ORF1a/1b region and provide alternative sources of nonstructural proteins. Multiple functional TRSs were identified for the majority of the SHFV 3' ORFs, and four previously identified TRSs were found not to be the predominant ones used. A third of the TRSs generated sg mRNAs with variant leader-body junction sequences. Sg mRNAs encoding E', GP2, or ORF5a as their 5' ORF as well as sg mRNAs encoding six previously unreported alternative frame ORFs or 14 previously unreported C-terminal ORFs of known proteins were also identified. Mutation of the start codon of two C-terminal ORFs in an infectious clone reduced virus yield. Mass spectrometry detected one previously unreported protein and suggested translation of some of the C-terminal ORFs. The results reveal the complexity of the transcriptional regulatory mechanism and expanded coding capacity for SHFV, which may also be characteristic of other nidoviruses.
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Nidovirales/genética , Nidovirales/patogenicidad , ARN Mensajero/genética , Secuencias Reguladoras de Ácido Ribonucleico , Animales , Northern Blotting , Chlorocebus aethiops , Codón Iniciador , Genoma Viral , Macaca , Mutación , Infecciones por Nidovirales/genética , Sistemas de Lectura Abierta , ARN Viral , Proteínas Virales/análisis , Proteínas Estructurales Virales/genéticaRESUMEN
Oxidative stress activates the cellular kinase HRI, which then phosphorylates eIF2α, resulting in stalled translation initiation and the formation of stress granules (SGs). SG assembly redirects cellular translation to stress response mRNAs and inhibits cap-dependent viral RNA translation. Flavivirus infections were previously reported to induce oxidative stress in infected cells but flavivirus-infected cells paradoxically develop resistance to arsenite (Ars)-induced SG formation with time after infection. This resistance was previously postulated to be due to sequestration of the SG protein Caprin1 by Japanese encephalitis virus capsid protein. However, Caprin1 did not co-localize with West Nile virus (WNV) capsid protein in infected cells. Other stressors induced SGs with equal efficiency in mock- and WNV-infected cells indicating the intrinsic ability of cells to assemble SGs was not disabled. Induction of both reactive oxygen species (ROS) and the antioxidant response was detected at early times after WNV-infection. The transcription factors, Nrf2 and ATF4, which activate antioxidant genes, were upregulated and translocated to the nucleus. Knockdown of Nrf2, ATF4 or apoptosis-inducing factor (AIF), a mitochondrial protein involved in regenerating intracellular reduced glutathione (GSH) levels, with siRNA or treatment of cells with buthionine sulphoximine, which induces oxidative stress by inhibiting GSH synthesis, decreased intracellular GSH levels and increased the number of SG-positive, infected cells. Mitochondria were protected from Ars-induced damage by WNV infection until late times in the infection cycle. The results indicate that the increase in virus-induced ROS levels is counterbalanced by a virus-induced antioxidant response that is sufficient to also overcome the increase in ROS induced by Ars treatment and prevent Ars-induced SG assembly and mitochondrial damage. The virus-induced alterations in the cellular redox status appear to provide benefits for the virus during its lifecycle.
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Antioxidantes/metabolismo , Glutatión/metabolismo , Estrés Oxidativo/fisiología , Virus del Nilo Occidental/patogenicidad , Animales , Arsenitos/metabolismo , Western Blotting , Línea Celular , Gránulos Citoplasmáticos/metabolismo , Humanos , Microscopía Fluorescente , ARN Interferente Pequeño , Especies Reactivas de Oxígeno/metabolismo , Transfección , Replicación Viral/fisiología , Fiebre del Nilo Occidental/metabolismo , Virus del Nilo Occidental/metabolismoRESUMEN
Interactions between nucleic acids and proteins are critical for many cellular processes, and their study is of utmost importance to many areas of biochemistry, cellular biology, and virology. Here, we introduce a new analytical method based on sedimentation velocity (SV) analytical ultracentrifugation, in combination with a novel multiwavelength detector to characterize such interactions. We identified the stoichiometry and molar mass of a complex formed during the interaction of a West Nile virus RNA stem loop structure with the human T cell-restricted intracellular antigen-1 related protein. SV has long been proven as a powerful technique for studying dynamic assembly processes under physiological conditions in solution. Here, we demonstrate, for the first time, how the new multiwavelength technology can be exploited to study protein-RNA interactions, and show how the spectral information derived from the new detector complements the traditional hydrodynamic information from analytical ultracentrifugation. Our method allows the protein and nucleic acid signals to be separated by spectral decomposition such that sedimentation information from each individual species, including any complexes, can be clearly identified based on their spectral signatures. The method presented here extends to any interacting system where the interaction partners are spectrally separable.
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Hidrodinámica , ARN Viral/análisis , Antígeno Intracelular 1 de las Células T/análisis , Ultracentrifugación , Virus del Nilo Occidental/química , HumanosRESUMEN
Simian hemorrhagic fever virus (SHFV) causes a fatal hemorrhagic fever in macaques but an asymptomatic, persistent infection in baboons. To investigate factors contributing to this differential infection outcome, the targets of SHFV infection, macrophages (MΦs) and myeloid dendritic cells (mDCs), were differentiated from macaque and baboon peripheral blood monocytes and used to compare viral replication and cell responses. SHFV replicated in >90% of macaque MΦs but in only â¼10% of baboon MΦs. Although SHFV infected â¼50% of macaque and baboon mDCs, virus replication was efficient in macaque but not in baboon mDCs. Both types of macaque cultures produced higher virus yields than baboon cultures. A more efficient type I interferon response and the production of proinflammatory cytokines, including interleukin-1ß (IL-1ß), IL-6, IL-12/23(p40), tumor necrosis factor alpha (TNF-α), and macrophage inflammatory protein 1α (MIP-1α), in response to SHFV infection were observed in macaque but not baboon cultures, suggesting less efficient counteraction of these responses by viral proteins in macaque cells. Baboon cultures produced higher levels of IL-10 than macaque cultures both prior to and after SHFV infection. In baboon but not macaque cell cultures, SHFV infection upregulated IL-10R1, a subunit of the IL-10 receptor (IL-10R), and also SOCS3, a negative regulator of proinflammatory cytokine production. Incubation of macaque cultures with human IL-10 before and/or after SHFV infection decreased production of IL-6, IL-1ß, and MIP-1α but not TNF-α, suggesting a role for IL-10 in suppressing SHFV-induced proinflammatory cytokine production in macaques.
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Infecciones por Arterivirus/inmunología , Arterivirus , Células Dendríticas/virología , Resistencia a la Enfermedad/inmunología , Macrófagos/virología , Células Mieloides/virología , Animales , Western Blotting , Citocinas/sangre , Cartilla de ADN/genética , Humanos , Macaca mulatta , Microscopía Confocal , Papio , Reacción en Cadena en Tiempo Real de la Polimerasa , Especificidad de la Especie , Replicación Viral/fisiologíaRESUMEN
UNLABELLED: The N-terminal region of simian hemorrhagic fever virus (SHFV) nonstructural polyprotein 1a is predicted to encode three papain-like proteases (PLP1α, PLP1ß, and PLP1γ). Catalytic residues and cleavage sites for each of the SHFV PLP1s were predicted by alignment of the SHFV PLP1 region sequences with each other as well as with those of other arteriviruses, and the predicted catalytic residues were shown to be proximal by homology modeling of the SHFV nsp1s on porcine respiratory and reproductive syndrome virus (PRRSV) nsp1 crystal structures. The functionality of the predicted catalytic Cys residues and cleavage sites was tested by analysis of the autoproteolytic products generated in in vitro transcription/translation reactions done with wild-type or mutant SHFV nsp1 constructs. Cleavage sites were also analyzed by mass spectroscopy analysis of selected immunoprecipitated cleavage products. The data showed that each of the three SHFV PLP1s is an active protease. Cys63 was identified as the catalytic Cys of SHFV PLP1α and is adjacent to an Ala instead of the canonical Tyr observed in other arterivirus PLP1s. SHFV PLP1γ is able to cleave at both downstream and upstream nsp1 junction sites. Although intermediate precursor polyproteins as well as alternative products generated by each of the SHFV PLP1s cleaving at sites within the N-terminal region of nsp1ß were produced in the in vitro reactions, Western blotting of SHFV-infected, MA104 cell lysates with SHFV nsp1 protein-specific antibodies detected only the three mature nsp1 proteins. IMPORTANCE: SHFV is unique among arteriviruses in having three N-terminal papain-like protease 1 (PLP1) domains. Other arteriviruses encode one or two active PLP1s. This is the first functional study of the SHFV PLP1s. Analysis of the products of in vitro autoprocessing of an N-terminal SHFV nonstructural 1a polypeptide fragment showed that each of the three SHFV PLP1s is active, and the predicted catalytic Cys residues and cleavage sites for each PLP1 were confirmed by testing mutant constructs. Several unique features of the SHFV PLP1s were discovered. The SHFV PLP1α catalytic Cys63 is unique among arterivirus PLP1s in being adjacent to an Ala instead of a Trp. Other arterivirus PLP1s cleave only in cis at a single downstream site, but SHFV PLP1γ can cleave at both the downstream nsp1γ-nsp2 and upstream nsp1ß-nsp1γ junctions. The three mature nsp1 proteins were produced both in the in vitro reactions and in infected cells.
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Arterivirus/genética , Papaína/genética , Papaína/metabolismo , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Secuencia de Aminoácidos , Animales , Arterivirus/metabolismo , Infecciones por Arterivirus/virología , Catálisis , Línea Celular , Datos de Secuencia Molecular , Poliproteínas/genética , Poliproteínas/metabolismo , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Virus del Síndrome Respiratorio y Reproductivo Porcino/metabolismo , Alineación de Secuencia , Porcinos/virologíaRESUMEN
UNLABELLED: The molecular mechanism of the hepatic tropism of hepatitis C virus (HCV) remains incompletely defined. In vitro hepatic differentiation of pluripotent stem cells produces hepatocyte-like cells (HLCs) permissive for HCV infection, providing an opportunity for studying liver development and host determinants of HCV susceptibility. We previously identified the transition stage of HCV permissiveness and now investigate whether a host protein whose expression is induced during this transition stage is important for HCV infection. We suppressed the expression of a liver-specific protein, cell death-inducing DFFA-like effector b (CIDEB), and performed hepatocyte function and HCV infection assays. We also used a variety of cell-based assays to dissect the specific step of the HCV life cycle that potentially requires CIDEB function. We found CIDEB to be an essential cofactor for HCV entry into hepatocytes. Genetic interference with CIDEB in stem cells followed by hepatic differentiation leads to HLCs that are refractory to HCV infection, and infection time course experiments revealed that CIDEB functions in a late step of HCV entry, possibly to facilitate membrane fusion. The role of CIDEB in mediating HCV entry is distinct from those of the well-established receptors, as it is not required for HCV pseudoparticle entry. Finally, HCV infection effectively downregulates CIDEB protein through a posttranscriptional mechanism. IMPORTANCE: This study identifies a hepatitis C virus (HCV) entry cofactor that is required for HCV infection of hepatocytes and potentially facilitates membrane fusion between viral and host membranes. CIDEB and its interaction with HCV may open up new avenues of investigation of lipid droplets and viral entry.
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Proteínas Reguladoras de la Apoptosis/metabolismo , Hepacivirus/fisiología , Hepatocitos/virología , Interacciones Huésped-Patógeno , Internalización del Virus , Proteínas Reguladoras de la Apoptosis/genética , Línea Celular , Técnicas de Silenciamiento del Gen , HumanosRESUMEN
The 3'-terminal nucleotides (nt) of West Nile virus (WNV) genomic RNA form a penultimate 16-nt small stem-loop (SSL) and an 80-nt terminal stem-loop (SL). These RNA structures are conserved in divergent flavivirus genomes. A previous in vitro study using truncated WNV 3' RNA structures predicted a putative tertiary interaction between the 5' side of the 3'-terminal SL and the loop of the SSL. Although substitution or deletion of the 3' G (nt 87) within the SSL loop, which forms the only G-C pair in the predicted tertiary interaction, in a WNV infectious clone was lethal, a finding consistent with the involvement in a functionally relevant pseudoknot interaction, extensive mutagenesis of nucleotides in the terminal SL did not identify a cis-acting pairing partner for this SSL 3' G. However, both the sequence and the structural context of two adjacent base pairs flanked by symmetrical internal loops in the 3'-terminal SL were shown to be required for efficient viral RNA replication. Nuclear magnetic resonance analysis confirmed the predicted SSL and SL structures but not the tertiary interaction. The SSL was previously reported to contain one of three eEF1A binding sites, and G87 in the SSL loop was shown to be involved in eEF1A binding. The nucleotides at the bottom part of the 3'-terminal SL switch between 3' RNA-RNA and 3'-5' RNA-RNA interactions. The data suggest that interaction of the 3' SL RNA with eEF1A at three sites and a unique metastable structural feature may participate in regulating structural changes in the 3'-terminal SL.
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Nucleótidos/genética , ARN Viral/genética , Replicación Viral/genética , Virus del Nilo Occidental/genética , Animales , Emparejamiento Base , Línea Celular , Cricetinae , Cartilla de ADN/genética , Ensayo de Cambio de Movilidad Electroforética , Inmunoprecipitación , Secuencias Invertidas Repetidas/genética , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Mutagénesis , Transfección , Replicación Viral/fisiología , Virus del Nilo Occidental/fisiologíaRESUMEN
Potential G-quadruplex sites have been identified in the genomes of DNA and RNA viruses and proposed as regulatory elements. The genus Orthoflavivirus contains arthropod-transmitted, positive-sense, single-stranded RNA viruses that cause significant human disease globally. Computational studies have identified multiple potential G-quadruplex sites that are conserved across members of this genus. Subsequent biophysical studies established that some G-quadruplexes predicted in Zika and tickborne encephalitis virus genomes can form and known quadruplex binders reduced viral yields from cells infected with these viruses. The susceptibility of RNA to degradation and the variability of loop regions have made structure determination challenging. Despite these difficulties, we report a high-resolution structure of the NS5-B quadruplex from the West Nile virus genome. Analysis reveals two stacked tetrads that are further stabilized by a stacked triad and transient noncanonical base pairing. This structure expands the landscape of solved RNA quadruplex structures and demonstrates the diversity and complexity of biological quadruplexes. We anticipate that the availability of this structure will assist in solving further viral RNA quadruplexes and provides a model for a conserved antiviral target in Orthoflavivirus genomes.
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G-Cuádruplex , Genoma Viral , ARN Viral , Virus del Nilo Occidental , ARN Viral/genética , ARN Viral/química , Virus del Nilo Occidental/genética , Conformación de Ácido Nucleico , Modelos Moleculares , Humanos , Emparejamiento BaseRESUMEN
Dengue is a major global health threat, and there are no approved antiviral agents. Prior research using Cas13 only demonstrated dengue mitigation in vitro. Here we demonstrate that systemic delivery of mRNA-encoded Cas13a and guide RNAs formulated in lipid nanoparticles can be used to treat dengue virus (DENV) 2 and 3 in mice. First, we identified guides against DENV 2 and 3 that demonstrated in vitro efficacy. Next, we confirmed that Cas13 enzymatic activity is necessary for DENV 2 or DENV 3 mitigation in vitro. Last, we show that a single dose of lipid-nanoparticle-formulated mRNA-encoded Cas13a and guide RNA, administered 1 day post-infection, promotes survival of all infected animals and serum viral titre decreases on days 2 and 3 post-infection after lethal challenge in mice. Off-target analysis in mice using RNA sequencing showed no collateral cleavage. Overall, these data demonstrate the potential of mRNA-encoded Cas13 as a pan-DENV drug.
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Antivirales , Virus del Dengue , Dengue , Modelos Animales de Enfermedad , Nanopartículas , ARN Mensajero , Animales , Dengue/tratamiento farmacológico , Ratones , Virus del Dengue/genética , Virus del Dengue/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Nanopartículas/química , Antivirales/farmacología , Antivirales/administración & dosificación , ARN Guía de Sistemas CRISPR-Cas/genética , Humanos , Proteínas Asociadas a CRISPR/metabolismo , Proteínas Asociadas a CRISPR/genética , Lípidos/química , Carga Viral/efectos de los fármacos , Femenino , LiposomasRESUMEN
During the 2015-2016 Zika virus (ZIKV) epidemic in the Americas, serological cross-reactivity with other flaviviruses and relatively high costs of nucleic acid testing in the region hindered the capacity for widespread diagnostic testing. In such cases where individual testing is not feasible, wastewater monitoring approaches may offer a means of community-level public health surveillance. To inform such approaches, we characterized the persistence and recovery of ZIKV RNA in experiments where we spiked cultured ZIKV into surface water, wastewater, and a combination of both to examine the potential for detection in open sewers serving communities most affected by the ZIKV outbreak, such as those in Salvador, Bahia, Brazil. We used reverse transcription droplet digital PCR to quantify ZIKV RNA. In our persistence experiments, we found that the persistence of ZIKV RNA decreased with increasing temperature, significantly decreased in surface water versus wastewater, and significantly decreased when the initial concentration of virus was lowered by one order of magnitude. In our recovery experiments, we found higher percent recovery of ZIKV RNA in pellets versus supernatants from the same sample, higher recoveries in pellets using skimmed milk flocculation, lower recoveries of ZIKV RNA in surface water versus wastewater, and lower recoveries from a freeze thaw. We also analyzed samples collected from Salvador, Brazil during the ZIKV outbreak (2015-2016) that consisted of archived samples obtained from open sewers or environmental waters thought to be contaminated by sewage. Although we did not detect any ZIKV RNA in the archived Brazil samples, results from these persistence and recovery experiments serve to inform future wastewater monitoring efforts in open sewers, an understudied and important application of wastewater monitoring.
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Infección por el Virus Zika , Virus Zika , Humanos , Virus Zika/genética , Infección por el Virus Zika/epidemiología , Infección por el Virus Zika/diagnóstico , Aguas Residuales , Brotes de Enfermedades , Brasil/epidemiología , ARNRESUMEN
Physiological blood-tissue barriers play a critical role in separating the circulation from immune-privileged sites and denying access to blood-borne viruses. The mechanism of virus restriction by these barriers is poorly understood. We utilize induced pluripotent stem cell (iPSC)-derived human brain microvascular endothelial cells (iBMECs) to study virus-blood-brain barrier (BBB) interactions. These iPSC-derived cells faithfully recapitulate a striking difference in in vivo neuroinvasion by two alphavirus isolates and are selectively permissive to neurotropic flaviviruses. A model of cocultured iBMECs and astrocytes exhibits high transendothelial electrical resistance and blocks non-neurotropic flaviviruses from getting across the barrier. We find that iBMECs constitutively express an interferon-induced gene, IFITM1, which preferentially restricts the replication of non-neurotropic flaviviruses. Barrier cells from blood-testis and blood-retinal barriers also constitutively express IFITMs that contribute to the viral resistance. Our application of a renewable human iPSC-based model for studying virus-BBB interactions reveals that intrinsic immunity at the barriers contributes to virus exclusion.
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Barrera Hematoencefálica , Células Madre Pluripotentes Inducidas , Antivirales , Encéfalo/fisiología , Células Endoteliales/fisiología , Humanos , Células Madre Pluripotentes Inducidas/fisiología , MasculinoRESUMEN
West Nile virus (WNV) infection leads to rapid and sustained Ca(2+) influx. This influx was observed with different strains of WNV and in different types of cells. Entry during virion endocytosis as well as through calcium channels contributed to the Ca(2+) influx observed in WNV-infected cells. Ca(2+) influx was not detected after infection with vesicular stomatitis virus (VSV) and occurred only through endocytosis in Sindbis virus-infected cells. Caspase 3 cleavage and activation of several kinases, including focal adhesion kinase (FAK), mitogen-activated extracellular signal-regulated protein kinase (ERK1/2), and protein-serine kinase B alpha (Akt), at early times after WNV infection were shown to be dependent on Ca(2+) influx. Although the activation of these kinases was sustained in virus-infected cells throughout infection, UV-inactivated WNV induced only a transient activation of FAK and ERK1/2 at early times after infection. The Ca(2+)-dependent FAK activation observed in WNV-infected cells was not mediated by alphavbeta3 integrins. Reduction of Ca(2+) influx at early times of infection by various treatments decreased the viral yield and delayed both the early transient caspase 3 cleavage and the activation of FAK, Akt, and ERK signaling. The results indicate that Ca(2+) influx is required for early infection events needed for efficient viral replication, possibly for virus-induced rearrangement of the endoplasmic reticulum (ER) membrane. Increased caspase 3 cleavage at both early (transient) and late times of infection correlated with decreased activation of the FAK and ERK1/2 pathways, indicating a role for these kinases in extending the survival of flavivirus-infected cells.
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Calcio/metabolismo , Fiebre del Nilo Occidental/metabolismo , Fiebre del Nilo Occidental/fisiopatología , Virus del Nilo Occidental/fisiología , Animales , Transporte Biológico , Caspasa 3/metabolismo , Línea Celular , Supervivencia Celular , Cricetinae , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Sistema de Señalización de MAP Quinasas , Ratones , Fiebre del Nilo Occidental/enzimología , Fiebre del Nilo Occidental/virología , Virus del Nilo Occidental/genéticaRESUMEN
The 2'-5'-oligoadenylate synthetase (2'-5'-OAS) family members are interferon-induced antiviral proteins. Twenty-three single nucleotide polymorphisms located within the OAS1, OAS2, OAS3, and OASL genes were analyzed in 142 patients with Russian tick-borne encephalitis. Statistically significant differences in genotype, allele, and haplotype frequencies for 3 OAS2 single nucleotide polymorphisms (rs1293762, rs15895, and rs1732778) and 2 OAS3 single nucleotide polymorphisms (rs2285932 and rs2072136) were detected between patients with central nervous system disease and both those with fever and/or meningitis and the control group. The data suggest a possible association between these 5 OAS single nucleotide polymorphisms and the outcome of tick-borne encephalitis virus infection in a Russian population.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/genética , Virus de la Encefalitis Transmitidos por Garrapatas/inmunología , Encefalitis Transmitida por Garrapatas/genética , Predisposición Genética a la Enfermedad , Familia de Multigenes , Frecuencia de los Genes , Humanos , Polimorfismo de Nucleótido Simple , Federación de RusiaRESUMEN
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection can cause neurological disease in humans, but little is known about the pathogenesis of SARS-CoV-2 infection in the central nervous system (CNS). Herein, using K18-hACE2 mice, we demonstrate that SARS-CoV-2 neuroinvasion and encephalitis is associated with mortality in these mice. Intranasal infection of K18-hACE2 mice with 105 plaque-forming units of SARS-CoV-2 resulted in 100% mortality by day 6 after infection. The highest virus titers in the lungs were observed on day 3 and declined on days 5 and 6 after infection. By contrast, very high levels of infectious virus were uniformly detected in the brains of all the animals on days 5 and 6. Onset of severe disease in infected mice correlated with peak viral levels in the brain. SARS-CoV-2-infected mice exhibited encephalitis hallmarks characterized by production of cytokines and chemokines, leukocyte infiltration, hemorrhage and neuronal cell death. SARS-CoV-2 was also found to productively infect cells within the nasal turbinate, eye and olfactory bulb, suggesting SARS-CoV-2 entry into the brain by this route after intranasal infection. Our data indicate that direct infection of CNS cells together with the induced inflammatory response in the brain resulted in the severe disease observed in SARS-CoV-2-infected K18-hACE2 mice.