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1.
RNA ; 28(2): 177-193, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-34759006

RESUMEN

The commitment to replicate the RNA genome of flaviviruses without a primer involves RNA-protein interactions that have been shown to include the recognition of the stem-loop A (SLA) in the 5' untranslated region (UTR) by the nonstructural protein NS5. We show that DENV2 NS5 arginine 888, located within the carboxy-terminal 18 residues, is completely conserved in all flaviviruses and interacts specifically with the top-loop of 3'SL in the 3'UTR which contains the pentanucleotide 5'-CACAG-3' previously shown to be critical for flavivirus RNA replication. We present virological and biochemical data showing the importance of this Arg 888 in virus viability and de novo initiation of RNA polymerase activity in vitro. Based on our binding studies, we hypothesize that ternary complex formation of NS5 with 3'SL, followed by dimerization, leads to the formation of the de novo initiation complex that could be regulated by the reversible zipping and unzipping of cis-acting RNA elements.


Asunto(s)
Virus del Dengue/fisiología , ARN/genética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral , Regiones no Traducidas 3' , Animales , Arginina/química , Línea Celular , Secuencia Conservada , Cricetinae , Cricetulus , ARN Polimerasas Dirigidas por ADN/metabolismo , Virus del Dengue/genética , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética
2.
Proc Natl Acad Sci U S A ; 117(20): 11038-11047, 2020 05 19.
Artículo en Inglés | MEDLINE | ID: mdl-32366663

RESUMEN

Dengue virus (DENV) is a global health threat, causing repeated epidemics throughout the tropical world. While low herd immunity levels to any one of the four antigenic types of DENV predispose populations to outbreaks, viral genetic determinants that confer greater fitness for epidemic spread is an important but poorly understood contributor of dengue outbreaks. Here we report that positive epistasis between the coding and noncoding regions of the viral genome combined to elicit an epidemiologic fitness phenotype associated with the 1994 DENV2 outbreak in Puerto Rico. We found that five amino acid substitutions in the NS5 protein reduced viral genomic RNA (gRNA) replication rate to achieve a more favorable and relatively more abundant subgenomic flavivirus RNA (sfRNA), a byproduct of host 5'-3' exoribonuclease activity. The resulting increase in sfRNA relative to gRNA levels not only inhibited type I interferon (IFN) expression in infected cells through a previously described mechanism, but also enabled sfRNA to compete with gRNA for packaging into infectious particles. We suggest that delivery of sfRNA to new susceptible cells to inhibit type I IFN induction before gRNA replication and without the need for further de novo sfRNA synthesis could form a "preemptive strike" strategy against DENV.


Asunto(s)
Regiones no Traducidas 3'/genética , Virus del Dengue/genética , Dengue/virología , Proteínas no Estructurales Virales/genética , Células A549 , Dengue/epidemiología , Epistasis Genética , Exorribonucleasas , Técnicas de Inactivación de Genes , Genoma Viral , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Interferón Tipo I/metabolismo , Proteínas Asociadas a Microtúbulos , Mutación , Puerto Rico/epidemiología , ARN Guía de Kinetoplastida/metabolismo , Replicación Viral
3.
Eur J Nucl Med Mol Imaging ; 49(13): 4516-4528, 2022 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-35876869

RESUMEN

PURPOSE: Zika (ZIKV) is a viral inflammatory disease affecting adults, children, and developing fetuses. It is endemic to tropical and sub-tropical countries, resulting in half the global population at risk of infection. Despite this, there are no approved therapies or vaccines against ZIKV disease. Non-invasive imaging biomarkers are potentially valuable tools for studying viral pathogenesis, prognosticating host response to disease, and evaluating in vivo efficacy of experimental therapeutic interventions. In this study, we evaluated [18F]fluorodeoxyglucose ([18F]FDG)-positron emission tomography (PET) as an imaging biomarker of ZIKV disease in a mouse model and correlated metabolic tracer tissue uptake with real-time biochemical, virological, and inflammatory features of tissue infection. METHODS: [18F]FDG-PET/CT imaging was performed in an acute, lethal ZIKV mouse infection model, at increasing stages of disease severity. [18F]FDG-PET findings were corroborated with ex vivo wholemount-tissue autoradiography and tracer biodistribution studies. Tracer uptake was also correlated with in situ tissue disease status, including viral burden and inflammatory response. Immune profiling of the spleen by flow cytometry was performed to identify the immune cell subsets driving tissue pathology and enhancing tracer uptake in ZIKV disease. RESULTS: Foci of increased [18F]FDG uptake were consistently detected in lymphoid tissues-particularly the spleen-of ZIKV-infected animals. Splenic uptake increased with disease severity, and corroborated findings in tissue pathology. Increased splenic uptake also correlated with increased viral replication and elevated expression of pro-inflammatory cytokines within these tissues. ZIKV-infected spleens were characterized by increased infiltration of myeloid cells, as well as increased proliferation of both myeloid and lymphoid cells. The increased cell proliferation correlated with increased tracer uptake in the spleen. Our findings support the use of [18F]FDG as an imaging biomarker to detect and track ZIKV disease in real time and highlight the dependency of affected tissue on the nature of the viral infection. CONCLUSION: [18F]FDG uptake in the spleen is a useful surrogate for interrogating in situ tissue viral burden and inflammation status in this ZIKV murine model.


Asunto(s)
Infección por el Virus Zika , Virus Zika , Animales , Ratones , Infección por el Virus Zika/diagnóstico por imagen , Infección por el Virus Zika/metabolismo , Infección por el Virus Zika/patología , Virus Zika/metabolismo , Fluorodesoxiglucosa F18/metabolismo , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , Distribución Tisular , Tomografía Computarizada por Rayos X , Tomografía de Emisión de Positrones , Tejido Linfoide/metabolismo , Tejido Linfoide/patología , Inflamación/diagnóstico por imagen , Inflamación/metabolismo , Modelos Animales de Enfermedad , Biomarcadores/metabolismo , Citocinas
4.
J Virol ; 94(21)2020 10 14.
Artículo en Inglés | MEDLINE | ID: mdl-32796069

RESUMEN

Zika virus (ZIKV) remains a potentially significant public health concern because it can cause teratogenic effects, such as microcephaly in newborns and neurological disease, like Guillain-Barré syndrome. Together with efforts to develop a vaccine, the discovery of antiviral molecules is important to control ZIKV infections and to prevent its most severe symptoms. Here, we report the development of small nonnucleoside inhibitors (NNIs) of ZIKV RNA-dependent RNA polymerase (RdRp) activity. These NNIs target an allosteric pocket (N pocket) located next to a putative hinge region between the thumb and the palm subdomains that was originally described for dengue virus (DENV) RdRp. We first tested the activity of DENV RdRp N-pocket inhibitors against ZIKV RdRp, introduced chemical modifications into these molecules, and assessed their potency using both enzymatic and cell-based assays. The most potent compound had a 50% inhibitory concentration value of 7.3 µM and inhibited ZIKV replication in a cell-based assay with a 50% effective concentration value of 24.3 µM. Importantly, we report four high-resolution crystal structures detailing how these NNIs insert into the N pocket of ZIKV RdRp. Our observations point to subtle differences in the size, shape, chemical environment, and hydration of the N pocket from ZIKV RdRp from those of the N pocket from DENV RdRp that are crucial for the design of improved antiviral inhibitors with activity against ZIKV.IMPORTANCE Zika virus belongs to the Flavivirus genus, which comprises several important human pathogens. There is currently neither an approved vaccine nor antiviral drugs available to prevent infection by ZIKV. The nonstructural protein 5 (NS5) polymerase, which is responsible for replicating the viral RNA genome, represents one of the most promising targets for antiviral drug development. Starting from compounds recently developed against dengue virus NS5, we designed and synthesized inhibitors targeting Zika virus NS5. We show that these novel compounds inhibit viral replication by targeting the polymerase activity. High-resolution X-ray crystallographic structures of protein-inhibitor complexes demonstrated specific binding to an allosteric site within the polymerase, called the N pocket. This work paves the way for the future structure-based design of potent compounds specifically targeting ZIKV RNA polymerase activity.


Asunto(s)
Antivirales/síntesis química , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Sulfonas/síntesis química , Tiofenos/síntesis química , Proteínas Virales/antagonistas & inhibidores , Regulación Alostérica , Sitio Alostérico/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Antivirales/farmacología , Sitios de Unión , Línea Celular Tumoral , Cricetulus , Diseño de Fármacos , Expresión Génica , Hepatocitos , Humanos , Cinética , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato , Sulfonas/farmacología , Tiofenos/farmacología , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral/efectos de los fármacos , Virus Zika/efectos de los fármacos , Virus Zika/enzimología , Virus Zika/genética , Virus Zika/aislamiento & purificación , Infección por el Virus Zika/virología
5.
J Gen Virol ; 101(9): 941-953, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32589122

RESUMEN

The dengue virus (DENV) replication complex is made up of its non-structural (NS) proteins and yet-to-be identified host proteins, but the molecular interactions between these proteins are not fully elucidated. In this work, we sought to uncover the interactions between DENV NS1 and its fellow NS proteins using a yeast two-hybrid (Y2H) approach, and found that domain II of NS1 binds to an N-terminal cytoplasmic fragment of NS4A. Mutations in amino acid residues 41 and 43 in this cytoplasmic region of NS4A disrupted the interaction between NS1 and the NS4A-2K-4B precursor protein. When the NS4A Y41F mutation was introduced into the context of the virus via a DENV2 infectious clone, this mutant virus exhibited impaired viral fitness and decreased infectious virus production. The NS4A Y41F mutant virus triggered a significantly muted transcriptional activation of interferon-stimulated genes compared to wild-type virus that is independent of NS4A's ability to antagonize type I interferon signalling. Taken together, we have identified a link between DENV NS1 and the cytoplasmic domain in NS4A that is important for its cellular and viral functions.


Asunto(s)
Virus del Dengue/genética , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Virus del Dengue/fisiología , Aptitud Genética , Humanos , Interferón Tipo I/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Técnicas del Sistema de Dos Híbridos , Proteínas no Estructurales Virales/química , Virión/metabolismo , Replicación Viral
6.
Int J Mol Sci ; 21(23)2020 Dec 04.
Artículo en Inglés | MEDLINE | ID: mdl-33291644

RESUMEN

Current methods to detect and monitor pathogens in biological systems are largely limited by the tradeoffs between spatial context and temporal detail. A new generation of molecular tracking that provides both information simultaneously involves in situ detection coupled with non-invasive imaging. An example is antisense imaging that uses antisense oligonucleotide probes complementary to a target nucleotide sequence. In this study, we explored the potential of repurposing antisense oligonucleotides initially developed as antiviral therapeutics as molecular probes for imaging of viral infections in vitro and in vivo. We employed nuclease-resistant phosphorodiamidate synthetic oligonucleotides conjugated with cell-penetrating peptides (i.e., PPMOs) previously established as antivirals for dengue virus serotype-2 (DENV2). As proof of concept, and before further development for preclinical testing, we evaluated its validity as in situ molecular imaging probe for tracking cellular DENV2 infection using live-cell fluorescence imaging. Although the PPMO was designed to specifically target the DENV2 genome, it was unsuitable as in situ molecular imaging probe. This study details our evaluation of the PPMOs to assess specific and sensitive molecular imaging of DENV2 infection and tells a cautionary tale for those exploring antisense oligonucleotides as probes for non-invasive imaging and monitoring of pathogen infections in experimental animal models.


Asunto(s)
Virus del Dengue/efectos de los fármacos , Virus del Dengue/fisiología , Hibridación in Situ , Imagen Molecular , Morfolinos/química , Péptidos/química , Replicación Viral/efectos de los fármacos , Animales , Chlorocebus aethiops , Humanos , Ratones , Oligonucleótidos Antisentido , Células Vero
7.
J Infect Dis ; 219(2): 223-233, 2019 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-30085051

RESUMEN

Preexisting immunity to Zika virus (ZIKV) or dengue virus (DENV) may alter the course of their infection, and here we use robust mouse models to examine pathological outcomes following passive immunization, sequential cross-infection, or vaccination with inactivated virus. DENV infection was enhanced (through antibody-dependent enhancement [ADE]) or was suppressed by both DENV and ZIKV immunity. Notably, inactivated ZIKV vaccination enhanced dengue disease severity, although it was highly protective against ZIKV infection. On the other hand, ADE was not observed upon ZIKV infection in mice that were passively immunized or preinfected with DENV. Surprisingly, however, we found that vaccination with inactivated DENV enhanced ZIKV infection, mainly in the mesenteric lymph node, indicating the potential for DENV immunity to cause ADE in vivo. Collectively, our data call for greater attention to detail in the design of ZIKV or DENV vaccines.


Asunto(s)
Reacciones Cruzadas/inmunología , Virus del Dengue/inmunología , Virus del Dengue/patogenicidad , Dengue/inmunología , Infección por el Virus Zika/inmunología , Virus Zika/inmunología , Virus Zika/patogenicidad , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Acrecentamiento Dependiente de Anticuerpo/inmunología , Línea Celular , Dengue/sangre , Dengue/patología , Vacunas contra el Dengue , Virus del Dengue/genética , Modelos Animales de Enfermedad , Genoma Viral , Humanos , Inmunidad , Inmunización , Ganglios Linfáticos , Ratones , Células THP-1 , Vacunación , Inactivación de Virus , Virus Zika/genética , Infección por el Virus Zika/sangre , Infección por el Virus Zika/patología
8.
Artículo en Inglés | MEDLINE | ID: mdl-30885901

RESUMEN

Globally, hepatitis E virus (HEV) causes significant morbidity and mortality each year. Despite this burden, there are no specific antivirals available to treat HEV patients, and the only licensed vaccine is not available outside China. Ribavirin and alpha interferon are used to treat chronic HEV infections; however, severe side effects and treatment failure are commonly reported. Therefore, this study aimed to identify potential antivirals for further development to combat HEV infection. We selected 16 compounds from the nucleoside and nonnucleoside antiviral classes that range in developmental status from late preclinical to FDA approved and evaluated them as potential antivirals for HEV infection, using genotype 1 replicon luminescence studies and replicon RNA quantification. Two potent inhibitors of HEV replication included NITD008 (half-maximal effective concentration [EC50], 0.03 µM; half-maximal cytotoxic concentration [CC50], >100 µM) and GPC-N114 (EC50, 1.07 µM, CC50, >100 µM), and both drugs reduced replicon RNA levels in cell culture (>50% reduction with either 10 µM GPC-N114 or 2.50 µM NITD008). Furthermore, GPC-N114 and NITD008 were synergistic in combinational treatment (combination index, 0.4) against HEV replication, allowing for dose reduction indices of 20.42 and 8.82 at 50% inhibition, respectively. Sofosbuvir has previously exhibited mixed results against HEV as an antiviral, both in vitro and in a few clinical applications; however, in this study it was effective against the HEV genotype 1 replicon (EC50, 1.97 µM; CC50, >100 µM) and reduced replicon RNA levels (47.2% reduction at 10 µM). Together these studies indicate drug repurposing may be a promising pathway for development of antivirals against HEV infection.


Asunto(s)
Adenosina/análogos & derivados , Antivirales/farmacología , Virus de la Hepatitis E/efectos de los fármacos , Hepatitis E/tratamiento farmacológico , Nitrilos/farmacología , Nitrocompuestos/farmacología , Adenosina/farmacología , Antivirales/uso terapéutico , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Genes Reporteros , Hepatitis E/virología , Humanos , ARN Viral/análisis , Replicón/efectos de los fármacos
9.
J Virol ; 92(7)2018 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-29321322

RESUMEN

A primary question in dengue virus (DENV) biology is the molecular strategy for recruitment of host cell protein synthesis machinery. Here, we combined cell fractionation, ribosome profiling, and transcriptome sequencing (RNA-seq) to investigate the subcellular organization of viral genome translation and replication as well as host cell translation and its response to DENV infection. We report that throughout the viral life cycle, DENV plus- and minus-strand RNAs were highly partitioned to the endoplasmic reticulum (ER), identifying the ER as the primary site of DENV translation. DENV infection was accompanied by an ER compartment-specific remodeling of translation, where ER translation capacity was subverted from host transcripts to DENV plus-strand RNA, particularly at late stages of infection. Remarkably, translation levels and patterns in the cytosol compartment were only modestly affected throughout the experimental time course of infection. Comparisons of ribosome footprinting densities of the DENV plus-strand RNA and host mRNAs indicated that DENV plus-strand RNA was only sparsely loaded with ribosomes. Combined, these observations suggest a mechanism where ER-localized translation and translational control mechanisms, likely cis encoded, are used to repurpose the ER for DENV virion production. Consistent with this view, we found ER-linked cellular stress response pathways commonly associated with viral infection, namely, the interferon response and unfolded protein response, to be only modestly activated during DENV infection. These data support a model where DENV reprograms the ER protein synthesis and processing environment to promote viral survival and replication while minimizing the activation of antiviral and proteostatic stress response pathways.IMPORTANCE DENV, a prominent human health threat with no broadly effective or specific treatment, depends on host cell translation machinery for viral replication, immune evasion, and virion biogenesis. The molecular mechanism by which DENV commandeers the host cell protein synthesis machinery and the subcellular organization of DENV replication and viral protein synthesis is poorly understood. Here, we report that DENV has an almost exclusively ER-localized life cycle, with viral replication and translation largely restricted to the ER. Surprisingly, DENV infection largely affects only ER-associated translation, with relatively modest effects on host cell translation in the cytosol. DENV RNA translation is very inefficient, likely representing a strategy to minimize disruption of ER proteostasis. Overall these findings demonstrate that DENV has evolved an ER-compartmentalized life cycle; thus, targeting the molecular signatures and regulation of the DENV-ER interaction landscape may reveal strategies for therapeutic intervention.


Asunto(s)
Virus del Dengue/fisiología , Dengue/inmunología , Retículo Endoplásmico/inmunología , Evasión Inmune , Biosíntesis de Proteínas/inmunología , ARN Mensajero/inmunología , ARN Viral/inmunología , Replicación Viral/inmunología , Línea Celular Tumoral , Dengue/patología , Retículo Endoplásmico/patología , Retículo Endoplásmico/virología , Humanos , Interferones/inmunología , Respuesta de Proteína Desplegada/inmunología
10.
Nucleic Acids Res ; 45(22): 12904-12920, 2017 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-29165589

RESUMEN

The protein-RNA interactions within the flavivirus replication complex (RC) are not fully understood. Our structure of dengue virus NS3 adenosine triphosphatase (ATPase)/helicase bound to the conserved 5' genomic RNA 5'-AGUUGUUAGUCU-3' reveals that D290 and R538 make specific interactions with G2 and G5 bases respectively. We show that single-stranded 12-mer RNA stimulates ATPase activity of NS3, however the presence of G2 and G5 leads to significantly higher activation. D290 is adjacent to the DEXH motif found in SF2 helicases like NS3 and interacts with R387, forming a molecular switch that activates the ATPase site upon RNA binding. Our structure guided mutagenesis revealed that disruption of D290-R387 interaction increases basal ATPase activity presumably as a result of higher conformational flexibility of the ATPase active site. Mutational studies also showed R538 plays a critical role in RNA interactions affecting translocation of viral RNA through dynamic interactions with bases at positions 4 and 5 of the ssRNA. Restriction of backbone flexibility around R538 through mutation of G540 to proline abolishes virus replication, indicating conformational flexibility around residue R538 is necessary for RNA translocation. The functionally critical sequence-specific contacts in NS3 RNA binding groove in subdomain III reveals potentially novel allosteric anti-viral drug targets.


Asunto(s)
Virus del Dengue/metabolismo , ARN Viral/metabolismo , Proteínas no Estructurales Virales/metabolismo , Replicación Viral , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Línea Celular , Virus del Dengue/genética , Cinética , Modelos Moleculares , Mutación , Conformación de Ácido Nucleico , Unión Proteica , Dominios Proteicos , ARN Helicasas/química , ARN Helicasas/genética , ARN Helicasas/metabolismo , ARN Viral/química , ARN Viral/genética , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Termodinámica , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética
11.
PLoS Pathog ; 12(9): e1005886, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27622521

RESUMEN

Dengue virus NS5 is the most highly conserved amongst the viral non-structural proteins and is responsible for capping, methylation and replication of the flavivirus RNA genome. Interactions of NS5 with host proteins also modulate host immune responses. Although replication occurs in the cytoplasm, an unusual characteristic of DENV2 NS5 is that it localizes to the nucleus during infection with no clear role in replication or pathogenesis. We examined NS5 of DENV1 and 2, which exhibit the most prominent difference in nuclear localization, employing a combination of functional and structural analyses. Extensive gene swapping between DENV1 and 2 NS5 identified that the C-terminal 18 residues (Cter18) alone was sufficient to direct the protein to the cytoplasm or nucleus, respectively. The low micromolar binding affinity between NS5 Cter18 and the nuclear import receptor importin-alpha (Impα), allowed their molecular complex to be purified, crystallised and visualized at 2.2 Å resolution using x-ray crystallography. Structure-guided mutational analysis of this region in GFP-NS5 clones of DENV1 or 2 and in a DENV2 infectious clone reveal residues important for NS5 subcellular localization. Notably, the trans conformation adopted by Pro-884 allows proper presentation for binding Impα and mutating this proline to Thr, as present in DENV1 NS5, results in mislocalizaion of NS5 to the cytoplasm without compromising virus fitness. In contrast, a single mutation to alanine at NS5 position R888, a residue conserved in all flaviviruses, resulted in a completely non-viable virus, and the R888K mutation led to a severely attenuated phentoype, even though NS5 was located in the nucleus. R888 forms a hydrogen bond with Y838 that is also conserved in all flaviviruses. Our data suggests an evolutionarily conserved function for NS5 Cter18, possibly in RNA interactions that are critical for replication, that is independent of its role in subcellular localization.


Asunto(s)
Núcleo Celular/metabolismo , Virus del Dengue/fisiología , Señales de Localización Nuclear/metabolismo , Proteínas no Estructurales Virales/metabolismo , Replicación Viral , Transporte Activo de Núcleo Celular , Sustitución de Aminoácidos , Animales , Línea Celular , Núcleo Celular/virología , Cricetinae , Humanos , Mutación Missense , Señales de Localización Nuclear/genética , Dominios Proteicos , Proteínas no Estructurales Virales/genética
12.
PLoS Pathog ; 12(1): e1005357, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26735137

RESUMEN

Dengue virus (DENV) is one of the most important arthropod-borne pathogens that cause life-threatening diseases in humans. However, no vaccine or specific antiviral is available for dengue. As seen in other RNA viruses, the innate immune system plays a key role in controlling DENV infection and disease outcome. Although the interferon (IFN) response, which is central to host protective immunity, has been reported to limit DENV replication, the molecular details of how DENV infection is modulated by IFN treatment are elusive. In this study, by employing a gain-of-function screen using a type I IFN-treated cell-derived cDNA library, we identified a previously uncharacterized gene, C19orf66, as an IFN-stimulated gene (ISG) that inhibits DENV replication, which we named Repressor of yield of DENV (RyDEN). Overexpression and gene knockdown experiments revealed that expression of RyDEN confers resistance to all serotypes of DENV in human cells. RyDEN expression also limited the replication of hepatitis C virus, Kunjin virus, Chikungunya virus, herpes simplex virus type 1, and human adenovirus. Importantly, RyDEN was considered to be a crucial effector molecule in the IFN-mediated anti-DENV response. When affinity purification-mass spectrometry analysis was performed, RyDEN was revealed to form a complex with cellular mRNA-binding proteins, poly(A)-binding protein cytoplasmic 1 (PABPC1), and La motif-related protein 1 (LARP1). Interestingly, PABPC1 and LARP1 were found to be positive modulators of DENV replication. Since RyDEN influenced intracellular events on DENV replication and, suppression of protein synthesis from DENV-based reporter construct RNA was also observed in RyDEN-expressing cells, our data suggest that RyDEN is likely to interfere with the translation of DENV via interaction with viral RNA and cellular mRNA-binding proteins, resulting in the inhibition of virus replication in infected cells.


Asunto(s)
Virus del Dengue/fisiología , Dengue/inmunología , Interferones/inmunología , Proteínas Virales/genética , Replicación Viral/inmunología , Línea Celular , Virus del Dengue/crecimiento & desarrollo , Técnicas de Silenciamiento del Gen , Humanos , Immunoblotting , Inmunoprecipitación , Espectrometría de Masas , Reacción en Cadena de la Polimerasa , Transfección
13.
Adv Exp Med Biol ; 1062: 147-163, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29845531

RESUMEN

Dengue virus (DENV) replication occurs in virus-induced vesicles that contain the replication complex (RC) where viral RNA, viral proteins and host proteins participate in RNA-RNA, RNA-protein and protein-protein interactions to ensure viral genome synthesis. However, the details of the multitude of interactions involved in the biogenesis of the infectious virion are not fully understood. In this review, we will focus on the interaction between non-structural (NS) proteins NS3 and NS5, as well as their interactions with viral RNA and briefly also the interaction of NS5 with the host nuclear transport receptor protein importin-α. The multifunctional NS3 protease/helicase and NS5 methyltransferase (MTase)/RNA-dependent RNA polymerase (RdRp) contain all the enzymatic activities required to synthesize the viral RNA genome. The success stories of drug discovery and development with Hepatitis C virus (HCV), a member of the Flaviviridae family, has led to the view that DENV NS3 and NS5 may be attractive antiviral drug targets. However, more than 10 years of intensive research effort by Novatis has revealed that they are not "low hanging fruits" and therefore, the search for potent directly acting antivirals (DAAs) remains a pipeline goal for several medium to large drug discovery enterprises. The effort to discover DAAs for DENV has been boosted by the epidemic outbreak of the closely related flavivirus member - Zika virus (ZIKV). Because the viral RNA replication occurs within a molecular machine that is composed several viral and host proteins, much interest has turned to characterising functionally essential protein-protein interactions in order to identify potential allosteric inhibitor binding sites within the RC.


Asunto(s)
Virus del Dengue/enzimología , Dengue/virología , ARN Viral/genética , Serina Endopeptidasas/metabolismo , Proteínas no Estructurales Virales/metabolismo , Replicación Viral , Animales , Dengue/genética , Dengue/metabolismo , Virus del Dengue/genética , Virus del Dengue/fisiología , Humanos , ARN Viral/metabolismo , Serina Endopeptidasas/genética , Proteínas no Estructurales Virales/genética , alfa Carioferinas/genética , alfa Carioferinas/metabolismo
14.
Adv Exp Med Biol ; 1062: 319-332, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29845542

RESUMEN

Dengue fever is a leading cause of illness and mortality in the tropics and subtropics. There are no therapeutics currently available and a recently approved vaccine is not very efficacious demanding an urgent need to develop an effective antiviral. The path to successful dengue drug development depends on availability of relevant preclinical testing models and better understanding of dengue pathogenesis. In recent years, efforts to develop dengue therapeutics have focused on both repurposing approved drugs as well as discovery of new chemical entities that act via virus or host targeted mechanisms. Here, we discuss the various innovative approaches, their outcome, and the lessons gleaned from the development efforts.


Asunto(s)
Antivirales/farmacología , Virus del Dengue/fisiología , Dengue/tratamiento farmacológico , Descubrimiento de Drogas/tendencias , Animales , Antivirales/química , Dengue/virología , Virus del Dengue/efectos de los fármacos , Virus del Dengue/genética , Descubrimiento de Drogas/métodos , Humanos
15.
Adv Exp Med Biol ; 1062: 115-129, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29845529

RESUMEN

Viruses from the Flavivirus family are the causative agents of dengue fever, Zika, Japanese encephalitis, West Nile encephalitis or Yellow fever and constitute major or emerging public health problems. A better understanding of the flavivirus replication cycle is likely to offer new opportunities for the design of antiviral therapies to treat severe conditions provoked by these viruses, but it should also help reveal fundamental biological mechanisms of the host cell. During virus replication, RNA synthesis is mediated by a dynamic and membrane-bound multi-protein assembly, named the replication complex (RC). The RC is composed of both viral and host-cell proteins that assemble within vesicles composed of the endoplasmic reticulum membrane, near the nucleus. At the heart of the flavivirus RC lies NS4B, a viral integral membrane protein that plays a role in virulence and in down-regulating the innate immune response. NS4B binds to the NS2B-NS3 protease-helicase, which itself interacts with the NS5 methyl-transferase polymerase. We present an overview of recent structural and functional data that augment our understanding of how viral RNA is replicated by dengue virus. We focus on structural data that illuminate the various roles played by proteins NS2B-NS3, NS4B and NS5. By participating in viral RNA cap methylation, the NS5 methyltransferase enables the virus to escape the host cell innate immune response. We present the molecular basis for this activity. We summarize what we know about the network of interactions established by NS2B-NS3, NS4B and NS5 (their "interactome"). This leads to a working model that is captured in the form of a rather naïve "cartoon", which we hope will be refined towards an atomic model in the near future.


Asunto(s)
Virus del Dengue/fisiología , Dengue/inmunología , Proteínas no Estructurales Virales/metabolismo , Replicación Viral , Animales , Dengue/virología , Virus del Dengue/genética , Humanos , Evasión Inmune , Inmunidad Innata , Proteínas no Estructurales Virales/genética
16.
Adv Exp Med Biol ; 1062: 107-113, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29845528

RESUMEN

A general discussion on viral entry and NS1 as potential drug targets was held at the Tofo Advanced Study Week (TASW) on Emerging Viral Diseases in September 2016. The opportunities and gaps for developing therapeutic countermeasures, to take advantage of the high-resolution cryo-electron microscopy structures of dengue and Zika viruses as well as the novel features of NS1 revealed by the 3D structures, were deliberated.


Asunto(s)
Antivirales/farmacología , Virus del Dengue/fisiología , Dengue/virología , Proteínas no Estructurales Virales/metabolismo , Internalización del Virus/efectos de los fármacos , Infección por el Virus Zika/virología , Virus Zika/fisiología , Animales , Virus del Dengue/química , Virus del Dengue/genética , Humanos , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Virus Zika/química , Virus Zika/genética
17.
Proc Natl Acad Sci U S A ; 112(48): 14834-9, 2015 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-26578813

RESUMEN

Dengue virus (DENV) causes several hundred million human infections and more than 20,000 deaths annually. Neither an efficacious vaccine conferring immunity against all four circulating serotypes nor specific drugs are currently available to treat this emerging global disease. Capping of the DENV RNA genome is an essential structural modification that protects the RNA from degradation by 5' exoribonucleases, ensures efficient expression of viral proteins, and allows escape from the host innate immune response. The large flavivirus nonstructural protein 5 (NS5) (105 kDa) has RNA methyltransferase activities at its N-terminal region, which is responsible for capping the virus RNA genome. The methyl transfer reactions are thought to occur sequentially using the strictly conserved flavivirus 5' RNA sequence as substrate (GpppAG-RNA), leading to the formation of the 5' RNA cap: G0pppAG-RNA → (m7)G0pppAG-RNA ("cap-0")→(m7)G0pppAm2'-O-G-RNA ("cap-1"). To elucidate how viral RNA is specifically recognized and methylated, we determined the crystal structure of a ternary complex between the full-length NS5 protein from dengue virus, an octameric cap-0 viral RNA substrate bearing the authentic DENV genomic sequence (5'-(m7)G0pppA1G2U3U4G5U6U7-3'), and S-adenosyl-l-homocysteine (SAH), the by-product of the methylation reaction. The structure provides for the first time, to our knowledge, a molecular basis for specific adenosine 2'-O-methylation, rationalizes mutagenesis studies targeting the K61-D146-K180-E216 enzymatic tetrad as well as residues lining the RNA binding groove, and offers previously unidentified mechanistic and evolutionary insights into cap-1 formation by NS5, which underlies innate immunity evasion by flaviviruses.


Asunto(s)
Virus del Dengue/enzimología , Metiltransferasas/química , Caperuzas de ARN/química , ARN Viral/química , Proteínas no Estructurales Virales/química , Cristalografía por Rayos X , Virus del Dengue/genética , Humanos , Metilación , Metiltransferasas/genética , Metiltransferasas/metabolismo , Estructura Terciaria de Proteína , Caperuzas de ARN/genética , Caperuzas de ARN/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo
18.
J Infect Dis ; 215(suppl_2): S96-S102, 2017 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-28403438

RESUMEN

Dengue is a significant global health problem. Even though a vaccine against dengue is now available, which is a notable achievement, its long-term protective efficacy against each of the 4 dengue virus serotypes remains to be definitively determined. Consequently, drugs directed at the viral targets or critical host mechanisms that can be used safely as prophylaxis or treatment to effectively ameliorate disease or reduce disease severity and fatalities are still needed to reduce the burden of dengue. This review will provide a brief account of the status of therapeutics research and development for dengue.


Asunto(s)
Anticuerpos Monoclonales/farmacología , Antivirales/farmacología , Dengue/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/uso terapéutico , Antivirales/uso terapéutico , Dengue/prevención & control , Descubrimiento de Drogas , Humanos
19.
PLoS Pathog ; 11(3): e1004682, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25775415

RESUMEN

Flavivirus RNA replication occurs within a replication complex (RC) that assembles on ER membranes and comprises both non-structural (NS) viral proteins and host cofactors. As the largest protein component within the flavivirus RC, NS5 plays key enzymatic roles through its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent-RNA polymerase (RdRp) domains, and constitutes a major target for antivirals. We determined a crystal structure of the full-length NS5 protein from Dengue virus serotype 3 (DENV3) at a resolution of 2.3 Å in the presence of bound SAH and GTP. Although the overall molecular shape of NS5 from DENV3 resembles that of NS5 from Japanese Encephalitis Virus (JEV), the relative orientation between the MTase and RdRp domains differs between the two structures, providing direct evidence for the existence of a set of discrete stable molecular conformations that may be required for its function. While the inter-domain region is mostly disordered in NS5 from JEV, the NS5 structure from DENV3 reveals a well-ordered linker region comprising a short 310 helix that may act as a swivel. Solution Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) analysis reveals an increased mobility of the thumb subdomain of RdRp in the context of the full length NS5 protein which correlates well with the analysis of the crystallographic temperature factors. Site-directed mutagenesis targeting the mostly polar interface between the MTase and RdRp domains identified several evolutionarily conserved residues that are important for viral replication, suggesting that inter-domain cross-talk in NS5 regulates virus replication. Collectively, a picture for the molecular origin of NS5 flexibility is emerging with profound implications for flavivirus replication and for the development of therapeutics targeting NS5.


Asunto(s)
Virus del Dengue/química , Virus del Dengue/fisiología , Proteínas no Estructurales Virales/química , Replicación Viral/fisiología , Cristalografía por Rayos X , Estructura Terciaria de Proteína , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo
20.
J Biol Chem ; 290(4): 2379-94, 2015 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-25488659

RESUMEN

Dengue virus multifunctional proteins NS3 protease/helicase and NS5 methyltransferase/RNA-dependent RNA polymerase form part of the viral replication complex and are involved in viral RNA genome synthesis, methylation of the 5'-cap of viral genome, and polyprotein processing among other activities. Previous studies have shown that NS5 residue Lys-330 is required for interaction between NS3 and NS5. Here, we show by competitive NS3-NS5 interaction ELISA that the NS3 peptide spanning residues 566-585 disrupts NS3-NS5 interaction but not the null-peptide bearing the N570A mutation. Small angle x-ray scattering study on NS3(172-618) helicase and covalently linked NS3(172-618)-NS5(320-341) reveals a rigid and compact formation of the latter, indicating that peptide NS5(320-341) engages in specific and discrete interaction with NS3. Significantly, NS3:Asn-570 to alanine mutation introduced into an infectious DENV2 cDNA clone did not yield detectable virus by plaque assay even though intracellular double-stranded RNA was detected by immunofluorescence. Detection of increased negative-strand RNA synthesis by real time RT-PCR for the NS3:N570A mutant suggests that NS3-NS5 interaction plays an important role in the balanced synthesis of positive- and negative-strand RNA for robust viral replication. Dengue virus infection has become a global concern, and the lack of safe vaccines or antiviral treatments urgently needs to be addressed. NS3 and NS5 are highly conserved among the four serotypes, and the protein sequence around the pinpointed amino acids from the NS3 and NS5 regions are also conserved. The identification of the functionally essential interaction between the two proteins by biochemical and reverse genetics methods paves the way for rational drug design efforts to inhibit viral RNA synthesis.


Asunto(s)
Virus del Dengue/fisiología , ARN Viral/química , Serina Endopeptidasas/química , Proteínas no Estructurales Virales/química , Replicación Viral , Animales , Antivirales/química , Sitios de Unión , Línea Celular , Línea Celular Tumoral , Cricetinae , ADN Complementario/metabolismo , Ensayo de Inmunoadsorción Enzimática , Humanos , Lisina/química , Mutagénesis Sitio-Dirigida , Mutación , Plásmidos/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , ARN Polimerasa Dependiente del ARN , Dispersión de Radiación
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