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1.
J Virol ; 97(6): e0034723, 2023 06 29.
Artículo en Inglés | MEDLINE | ID: mdl-37199644

RESUMEN

Multiple mechanisms exist in a cell to cope with stress. Four independent stress-sensing kinases constitute the integrated stress response machinery of the mammalian cell, and they sense the stress signals and act by phosphorylating the eukaryotic initiation factor 2α (eIF2α) to arrest cellular translation. Eukaryotic initiation factor 2 alpha kinase 4 (eIF2AK4) is one of the four kinases and is activated under conditions of amino acid starvation, UV radiation, or RNA virus infection, resulting in shutdown of global translation. An earlier study in our laboratory constructed the protein interaction network of the hepatitis E virus (HEV) and identified eIF2AK4 as a host interaction partner of the genotype 1 (g1) HEV protease (PCP). Here, we report that PCP's association with the eIF2AK4 results in inhibition of self-association and concomitant loss of kinase activity of eIF2AK4. Site-directed mutagenesis of the 53rd phenylalanine residue of PCP abolishes its interaction with the eIF2AK4. Further, a genetically engineered HEV-expressing F53A mutant PCP shows poor replication efficiency. Collectively, these data identify an additional property of the g1-HEV PCP protein, through which it helps the virus in antagonizing eIF2AK4-mediated phosphorylation of the eIF2α, thus contributing to uninterrupted synthesis of viral proteins in the infected cells. IMPORTANCE Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in humans. It causes chronic infection in organ transplant patients. Although the disease is self-limiting in normal individuals, it is associated with high mortality (~30%) in pregnant women. In an earlier study, we identified the interaction between the genotype 1 HEV protease (PCP) and cellular eukaryotic initiation factor 2 alpha kinase 4 (eIF2AK4). Since eIF2AK4 is a sensor of the cellular integrated stress response machinery, we evaluated the significance of the interaction between PCP and eIF2AK4. Here, we show that PCP competitively associates with and interferes with self-association of the eIF2AK4, thereby inhibiting its kinase activity. Lack of eIF2AK4 activity prevents phosphorylation-mediated inactivation of the cellular eIF2α, which is essential for initiation of cap-dependent translation. Thus, PCP behaves as a proviral factor, promoting uninterrupted synthesis of viral proteins in infected cells, which is crucial for survival and proliferation of the virus.


Asunto(s)
Endopeptidasas , Virus de la Hepatitis E , Proteínas Serina-Treonina Quinasas , Proteínas Virales , Femenino , Humanos , Embarazo , Endopeptidasas/genética , Endopeptidasas/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Hepatitis E/virología , Virus de la Hepatitis E/enzimología , Fosforilación , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Mutación , Aminoácidos/genética , Aminoácidos/metabolismo
2.
J Virol ; 92(20)2018 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-30068652

RESUMEN

Hepatitis E virus (HEV) generally causes self-limiting acute viral hepatitis in normal individuals. It causes a more severe disease in immunocompromised persons and pregnant women. Due to the lack of an efficient cell culture system or animal model, the life cycle of the virus is understudied, few antiviral targets are known, and very few antiviral candidates against HEV infection have been identified. Inhibition of virus release is one possible antiviral development strategy, which limits the spread of the virus. Previous studies have demonstrated the essential role of the interaction between the PSAP motif of the viral open reading frame 3 protein (ORF3-PSAP) and the UEV domain of the host tumor susceptibility gene 101 (TSG101) protein (UEV-TSG101) in mediating the release of genotype 3 HEV. Cyclic peptide (CP) inhibitors of the interaction between the human immunodeficiency virus (HIV) gag-PTAP motif and UEV-TSG101 are known to block the release of HIV. Using a molecular dynamic simulation, we observed that both gag-PTAP and ORF3-PSAP motifs bind to the same site in UEV-TSG101 by hydrogen bonding. HIV-released inhibitory CPs also displayed binding to the same site in UEV-TSG101, indicating that they may compete with ORF3-PSAP or gag-PTAP for binding to UEV-TSG101. Two independent assays confirmed the ability of a cyclic peptide (CP11) to inhibit the ORF3-TSG101 interaction. CP11 treatment also reduced the release of both genotype 1 and genotype 3 HEV by approximately 90%, with a 50% inhibitory concentration (IC50) of 2 µM. Thus, CP11 appears to be an attractive candidate for further validation of its anti-HEV properties.IMPORTANCE There is no specific therapy against hepatitis E virus (HEV)-induced hepatic and nonhepatic health problems. Prevention of the release of the progeny viruses from infected cells is an attractive strategy to limit the spread of the virus. Interactions between the viral open reading frame 3 and the host tumor susceptibility gene 101 proteins have been shown to be essential for the release of genotype 3 HEV from infected cells. In this study, we have identified a cyclic peptide inhibitor of the above-mentioned interaction and demonstrate the efficiency of the inhibitor in preventing virus release from infected cells. Thus, our findings uncover the possibility of developing a specific antiviral agent against HEV by blocking its release from infected cells.


Asunto(s)
Antivirales/metabolismo , Proteínas de Unión al ADN/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Virus de la Hepatitis E/efectos de los fármacos , Virus de la Hepatitis E/fisiología , Péptidos Cíclicos/metabolismo , Factores de Transcripción/metabolismo , Proteínas Virales/metabolismo , Liberación del Virus/efectos de los fármacos , Línea Celular , Proteínas de Unión al ADN/antagonistas & inhibidores , Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Hepatocitos/virología , Humanos , Concentración 50 Inhibidora , Unión Proteica/efectos de los fármacos , Factores de Transcripción/antagonistas & inhibidores , Proteínas Virales/antagonistas & inhibidores
3.
J Virol ; 91(21)2017 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-28814517

RESUMEN

Hepatitis E virus (HEV) causes an acute, self-limiting hepatitis in healthy individuals and leads to chronic disease in immunocompromised individuals. HEV infection in pregnant women results in a more severe outcome, with the mortality rate going up to 30%. Though the virus usually causes sporadic infection, epidemics have been reported in developing and resource-starved countries. No specific antiviral exists against HEV. A combination of interferon and ribavirin therapy has been used to control the disease with some success. Zinc is an essential micronutrient that plays crucial roles in multiple cellular processes. Zinc salts are known to be effective in reducing infections caused by few viruses. Here, we investigated the effect of zinc salts on HEV replication. In a human hepatoma cell (Huh7) culture model, zinc salts inhibited the replication of genotype 1 (g-1) and g-3 HEV replicons and g-1 HEV infectious genomic RNA in a dose-dependent manner. Analysis of a replication-defective mutant of g-1 HEV genomic RNA under similar conditions ruled out the possibility of zinc salts acting on replication-independent processes. An ORF4-Huh7 cell line-based infection model of g-1 HEV further confirmed the above observations. Zinc salts did not show any effect on the entry of g-1 HEV into the host cell. Furthermore, our data reveal that zinc salts directly inhibit the activity of viral RNA-dependent RNA polymerase (RdRp), leading to inhibition of viral replication. Taken together, these studies unravel the ability of zinc salts in inhibiting HEV replication, suggesting their possible therapeutic value in controlling HEV infection.IMPORTANCE Hepatitis E virus (HEV) is a public health concern in resource-starved countries due to frequent outbreaks. It is also emerging as a health concern in developed countries owing to its ability to cause acute and chronic infection in organ transplant and immunocompromised individuals. Although antivirals such as ribavirin have been used to treat HEV cases, there are known side effects and limitations of such therapy. Our discovery of the ability of zinc salts to block HEV replication by virtue of their ability to inhibit the activity of viral RdRp is important because these findings pave the way to test the efficacy of zinc supplementation therapy in HEV-infected patients. Since zinc supplementation therapy is known to be safe in healthy individuals and since high-dose zinc is used in the treatment of Wilson's disease, it may be possible to control HEV-associated health problems following a similar treatment regimen.


Asunto(s)
Antivirales/farmacología , Virus de la Hepatitis E/efectos de los fármacos , Hepatitis E/tratamiento farmacológico , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Compuestos de Zinc/farmacología , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Hepatitis E/virología , Virus de la Hepatitis E/enzimología , Virus de la Hepatitis E/genética , Humanos , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , ARN Viral/genética , Células Tumorales Cultivadas
4.
PLoS Pathog ; 12(4): e1005521, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-27035822

RESUMEN

Hepatitis E virus (HEV) causes acute hepatitis in many parts of the world including Asia, Africa and Latin America. Though self-limiting in normal individuals, it results in ~30% mortality in infected pregnant women. It has also been reported to cause acute and chronic hepatitis in organ transplant patients. Of the seven viral genotypes, genotype-1 virus infects humans and is a major public health concern in South Asian countries. Sporadic cases of genotype-3 and 4 infection in human and animals such as pigs, deer, mongeese have been reported primarily from industrialized countries. Genotype-5, 6 and 7 viruses are known to infect animals such as wild boar and camel, respectively. Genotype-3 and 4 viruses have been successfully propagated in the laboratory in mammalian cell culture. However, genotype-1 virus replicates poorly in mammalian cell culture and no other efficient model exists to study its life cycle. Here, we report that endoplasmic reticulum (ER) stress promotes genotype-1 HEV replication by inducing cap-independent, internal initiation mediated translation of a novel viral protein (named ORF4). Importantly, ORF4 expression and stimulatory effect of ER stress inducers on viral replication is specific to genotype-1. ORF4 protein sequence is mostly conserved among genotype-1 HEV isolates and ORF4 specific antibodies were detected in genotype-1 HEV patient serum. ORF4 interacted with multiple viral and host proteins and assembled a protein complex consisting of viral helicase, RNA dependent RNA polymerase (RdRp), X, host eEF1α1 (eukaryotic elongation factor 1 isoform-1) and tubulinß. In association with eEF1α1, ORF4 stimulated viral RdRp activity. Furthermore, human hepatoma cells that stably express ORF4 or engineered proteasome resistant ORF4 mutant genome permitted enhanced viral replication. These findings reveal a positive role of ER stress in promoting genotype-1 HEV replication and pave the way towards development of an efficient model of the virus.


Asunto(s)
Replicación del ADN/genética , Estrés del Retículo Endoplásmico/genética , Virus de la Hepatitis E/fisiología , ARN Viral/genética , Replicación Viral/genética , Células Cultivadas , Genotipo , Humanos , ARN Polimerasa Dependiente del ARN/genética , Proteínas Virales/genética
5.
Virus Genes ; 51(1): 1-11, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-25895103

RESUMEN

The Hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), nonstructural protein 5B (NS5B), is anchored in the membrane through a C-terminal helix. A sequence of ca. 12 residues that connects the catalytically competent portion of the RdRp and the C-terminal helix, the juxtamembrane sequence (JMS), has a poorly defined role in RdRp function in a large part since it is translated from a cis-acting RNA element (CRE) that is essential for HCV replication. Using a HCV replicon that transposed a second copy of CRE to the 3' UTR of the HCV replicon, we demonstrate that amino acid substitutions in the JMS were detrimental for HCV replicon replication. Substitutions in the JMS also resulted in a defect in de novo-initiated RNAs synthesis in vitro and in a cell-based reporter assay. A nonnucleoside inhibitor of the NS5B that binds to the catalytic pocket was less potent in inhibiting NS5B in the presence of JMS mutations. The JMS mutants exhibit reduced stability in thermodenaturation assays, suggesting that the JMS helps confer a more stable conformation to NS5B that could impact RNA synthesis.


Asunto(s)
Inhibidores Enzimáticos/metabolismo , Hepacivirus/enzimología , ARN Viral/biosíntesis , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismo , Sustitución de Aminoácidos , Línea Celular , Hepacivirus/genética , Calor , Humanos , Modelos Moleculares , Proteínas Mutantes/antagonistas & inhibidores , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformación Proteica , Estabilidad Proteica , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética
6.
J Virol ; 86(8): 4317-27, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22318148

RESUMEN

Brome mosaic virus (BMV) is a model positive-strand RNA virus whose replication has been studied in a number of surrogate hosts. In transiently transfected human cells, the BMV polymerase 2a activated signaling by the innate immune receptor RIG-I, which recognizes de novo-initiated non-self-RNAs. Active-site mutations in 2a abolished RIG-I activation, and coexpression of the BMV 1a protein stimulated 2a activity. Mutations previously shown to abolish 1a and 2a interaction prevented the 1a-dependent enhancement of 2a activity. New insights into 1a-2a interaction include the findings that helicase active site of 1a is required to enhance 2a polymerase activity and that negatively charged amino acid residues between positions 110 and 120 of 2a contribute to interaction with the 1a helicase-like domain but not to the intrinsic polymerase activity. Confocal fluorescence microscopy revealed that the BMV 1a and 2a colocalized to perinuclear region in human cells. However, no perinuclear spherule-like structures were detected in human cells by immunoelectron microscopy. Sequencing of the RNAs coimmunoprecipitated with RIG-I revealed that the 2a-synthesized short RNAs are derived from the message used to translate 2a. That is, 2a exhibits a strong cis preference for BMV RNA2. Strikingly, the 2a RNA products had initiation sequences (5'-GUAAA-3') identical to those from the 5' sequence of the BMV genomic RNA2 and RNA3. These results show that the BMV 2a polymerase does not require other BMV proteins to initiate RNA synthesis but that the 1a helicase domain, and likely helicase activity, can affect RNA synthesis by 2a.


Asunto(s)
Bromovirus/enzimología , Bromovirus/genética , ARN Viral/biosíntesis , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Virales/metabolismo , Secuencia de Bases , Bromovirus/inmunología , Línea Celular , Humanos , Inmunidad Innata , Datos de Secuencia Molecular , Unión Proteica , Transporte de Proteínas , ARN Polimerasa Dependiente del ARN/genética , Receptores Inmunológicos/metabolismo , Transducción de Señal
7.
Nucleic Acids Res ; 39(4): 1565-75, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20961956

RESUMEN

RIG-I recognizes molecular patterns in viral RNA to regulate the induction of type I interferons. The C-terminal domain (CTD) of RIG-I exhibits high affinity for 5' triphosphate (ppp) dsRNA as well as blunt-ended dsRNA. Structures of RIG-I CTD bound to 5'-ppp dsRNA showed that RIG-I recognizes the termini of dsRNA and interacts with the ppp through electrostatic interactions. However, the structural basis for the recognition of non-phosphorylated dsRNA by RIG-I is not fully understood. Here, we show that RIG-I CTD binds blunt-ended dsRNA in a different orientation compared to 5' ppp dsRNA and interacts with both strands of the dsRNA. Overlapping sets of residues are involved in the recognition of blunt-ended dsRNA and 5' ppp dsRNA. Mutations at the RNA-binding surface affect RNA binding and signaling by RIG-I. These results provide the mechanistic basis for how RIG-I recognizes different RNA ligands.


Asunto(s)
ARN Helicasas DEAD-box/química , ARN Bicatenario/química , Proteínas de Unión al ARN/química , Sitios de Unión , Cristalografía , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Mutación , Fosfatos/química , Unión Proteica , Estructura Terciaria de Proteína , ARN Bicatenario/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Receptores Inmunológicos , Transducción de Señal
8.
Front Microbiol ; 14: 1218654, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37908540

RESUMEN

Viral hepatitis is a major public health concern globally. World health organization aims at eliminating viral hepatitis as a public health threat by 2030. Among the hepatitis causing viruses, hepatitis B and C are primarily transmitted via contaminated blood. Hepatitis A and E, which gets transmitted primarily via the feco-oral route, are the leading cause of acute viral hepatitis. Although vaccines are available against some of these viruses, new cases continue to be reported. There is an urgent need to devise a potent yet economical antiviral strategy against the hepatitis-causing viruses (denoted as hepatitis viruses) for achieving global elimination of viral hepatitis. Although zinc was known to mankind for a long time (since before Christ era), it was identified as an element in 1746 and its importance for human health was discovered in 1963 by the pioneering work of Dr. Ananda S. Prasad. A series of follow up studies involving zinc supplementation as a therapy demonstrated zinc as an essential element for humans, leading to establishment of a recommended dietary allowance (RDA) of 15 milligram zinc [United States RDA for zinc]. Being an essential component of many cellular enzymes and transcription factors, zinc is vital for growth and homeostasis of most living organisms, including human. Importantly, several studies indicate potent antiviral activity of zinc. Multiple studies have demonstrated antiviral activity of zinc against viruses that cause hepatitis. This article provides a comprehensive overview of the findings on antiviral activity of zinc against hepatitis viruses, discusses the mechanisms underlying the antiviral properties of zinc and summarizes the prospects of harnessing the therapeutic benefit of zinc supplementation therapy in reducing the disease burden due to viral hepatitis.

9.
Microbiol Spectr ; 11(4): e0282722, 2023 08 17.
Artículo en Inglés | MEDLINE | ID: mdl-37382527

RESUMEN

Multiple processes exist in a cell to ensure continuous production of essential proteins either through cap-dependent or cap-independent translation processes. Viruses depend on the host translation machinery for viral protein synthesis. Therefore, viruses have evolved clever strategies to use the host translation machinery. Earlier studies have shown that genotype 1 hepatitis E virus (g1-HEV) uses both cap-dependent and cap-independent translation machineries for its translation and proliferation. Cap-independent translation in g1-HEV is driven by an 87-nucleotide-long RNA element that acts as a noncanonical, internal ribosome entry site-like (IRESl) element. Here, we have identified the RNA-protein interactome of the HEV IRESl element and characterized the functional significance of some of its components. Our study identifies the association of HEV IRESl with several host ribosomal proteins, demonstrates indispensable roles of ribosomal protein RPL5 and DHX9 (RNA helicase A) in mediating HEV IRESl activity, and establishes the latter as a bona fide internal translation initiation site. IMPORTANCE Protein synthesis is a fundamental process for survival and proliferation of all living organisms. The majority of cellular proteins are produced through cap-dependent translation. Cells also use a variety of cap-independent translation processes to synthesize essential proteins during stress. Viruses depend on the host cell translation machinery to synthesize their own proteins. Hepatitis E virus (HEV) is a major cause of hepatitis worldwide and has a capped positive-strand RNA genome. Viral nonstructural and structural proteins are synthesized through a cap-dependent translation process. An earlier study from our laboratory reported the presence of a fourth open reading frame (ORF) in genotype 1 HEV, which produces the ORF4 protein using a cap-independent internal ribosome entry site-like (IRESl) element. In the current study, we identified the host proteins that associate with the HEV-IRESl RNA and generated the RNA-protein interactome. Through a variety of experimental approaches, our data prove that HEV-IRESl is a bona fide internal translation initiation site.


Asunto(s)
Virus de la Hepatitis E , Virus de la Hepatitis E/genética , Sitios Internos de Entrada al Ribosoma , Proteínas Ribosómicas/genética , ARN Viral/genética , ARN Viral/metabolismo
10.
Antimicrob Agents Chemother ; 56(2): 830-7, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22143520

RESUMEN

Filibuvir and VX-222 are nonnucleoside inhibitors (NNIs) that bind to the thumb II allosteric pocket of the hepatitis C virus (HCV) RNA-dependent RNA polymerase. Both compounds have shown significant promise in clinical trials and, therefore, it is relevant to better understand their mechanisms of inhibition. In our study, filibuvir and VX-222 inhibited the 1b/Con1 HCV subgenomic replicon, with 50% effective concentrations (EC(50)s) of 70 nM and 5 nM, respectively. Using several RNA templates in biochemical assays, we found that both compounds preferentially inhibited primer-dependent RNA synthesis but had either no or only modest effects on de novo-initiated RNA synthesis. Filibuvir and VX-222 bind to the HCV polymerase with dissociation constants of 29 and 17 nM, respectively. Three potential resistance mutations in the thumb II pocket were analyzed for effects on inhibition by the two compounds. The M423T substitution in the RNA polymerase was at least 100-fold more resistant to filibuvir in the subgenomic replicon and in the enzymatic assays. This resistance was the result of a 250-fold loss in the binding affinity (K(d)) of the mutated enzyme to filibuvir. In contrast, the inhibitory activity of VX-222 was only modestly affected by the M423T substitution but more significantly affected by an I482L substitution.


Asunto(s)
Antivirales/farmacología , Ciclohexanoles/farmacología , Inhibidores Enzimáticos/farmacología , Hepacivirus/efectos de los fármacos , Pironas/farmacología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/metabolismo , Tiofenos/farmacología , Triazoles/farmacología , Antivirales/metabolismo , Sitios de Unión/efectos de los fármacos , Línea Celular Tumoral , Ciclohexanoles/metabolismo , Farmacorresistencia Viral , Inhibidores Enzimáticos/metabolismo , Hepacivirus/enzimología , Humanos , Modelos Moleculares , Mutación/efectos de los fármacos , Pironas/química , Pironas/metabolismo , ARN Viral/genética , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/genética , Replicón/efectos de los fármacos , Moldes Genéticos , Tiofenos/metabolismo , Triazoles/química , Triazoles/metabolismo
11.
Front Microbiol ; 13: 881595, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35814711

RESUMEN

Hepatitis E virus (HEV) causes an acute, self-limiting hepatitis. The disease takes a severe form in pregnant women, leading to around 30% mortality. Zinc is an essential micronutrient that plays a crucial role in multiple cellular processes. Our earlier findings demonstrated the antiviral activity of zinc salts against HEV infection. Zinc oxide (ZnO) and its nanostructures have attracted marked interest due to their unique characteristics. Here we synthesized ZnO nanoparticles [ZnO(NP)] and tetrapod-shaped ZnO nanoparticles [ZnO(TP)] and evaluated their antiviral activity. Both ZnO(NP) and ZnO(TP) displayed potent antiviral activity against hepatitis E and hepatitis C viruses, with the latter being more effective. Measurement of cell viability and intracellular reactive oxygen species levels revealed that both ZnO(NP) and ZnO(TP) are noncytotoxic to the cells even at significantly higher doses, compared to a conventional zinc salt (ZnSO4). Our study paves the way for evaluation of the potential therapeutic benefit of ZnO(TP) against HEV and HCV.

12.
J Biol Chem ; 285(47): 36635-44, 2010 Nov 19.
Artículo en Inglés | MEDLINE | ID: mdl-20855885

RESUMEN

The innate immune receptor Toll-like receptor 3 (TLR3) can be present on the surface of the plasma membranes of cells and in endolysosomes. The Unc93b1 protein has been reported to facilitate localization of TLR7 and 9 and is required for TLR3, -7, and -9 signaling. We demonstrate that siRNA knockdown of Unc93b1 reduced the abundance of TLR3 on the cell surface without altering total TLR3 accumulation. In addition, siRNA to Unc93b1 reduced the secretion of the TLR3 ectodomain (T3ECD) into the cell medium. Furthermore, two human single nucleotide polymorphisms that affected herpesvirus and influenza virus encephalopathy as well as a natural isoform generated by alternative splicing were found to be impaired for T3ECD secretion and decreased the abundance of TLR3 on the cell surface. The locations of the SNP P554S and the deletion in the isoform led to the identification of a loop in the TLR3 ectodomain that is required for secretion and a second whose presence decreased secretion. Finally, a truncated protein containing the N-terminal 10 leucine-rich repeats of T3ECD was sufficient for secretion in an Unc93b1-dependent manner.


Asunto(s)
Proteínas de Transporte de Membrana/metabolismo , Polimorfismo de Nucleótido Simple/genética , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/metabolismo , Secuencia de Aminoácidos , Western Blotting , Membrana Celular/metabolismo , Células Cultivadas , Glicosilación , Humanos , Riñón/citología , Riñón/metabolismo , Luciferasas/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Transducción de Señal
13.
Front Microbiol ; 11: 656, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32351484

RESUMEN

Understanding the dynamics of host innate immune responses against a pathogen marks the first step toward developing intervention strategies against the pathogen. The cytosolic pattern recognition receptor retinoic acid-inducible gene I (RIG-I) has been shown to be the major innate immune sensor for hepatitis E virus (HEV). Here, we show that HEV capsid protein (ORF2), a 660 amino acid long protein, interferes with the RIG-I signaling. Interestingly, only the full length ORF2 protein but not the 112-608 ORF2 protein inhibited RIG-I dependent interferon response. Both synthetic agonist and virus induced RIG-I activation was modulated by ORF2. Interference of interferon response was confirmed by reporter assays involving different interferon inducible promoters, qRT PCR, ELISA, and immunofluorescence microscopy. Neither glycosylation nor dimerization of the ORF2 protein had any effect on the observed inhibition. Further analyses revealed that the ORF2 protein antagonized Toll-like receptor (TLR) pathways as well. ORF2 inhibited signaling by RIG-I and TLR adapters, IPS-1, MyD88, and TRIF but was unable to inhibit activation by ectopically expressed IRF3 suggesting that it may be acting at a site upstream of IRF3 and downstream of adapter proteins. Our data uncover a new mechanism by which HEV may interfere with the host antiviral signaling.

14.
Front Microbiol ; 11: 141, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32117160

RESUMEN

Hepatitis E virus (HEV) is associated with acute hepatitis disease, which may lead to chronic disease in immunocompromised individuals. The disease is particularly severe among pregnant women (20-30% mortality). The only licensed vaccine against HEV, which is available in China, is the Escherichia coli purified recombinant virus-like particles (VLPs) encompassing the 368-660 amino acids (aa) of the viral ORF2 protein. The viral capsid is formed by the ORF2 protein, which harbors three glycosylation sites. Baculo virus expression system has been employed to generate a glycosylated VLP, which encompasses 112-608aa of the ORF2 protein. Here, we sought to produce a recombinant VLP containing 112-608aa of the ORF2 protein in Pichia pastoris (P. pastoris) expression system. The cDNA sequence encoding 112-608aa of the ORF2 protein was fused with the α-mating factor secretion signal coding sequence (for release of the fusion protein to the culture medium) and cloned into the yeast vector pPICZα. Optimum expression of recombinant protein was obtained at 72 h induction in 1.5% methanol using inoculum density (A600) of 80 and at pH-3.0 of the culture medium. Identity of the purified protein was confirmed by mass spectrometry analysis. Further studies revealed the glycosylation pattern and VLP nature of the purified protein. Immunization of BALB/c mice with these VLPs induced potent immune response as evidenced by the high ORF2 specific IgG titer and augmented splenocyte proliferation in a dose dependent manner. 112-608aa ORF2 VLPs produced in P. pastoris appears to be a suitable candidate for development of diagnostic and prophylactic reagents against the hepatitis E.

15.
Enzyme Microb Technol ; 128: 49-58, 2019 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-31186110

RESUMEN

Human interferon alpha 2b (IFN α2b) is a type I interferon exhibiting antiviral, anti-proliferative and immunomodulatory activities. The clinical outcome of the approved recombinant human IFN α2b drugs in the market suffers from short plasma half-life, rapid clearance and other side effects. Human IFN α2b expression in mammalian cell lines results in significant heterogeneity in glycan moieties, inconsistent product quality and high production cost. Potential scope exists for the design and development of a successful expression platform for enhanced human IFN α2b production with improved pharmacokinetic property. Glycoengineering strategy was employed to construct IFN α2b with potential N-glycosylation site to evade the drawbacks of approved recombinant human IFN α2b drugs. Heterogeneity of glycosylation and hypermannosylation in the wild-type strains of Pichia pastoris was circumvented by employing glycoengineered strain (SuperMan5) to produce glycosylated IFN α2b with human type N-glycans. Recombinant SuperMan5 strain expressed human type N-glycosylated IFN α2b with greater homogeneity elucidated by glycan analysis (MALDI-TOF/MS). The purified glycosylated IFN α2b was biologically active, inhibiting the viral replication of HCV and HEV at 85% and 66%, respectively. Pharmacokinetic studies in Wistar rats revealed 1.3 fold increase in plasma half-life for glycosylated IFN α2b compared to standard IFN α2b produced by E. coli.


Asunto(s)
Expresión Génica , Factores Inmunológicos/metabolismo , Interferón alfa-2/metabolismo , Ingeniería Metabólica/métodos , Pichia/metabolismo , Animales , Glicosilación , Semivida , Hepacivirus/efectos de los fármacos , Virus de la Hepatitis E/efectos de los fármacos , Humanos , Factores Inmunológicos/química , Factores Inmunológicos/farmacocinética , Factores Inmunológicos/farmacología , Interferón alfa-2/química , Interferón alfa-2/farmacocinética , Interferón alfa-2/farmacología , Pichia/genética , Plasma/química , Polisacáridos/análisis , Ratas Wistar , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Replicación Viral/efectos de los fármacos
16.
mSystems ; 3(1)2018.
Artículo en Inglés | MEDLINE | ID: mdl-29404423

RESUMEN

Comprehensive knowledge of host-pathogen interactions is central to understand the life cycle of a pathogen and devise specific therapeutic strategies. Protein-protein interactions (PPIs) are key mediators of host-pathogen interactions. Hepatitis E virus (HEV) is a major cause of viral hepatitis in humans. Recent reports also demonstrate its extrahepatic manifestations in the brain. Toward understanding the molecular details of HEV life cycle, we screened human liver and fetal brain cDNA libraries to identify the host interaction partners of proteins encoded by genotype 1 HEV and constructed the virus-host PPI network. Analysis of the network indicated a role of HEV proteins in modulating multiple host biological processes such as stress and immune responses, the ubiquitin-proteasome system, energy and iron metabolism, and protein translation. Further investigations revealed the presence of multiple host translation regulatory factors in the viral translation/replication complex. Depletion of host translation factors such as eIF4A2, eIF3A, and RACK1 significantly reduced the viral replication, whereas eIF2AK4 depletion had no effect. These findings highlight the ingenuity of the pathogen in manipulating the host machinery to its own benefit, a clear understanding of which is essential for the identification of strategic targets and development of specific antivirals against HEV. IMPORTANCE Hepatitis E virus (HEV) is a pathogen that is transmitted by the fecal-oral route. Owing to the lack of an efficient laboratory model, the life cycle of the virus is poorly understood. During the course of infection, interactions between the viral and host proteins play essential roles, a clear understanding of which is essential to decode the life cycle of the virus. In this study, we identified the direct host interaction partners of all HEV proteins and generated a PPI network. Our functional analysis of the HEV-human PPI network reveals a role of HEV proteins in modulating multiple host biological processes such as stress and immune responses, the ubiquitin-proteasome system, energy and iron metabolism, and protein translation. Further investigations revealed an essential role of several host factors in HEV replication. Collectively, the results from our study provide a vast resource of PPI data from HEV and its human host and identify the molecular components of the viral translation/replication machinery.

17.
Sci Rep ; 7(1): 5816, 2017 07 19.
Artículo en Inglés | MEDLINE | ID: mdl-28725041

RESUMEN

Hepatitis C virus (HCV) is a global pathogen and infects more than 185 million individuals worldwide. Although recent development of direct acting antivirals (DAA) has shown promise in HCV therapy, there is an urgent need for the development of more affordable treatment options. We initiated this study to identify novel inhibitors of HCV through screening of compounds from the National Cancer Institute (NCI) diversity dataset. Using cell-based assays, we identified NSC-320218 as a potent inhibitor against HCV with an EC50 of 2.5 µM and CC50 of 75 µM. The compound inhibited RNA dependent RNA polymerase (RdRp) activity of all six major HCV genotypes indicating a pan-genotypic effect. Limited structure-function analysis suggested that the entire molecule is necessary for the observed antiviral activity. However, the compound failed to inhibit HCV NS5B activity in vitro, suggesting that it may not be directly acting on the NS5B protein but could be interacting with a host protein. Importantly, the antiviral compound also inhibited dengue virus and hepatitis E virus replication in hepatocytes. Thus, our study has identified a broad-spectrum antiviral therapeutic agent against multiple viral infections.


Asunto(s)
Antivirales/farmacología , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/farmacología , Hepacivirus/enzimología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Antivirales/química , Línea Celular Tumoral , Virus del Dengue/efectos de los fármacos , Virus del Dengue/fisiología , Inhibidores Enzimáticos/química , Genotipo , Hepacivirus/efectos de los fármacos , Hepacivirus/genética , Hepacivirus/fisiología , Humanos , ARN Polimerasa Dependiente del ARN/metabolismo , Recombinación Genética/genética , Replicón/genética , Bibliotecas de Moléculas Pequeñas , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacos
19.
Sci Rep ; 6: 25133, 2016 04 26.
Artículo en Inglés | MEDLINE | ID: mdl-27113483

RESUMEN

Hepatitis E virus (HEV) is a major cause of hepatitis in normal and organ transplant individuals. HEV open reading frame-1 encodes a polypeptide comprising of the viral nonstructural proteins as well as domains of unknown function such as the macro domain (X-domain), V, DUF3729 and Y. The macro domain proteins are ubiquitously present from prokaryotes to human and in many positive-strand RNA viruses, playing important roles in multiple cellular processes. Towards understanding the function of the HEV macro domain, we characterized its interaction partners among other HEV encoded proteins. Here, we report that the HEV X-domain directly interacts with the viral methyltransferase and the ORF3 proteins. ORF3 association with the X-domain was mediated through two independent motifs, located within its N-terminal 35aa (amino acids) and C-terminal 63-123aa. Methyltransferase interaction domain was mapped to N-terminal 30-90aa. The X-domain interacted with both ORF3 and methyltransferase through its C-terminal region, involving 66(th),67(th) isoleucine and 101(st),102(nd) leucine, conserved across HEV genotypes. Furthermore, ORF3 and methyltransferase competed with each other for associating with the X-domain. These findings provide molecular understanding of the interaction between the HEV macro domain, methyltransferase and ORF3, suggesting an important role of the macro domain in the life cycle of HEV.


Asunto(s)
Virus de la Hepatitis E/fisiología , Poliproteínas/metabolismo , Proteínas Virales/metabolismo , Análisis Mutacional de ADN , Virus de la Hepatitis E/genética , Humanos , Unión Proteica , Mapeo de Interacción de Proteínas
20.
J Mol Biol ; 330(4): 675-85, 2003 Jul 18.
Artículo en Inglés | MEDLINE | ID: mdl-12850139

RESUMEN

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) initiates RNA synthesis in vivo by a de novo mechanism. In vitro, however, the HCV RdRp can initiate de novo or extend from a primed template. A novel beta-loop near the RdRp active site was previously found to prevent the use of primed templates. We found that, in addition to the beta-loop, the C-terminal tail of the HCV RdRp and the de novo initiation GTP are required to exclude the use of primed-templates. GTP binding to the NTPi site of the HCV RdRp orchestrates the participation of other structures. The interactions of the beta-loop, C-terminal tail, and GTP provide an elegant solution to ensure de novo initiation of HCV RNA synthesis.


Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Hepacivirus/enzimología , ARN Viral , ARN/biosíntesis , Secuencia de Bases , Relación Dosis-Respuesta a Droga , Escherichia coli/metabolismo , Guanosina Trifosfato/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Estructura Terciaria de Proteína , Proteínas no Estructurales Virales/química
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