Hepatitis A Virus Picornain 3C
Ontology highlight
ABSTRACT:
SUBMITTER: Yin J
PROVIDER: S-EPMC7149673 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We employed a highly sensitive technology, 3C-HTGTS (3C-high-throughput genome-wide sequencing), to globally identify HBV DNA-host DNA contacts in cellular models of HBV infection. We found that HBV cccDNA does not randomly position in host genome, but instead preferentially establishes contacts with the host DNA at active chromatin regions. In contrast, the organization of integrated HBV DNA is largely regulated by the local genome epigenetic environment, which particularly forms chromosome loop with host genome where gene promoters together with active histone modifications are enriched. Our study provides a high throughput 3D landscape for the spatial organizations of cccDNA and integrated HBV DNA within the human genome, and provides a foundation to further understand the mechanisms of HBV modulation of liver disease development and progression in the future.
2022-05-31 | GSE146497 | GEO
Project description:Oligonucleotides as short as 6?nt in length have been shown to bind specifically and tightly to proteins and affect their biological function. Yet, sparse structural data are available for corresponding complexes. Employing a recently developed hexanucleotide array, we identified hexadeoxyribonucleotides that bind specifically to the 3C protease of hepatitis A virus (HAV 3C(pro)). Inhibition assays in vitro identified the hexanucleotide 5'-GGGGGT-3' (G(5)T) as a 3C(pro) protease inhibitor. Using (1)H NMR spectroscopy, G(5)T was found to form a G-quadruplex, which might be considered as a minimal aptamer. With the help of (1)H, (15)N-HSQC experiments the binding site for G(5)T was located to the C-terminal ?-barrel of HAV 3C(pro). Importantly, the highly conserved KFRDI motif, which has previously been identified as putative viral RNA binding site, is not part of the G(5)T-binding site, nor does G(5)T interfere with the binding of viral RNA. Our findings demonstrate that sequence-specific nucleic acid-protein interactions occur with oligonucleotides as small as hexanucleotides and suggest that these compounds may be of pharmaceutical relevance.
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Project description:During viral infections, some viruses subvert the host proteins to promote the translation or RNA replication with their protease-mediated cleavage. Poly (A)-binding protein (PABP) is a target for several RNA viruses; however, the impact of duck hepatitis A virus (DHAV) on PABP remains unknown. In this study, we demonstrated for the first time that DHAV infection stimulates a decrease in endogenous PABP and generates two cleavage fragments. On the basis of in vitro cleavage assays, an accumulation of PABP cleavage fragments was detected in duck embryo fibroblast (DEF) cell extracts incubated with functional DHAV 3C protease. In addition, DHAV 3C protease was sufficient for the cleavage of recombinant PABP without the assistance of other eukaryotic cellular cofactors. Furthermore, using site-directed mutagenesis, our data demonstrated a 3C protease cleavage site located between Q367 and G368 in duck PABP. Moreover, the knockdown of PABP inhibited the production of viral RNA, and the C-terminal domain of PABP caused a reduction in viral replication compared to the N-terminal domain. Taken together, these findings suggested that DHAV 3C protease mediates the cleavage of PABP, which may be a strategy to manipulate viral replication.
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Project description:Hepatitis A virus (HAV) infection is a major cause of acute hepatitis worldwide and occasionally causes acute liver failure and can lead to death in the absence of liver transplantation. Although HAV vaccination is available, the prevalence of HAV vaccination is not adequate in some countries. Additionally, the improvements in public health reduced our immunity to HAV infection. These situations motivated us to develop potentially new anti-HAV therapeutic options. We carried out the in silico screening of anti-HAV compounds targeting the 3C protease enzyme using the Schrodinger Modeling software from the antiviral library of 25,000 compounds to evaluate anti-HAV 3C protease inhibitors. Additionally, in vitro studies were introduced to examine the inhibitory effects of HAV subgenomic replicon replication and HAV HA11-1299 genotype IIIA replication in hepatoma cell lines using luciferase assays and real-time RT-PCR. In silico studies enabled us to identify five lead candidates with optimal binding interactions in the active site of the target HAV 3C protease using the Schrodinger Glide program. In vitro studies substantiated our hypothesis from in silico findings. One of our lead compounds, Z10325150, showed 47% inhibitory effects on HAV genotype IB subgenomic replicon replication and 36% inhibitory effects on HAV genotype IIIA HA11-1299 replication in human hepatoma cell lines, with no cytotoxic effects at concentrations of 100 μg/mL. The effects of the combination therapy of Z10325150 and RNA-dependent RNA polymerase inhibitor, favipiravir on HAV genotype IB HM175 subgenomic replicon replication and HAV genotype IIIA HA11-1299 replication showed 64% and 48% inhibitory effects of HAV subgenomic replicon and HAV replication, respectively. We identified the HAV 3C protease inhibitor Z10325150 through in silico screening and confirmed the HAV replication inhibitory activity in human hepatocytes. Z10325150 may offer the potential for a useful HAV inhibitor in severe hepatitis A.
| S-EPMC9181686 | biostudies-literature
Project description:Hepatitis A is an acute infection caused by Hepatitis A virus (HAV), which is widely distributed throughout the world. The HAV 3C cysteine protease (3Cpro), an important nonstructural protein, is responsible for most cleavage within the viral polyprotein and is critical for the processes of viral replication. Our group has previously demonstrated that HAV 3Cpro cleaves human NF-?B essential modulator (NEMO), a kinase required in interferon signaling. Based on this finding, we generated four luciferase-based biosensors containing the NEMO sequence (PVLKAQ?ADIYKA) that is cleaved by HAV 3Cpro and/or the Nostoc punctiforme DnaE intein, to monitor the activity of HAV 3Cpro in human embryonic kidney cells (HEK-293T). Western blotting showed that HAV 3Cpro recognized and cleaved the NEMO cleavage sequence incorporated in the four biosensors, whereas only one cyclized luciferase-based biosensor (233-DnaE-HAV, 233DH) showed a measurable and reliable increase in firefly luciferase activity, with very low background, in the presence of HAV 3Cpro. With this biosensor (233DH), we monitored HAV 3Cpro activity in HEK-293T cells, and tested it against a catalytically deficient mutant HAV 3Cpro and other virus-encoded proteases. The results showed that the activity of this luciferase biosensor is specifically dependent on HAV 3Cpro. Collectively, our data demonstrate that the luciferase biosensor developed here might provide a rapid, sensitive, and efficient evaluation of HAV 3Cpro activity, and should extend our better understanding of the biological relevance of HAV 3Cpro.
| S-EPMC7092888 | biostudies-literature
Project description:The related 3C and 3C-like proteinase (3C(pro) and 3CL(pro)) of picornaviruses and coronaviruses, respectively, are good drug targets. As part of an effort to generate broad-spectrum inhibitors of these enzymes, we screened a library of inhibitors based on a halopyridinyl ester from a previous study of the severe acute respiratory syndrome (SARS) 3CL proteinase against Hepatitis A virus (HAV) 3C(pro). Three of the compounds, which also had furan rings, inhibited the cleavage activity of HAV 3C(pro) with K(ic)s of 120-240nM. HPLC-based assays revealed that the inhibitors were slowly hydrolyzed by both HAV 3C(pro) and SARS 3CL(pro), confirming the identity of the expected products. Mass spectrometric analyses indicated that this hydrolysis proceeded via an acyl-enzyme intermediate. Modeling studies indicated that the halopyridinyl moiety of the inhibitor fits tightly into the S1-binding pocket, consistent with the lack of tolerance of the inhibitors to modification in this portion of the molecule. These compounds are among the most potent non-peptidic inhibitors reported to date against a 3C(pro).
| S-EPMC7125897 | biostudies-literature
Project description:The 3C protease is a key factor in picornavirus-induced pathologies with a comprehensive action on cell targets. However, the effects induced by the enzyme have not been described at the organismic level. Here, the model of developing Danio rerio embryos was used to analyze possible toxic effects of the 3C protease of human hepatitis A virus (3Cpro) at the whole-body level. The transient 3Cpro expression had a notable lethal effect and induced a number of specific abnormalities in Danio rerio embryos within 24 h. These effects are due to the proteolytic activity of the enzyme. At the same time, the 3Cpro variant with reduced catalytic activity (3Cmut) increased the incidence of embryonic abnormalities; however, this effect was smaller compared to the native enzyme form. While the expression of 3Cmut increased the overall rate of abnormalities, no predominance of specific ones was observed. The data obtained point to a presence significant impact of picornavirus 3Cprotease at the whole-organism level and make contribution to the study of the infectious process caused by human hepatitis A virus.
| S-EPMC8440601 | biostudies-literature
Project description:Proteinase 3C of hepatitis A virus (HAV) plays a key role in the viral life cycle by generating mature viral proteins from the precursor polyprotein. In addition to its proteolytic activity, 3C binds to viral RNA, and thus influences viral genome replication. In order to investigate the interplay between proteolytic activity and RNA binding at the molecular level, we subjected HAV 3C and three variants carrying mutations of the cysteine residues [C24S (Cys-24-->Ser), C172A and C24S/C172A] to proteolysis assays with peptide substrates, and to surface plasmon resonance binding studies with peptides and viral RNA. We report that the enzyme readily forms dimers via disulphide bridges involving Cys-24. Dissociation constants (K(D)) for peptides were in the millimolar range. The binding kinetics for the peptides were characterized by k(on) and k(off) values of the order of 10(2) M(-1) x s(-1) and 10(-2) to 10(-1) s(-1) respectively. In contrast, 3C binding to immobilized viral RNA, representing the structure of the 5'-terminal domain, followed fast binding kinetics with k(on) and k(off) values beyond the limits of the kinetic resolution of the technique. The affinity of viral RNA depended strongly on the dimerization status of 3C. Whereas monomeric 3C bound to the viral RNA with a K(D) in the millimolar range, dimeric 3C had a significantly increased binding affinity with K(D) values in the micromolar range. A model of the 3C dimer suggests that spatial proximity of the presumed RNA-binding motifs KFRDI is possible. 3C binding to RNA was also promoted in the presence of substrate peptides, indicating co-operativity between RNA binding and protease activity. The data imply that the dual functions of 3C are mutually dependent, and regulate protein and RNA synthesis during the viral life cycle.
| S-EPMC1134706 | biostudies-literature
Project description:It is widely accepted that Hepatitis A virus (HAV) is responsible for liver failure and even death in older people and in people with other serious health issues; so, proposing new compounds with inhibitory activity can help to treated of these disease's. In current study, a new class of quinolines is proposed with inhibitor activity of the HAV proteinase. So, in the first step, fused quinoline derivatives has been synthesized in short reaction time (12.0 min) and high efficiency yields (94%) in presence of 1-carboxymethyl-2,3-dimethylimidazolium iodide ([cmdmim]I) ionic liquid catalyst using a new method. In the following, chemical reactivity and inhibitory activity of synthesized quinolines were evaluated in density functional theory (DFT) framework and molecular docking methodologies. High global softness (0.67 eV), low HOMOSWBNNT-LUMO4a gap (4.78 eV), and more negative adsorption energy (- 87.9 kJ mol-1) in these quinolines reveal that the 4a and 4b compounds have better delivery than other quinolines using SWBNNT as suitable carrier to target cells. Molecular docking shows that the best cavity of the HAV has - 134.2 kJ mol-1 interaction energy involving bonding and non-bonding interactions. In fact, these interactions are between fused quinolines with especial geometries and sidechain flexibility amino acids residues inside the best binding site of the HAV, as hydrogen bonding, steric, and electrostatic interactions. So, these interactions imply that proposed fused quinolines have good inhibitor activity for the HAV.
| S-EPMC8460796 | biostudies-literature
Project description:3C proteases, the main proteases of picornaviruses, play the key role in viral life cycle by processing polyproteins. In addition, 3C proteases digest certain host cell proteins to suppress antiviral defense, transcription, and translation. The activity of 3C proteases per se induces host cell death, which makes them critical factors of viral cytotoxicity. To date, cytotoxic effects have been studied for several 3C proteases, all of which induce apoptosis. This study for the first time describes the cytotoxic effect of 3C protease of human hepatitis A virus (3Cpro), the only proteolytic enzyme of the virus.Individual expression of 3Cpro induced catalytic activity-dependent cell death, which was not abrogated by the pan-caspase inhibitor (z-VAD-fmk) and was not accompanied by phosphatidylserine externalization in contrast to other picornaviral 3C proteases. The cell survival was also not affected by the inhibitors of cysteine proteases (z-FA-fmk) and RIP1 kinase (necrostatin-1), critical enzymes involved in non-apoptotic cell death. A substantial fraction of dying cells demonstrated numerous non-acidic cytoplasmic vacuoles with not previously described features and originating from several types of endosomal/lysosomal organelles. The lysosomal protein Lamp1 and GTPases Rab5, Rab7, Rab9, and Rab11 were associated with the vacuolar membranes. The vacuolization was completely blocked by the vacuolar ATPase inhibitor (bafilomycin A1) and did not depend on the activity of the principal factors of endosomal transport, GTPases Rab5 and Rab7, as well as on autophagy and macropinocytosis.3Cpro, apart from other picornaviral 3C proteases, induces caspase-independent cell death, accompanying by cytoplasmic vacuolization. 3Cpro-induced vacuoles have unique properties and are formed from several organelle types of the endosomal/lysosomal compartment. The data obtained demonstrate previously undocumented morphological characters of the 3Cpro-induced cell death, which can reflect unknown aspects of the human hepatitis A virus-host cell interaction.
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