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1.
PLoS Pathog ; 20(2): e1011999, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38306394

RESUMEN

Hepatitis B virus (HBV) chronically infects 296 million people worldwide, posing a major global health threat. Export of HBV RNAs from the nucleus to the cytoplasm is indispensable for viral protein translation and genome replication, however the mechanisms regulating this critical process remain largely elusive. Here, we identify a key host factor embryonic lethal, abnormal vision, Drosophila-like 1 (ELAVL1) that binds HBV RNAs and controls their nuclear export. Using an unbiased quantitative proteomics screen, we demonstrate direct binding of ELAVL1 to the HBV pregenomic RNA (pgRNA). ELAVL1 knockdown inhibits HBV RNAs posttranscriptional regulation and suppresses viral replication. Further mechanistic studies reveal ELAVL1 recruits the nuclear export receptor CRM1 through ANP32A and ANP32B to transport HBV RNAs to the cytoplasm via specific AU-rich elements, which can be targeted by a compound CMLD-2. Moreover, ELAVL1 protects HBV RNAs from DIS3+RRP6+ RNA exosome mediated nuclear RNA degradation. Notably, we find HBV core protein is dispensable for HBV RNA-CRM1 interaction and nuclear export. Our results unveil ELAVL1 as a crucial host factor that regulates HBV RNAs stability and trafficking. By orchestrating viral RNA nuclear export, ELAVL1 is indispensable for the HBV life cycle. Our study highlights a virus-host interaction that may be exploited as a new therapeutic target against chronic hepatitis B.


Asunto(s)
Virus de la Hepatitis B , ARN Viral , Animales , Humanos , Virus de la Hepatitis B/metabolismo , Transporte Activo de Núcleo Celular , ARN Viral/genética , ARN Viral/metabolismo , Drosophila/genética , Replicación Viral/genética , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteína 1 Similar a ELAV/genética , Proteína 1 Similar a ELAV/metabolismo
2.
PLoS Pathog ; 19(12): e1011808, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-38048324

RESUMEN

Chronic hepatitis B virus (HBV) infection is a major cause of liver cirrhosis and liver cancer, despite strong prevention and treatment efforts. The study of the epigenetic modification of HBV has become a research hotspot, including the N6-methyladenosine (m6A) modification of HBV RNA, which plays complex roles in the HBV life cycle. In addition to m6A modification, 5-methylcytosine (m5C) is another major modification of eukaryotic mRNA. In this study, we explored the roles of m5C methyltransferase and demethyltransferase in the HBV life cycle. The results showed that m5C methyltransferase NSUN2 deficiency could negatively regulate the expression of HBV while m5C demethyltransferase TET2 deficiency positively regulates the expression of HBV. Subsequently, we combined both in vitro bisulfite sequencing and high-throughput bisulfite sequencing methods to determine the distribution and stoichiometry of m5C modification in HBV RNA. Two sites: C2017 and C131 with the highest-ranking methylation rates were identified, and mutations at these two sites could lead to the decreased expression and replication of HBV, while the mutation of the "fake" m5C site had no effect. Mechanistically, NSUN2-mediated m5C modification promotes the stability of HBV RNA. In addition, compared with wild-type HepG2-NTCP cells and primary human hepatocytes, the replication level of HBV after NSUN2 knockdown decreased, and the ability of the mutant virus to infect and replicate in wild-type HepG2-NTCP cells and PHHs was substantially impaired. Similar results were found in the experiments using C57BL/6JGpt-Nsun2+/- mice. Interestingly, we also found that HBV expression and core protein promoted the endogenous expression of NSUN2, which implied a positive feedback loop. In summary, our study provides an accurate and high-resolution m5C profile of HBV RNA and reveals that NSUN2-mediated m5C modification of HBV RNA positively regulates HBV replication by maintaining RNA stability.


Asunto(s)
Virus de la Hepatitis B , Hepatitis B Crónica , Animales , Humanos , Ratones , Virus de la Hepatitis B/genética , Hepatitis B Crónica/genética , Metiltransferasas/genética , Ratones Endogámicos C57BL , ARN
3.
PLoS Pathog ; 19(5): e1011382, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-37224147

RESUMEN

Hepatitis B virus (HBV) chronically infects 296 million individuals and there is no cure. As an important step of viral life cycle, the mechanisms of HBV egress remain poorly elucidated. With proteomic approach to identify capsid protein (HBc) associated host factors and siRNA screen, we uncovered tumor susceptibility gene 101 (TSG101). Knockdown of TSG101 in HBV-producing cells, HBV-infected cells and HBV transgenic mice suppressed HBV release. Co-immunoprecipitation and site mutagenesis revealed that VFND motif in TSG101 and Lys-96 ubiquitination in HBc were essential for TSG101-HBc interaction. In vitro ubiquitination experiment demonstrated that UbcH6 and NEDD4 were potential E2 ubiquitin-conjugating enzyme and E3 ligase that catalyzed HBc ubiquitination, respectively. PPAY motif in HBc and Cys-867 in NEDD4 were required for HBc ubiquitination, TSG101-HBc interaction and HBV egress. Transmission electron microscopy confirmed that TSG101 or NEDD4 knockdown reduces HBV particles count in multivesicular bodies (MVBs). Our work indicates that TSG101 recognition for NEDD4 ubiquitylated HBc is critical for MVBs mediated HBV egress.


Asunto(s)
Virus de la Hepatitis B , Proteómica , Animales , Ratones , Virus de la Hepatitis B/genética , Factores de Transcripción/genética , Proteínas de Unión al ADN/genética , Ratones Transgénicos
4.
J Biol Chem ; 299(9): 105151, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37567479

RESUMEN

Hepatitis B virus (HBV) is a hepatotropic DNA virus that has a very compact genome. Due to this genomic density, several distinct mechanisms are used to facilitate the viral life cycle. Recently, accumulating evidence show that G-quadruplex (G4) in different viruses play essential regulatory roles in key steps of the viral life cycle. Although G4 structures in the HBV genome have been reported, their function in HBV replication remains elusive. In this study, we treated an HBV replication-competent cell line and HBV-infected cells with the G4 structure stabilizer pyridostatin (PDS) and evaluated different HBV replication markers to better understand the role played by the G4. In both models, we found PDS had no effect on viral precore RNA (pcRNA) or pre-genomic RNA (pgRNA), but treatment did increase HBeAg/HBc ELISA reads and intracellular levels of viral core/capsid protein (HBc) in a dose-dependent manner, suggesting post-transcriptional regulation. To further dissect the mechanism of G4 involvement, we used in vitro-synthesized HBV pcRNA and pgRNA. Interestingly, we found PDS treatment only enhanced HBc expression from pgRNA but not HBeAg expression from pcRNA. Our bioinformatic analysis and CD spectroscopy revealed that pgRNA harbors a conserved G4 structure. Finally, we introduced point mutations in pgRNA to disrupt its G4 structure and observed the resulting mutant failed to respond to PDS treatment and decreased HBc level in in vitro translation assay. Taken together, our data demonstrate that HBV pgRNA contains a G4 structure that plays a vital role in the regulation of viral mRNA translation.


Asunto(s)
G-Cuádruplex , Virus de la Hepatitis B , Hepatitis B , Humanos , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Hepatitis B/virología , Antígenos e de la Hepatitis B/metabolismo , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/metabolismo , Replicación Viral/genética , Línea Celular , G-Cuádruplex/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/genética , Mutación , Aminoquinolinas/farmacología
5.
J Virol ; 97(7): e0051223, 2023 07 27.
Artículo en Inglés | MEDLINE | ID: mdl-37347173

RESUMEN

Nonstructural protein 13 (nsp13), the helicase of SARS-CoV-2, has been shown to possess multiple functions that are essential for viral replication, and is considered an attractive target for the development of novel antivirals. We were initially interested in the interplay between nsp13 and interferon (IFN) signaling, and found that nsp13 inhibited reporter signal in an IFN-ß promoter assay. Surprisingly, the ectopic expression of different components of the RIG-I/MDA5 pathway, which were used to stimulate IFN-ß promoter, was also mitigated by nsp13. However, endogenous expression of these genes was not affected by nsp13. Interestingly, nsp13 restricted the expression of foreign genes originating from plasmid transfection, but failed to inhibit them after chromosome integration. These data, together with results from a runoff transcription assay and RNA sequencing, suggested a specific inhibition of episomal but not chromosomal gene transcription by nsp13. By using different truncated and mutant forms of nsp13, we demonstrated that its NTPase and helicase activities contributed to the inhibition of episomal DNA transcription, and that this restriction required direct interaction with episomal DNA. Based on these findings, we developed an economical and convenient high-throughput drug screening method targeting nsp13. We evaluated the inhibitory effects of various compounds on nsp13 by the expression of reporter gene plasmid after co-transfection with nsp13. In conclusion, we found that nsp13 can specifically inhibit episomal DNA transcription and developed a high-throughput drug screening method targeting nsp13 to facilitate the development of new antiviral drugs. IMPORTANCE To combat COVID-19, we need to understand SARS-CoV-2 and develop effective antiviral drugs. In our study, we serendipitously found that SARS-CoV-2 nsp13 could suppress episomal DNA transcription without affecting chromosomal DNA. Detailed characterization revealed that nsp13 suppresses episomal gene expression through its NTPase and helicase functions following DNA binding. Furthermore, we developed a high-throughput drug screening system targeting SARS-CoV-2 nsp13. Compared to traditional SARS-CoV-2 drug screening methods, our system is more economical and convenient, facilitating the development of more potent and selective nsp13 inhibitors and enabling the discovery of new antiviral therapies.


Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Nucleósido-Trifosfatasa/genética , ARN Helicasas/metabolismo , Proteínas no Estructurales Virales/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Antivirales/farmacología , ADN , Plásmidos/genética
6.
Hepatology ; 77(4): 1366-1381, 2023 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-35718932

RESUMEN

BACKGROUND AND AIMS: Murine hepatic cells cannot support hepatitis B virus (HBV) infection even with supplemental expression of viral receptor, human sodium taurocholate cotransporting polypeptide (hNTCP). However, the specific restricted step remains elusive. In this study, we aimed to dissect HBV infection process in murine hepatic cells. APPROACH AND RESULTS: Cells expressing hNTCP were inoculated with HBV or hepatitis delta virus (HDV). HBV pregenomic RNA (pgRNA), covalently closed circular DNA (cccDNA), and different relaxed circular DNA (rcDNA) intermediates were produced in vitro . The repair process from rcDNA to cccDNA was assayed by in vitro repair experiments and in mouse with hydrodynamic injection. Southern blotting and in situ hybridization were used to detect HBV DNA. HBV, but not its satellite virus HDV, was restricted from productive infection in murine hepatic cells expressing hNTCP. Transfection of HBV pgRNA could establish HBV replication in human, but not in murine, hepatic cells. HBV replication-competent plasmid, cccDNA, and recombinant cccDNA could support HBV transcription in murine hepatic cells. Different rcDNA intermediates could be repaired to form cccDNA both in vitro and in vivo . In addition, rcDNA could be detected in the nucleus of murine hepatic cells, but cccDNA could not be formed. Interestingly, nuclease sensitivity assay showed that the protein-linked rcDNA isolated from cytoplasm was completely nuclease resistant in murine, but not in human, hepatic cells. CONCLUSIONS: Our results imply that the disassembly of cytoplasmic HBV nucleocapsids is restricted in murine hepatic cells. Overcoming this limitation may help to establish an HBV infection mouse model.


Asunto(s)
Virus de la Hepatitis B , Hepatitis B , Ratones , Humanos , Animales , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/metabolismo , ADN Viral/genética , Replicación Viral/genética , Hepatocitos/metabolismo , Nucleocápside/metabolismo , Hepatitis B/genética , Citoplasma/metabolismo , ADN Circular/metabolismo
7.
J Virol ; 96(21): e0136222, 2022 11 09.
Artículo en Inglés | MEDLINE | ID: mdl-36226986

RESUMEN

Hepatitis B virus (HBV) infection is a major health burden worldwide, and currently there is no cure. The persistence of HBV covalently closed circular DNA (cccDNA) is the major obstacle for antiviral trement. HBV core protein (HBc) has emerged as a promising antiviral target, as it plays important roles in critical steps of the viral life cycle. However, whether HBc could regulate HBV cccDNA transcription remains under debate. In this study, different approaches were used to address this question. In synthesized HBV cccDNA and HBVcircle transfection assays, lack of HBc showed no effect on transcription of HBV RNA as well as HBV surface antigen (HBsAg) production in a hepatoma cell line and primary human hepatocytes. Reconstitution of HBc did not alter the expression of cccDNA-derived HBV markers. Similar results were obtained from an in vivo mouse model harboring cccDNA. Chromatin immunoprecipitation (ChIP) or ChIP sequencing assays revealed transcription regulation of HBc-deficient cccDNA chromatin similar to that of wild-type cccDNA. Furthermore, treatment with capsid assembly modulators (CAMs) dramatically reduced extracellular HBV DNA but could not alter viral RNA and HBsAg. Our results demonstrate that HBc neither affects histone modifications and transcription factor binding of cccDNA nor directly influences cccDNA transcription. Although CAMs could reduce HBc binding to cccDNA, they do not suppress cccDNA transcriptional activity. Thus, therapeutics targeting capsid or HBc should not be expected to sufficiently reduce cccDNA transcription. IMPORTANCE Hepatitis B virus (HBV) core protein (HBc) has emerged as a promising antiviral target. However, whether HBc can regulate HBV covalently closed circular DNA (cccDNA) transcription remains elusive. This study illustrated that HBc has no effect on epigenetic regulation of cccDNA, and it does not participate in cccDNA transcription. Given that HBc is dispensable for cccDNA transcription, novel cccDNA-targeting therapeutics are needed for an HBV cure.


Asunto(s)
ADN Circular , Hepatitis B , Animales , Humanos , Ratones , Antivirales , Proteínas de la Cápside/genética , ADN Circular/genética , ADN Viral/genética , Epigénesis Genética , Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B , Virus de la Hepatitis B/fisiología , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/metabolismo , Replicación Viral/genética , Transcripción Genética
8.
J Hepatol ; 75(5): 1072-1082, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34242702

RESUMEN

BACKGROUND & AIMS: Our understanding of the interactions between HBV and its host cells is still quite limited. Spliceosome associated factor 1 (SART1) has recently been found to restrict HCV. Thus, we aimed to dissect its role in HBV infection. METHODS: SART1 was knocked down by RNA interference and over-expressed by lentiviral or adeno-associated virus (AAV) vectors in HBV-infected cell cultures and in vivo in HBV-infected mice. Luciferase reporter assays were used to determine viral or host factor promoter activities, and chromatin immunoprecipitation (ChIP) was used to investigate protein-DNA interactions. RESULTS: In HBV-infected cell cultures, downregulation of SART1 did not affect covalently closed circular HBV DNA but resulted in markedly enhanced HBV RNA, antigen expression and progeny virus production. On the other hand, HBV transcription and replication were significantly inhibited by overexpression of SART1. Similar results were observed in AAV-HBV-infected mice persistently replicating HBV. Inhibition of Janus kinases had no effect on SART1-mediated inhibition of HBV replication. HBV promoter assays revealed that SART1 reduced HBV core promoter activity. By screening known HBV transcription factors, we found that SART1 specifically suppressed the expression of hepatocyte nuclear factor 4α (HNF4α). Luciferase reporter and ChIP assays demonstrated a direct downregulation of HNF4α expression by association of SART1 with the HNF4α proximal P1 promoter element. CONCLUSIONS: We identify SART1 as a novel host factor suppressing HBV cccDNA transcription. Besides its effect on interferon-stimulated genes, SART1 exerts an anti-HBV activity by suppressing HNF4α expression, which is essential for transcription of HBV cccDNA. LAY SUMMARY: Hepatitis B virus (HBV) infects hepatocytes and persists in the form of covalently closed circular DNA (cccDNA), which remains a major obstacle to successful antiviral treatment. In this study, using various HBV models, we demonstrate that the protein SART1 restricts HBV cccDNA transcription by suppressing a key transcription factor, HNF4α.


Asunto(s)
Antivirales/metabolismo , Redes Reguladoras de Genes/genética , Hepatitis B/tratamiento farmacológico , Factor Nuclear 4 del Hepatocito/antagonistas & inhibidores , Ribonucleoproteínas Nucleares Pequeñas/farmacología , Antivirales/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Hepatitis B/fisiopatología , Factor Nuclear 4 del Hepatocito/metabolismo , Humanos , Ribonucleoproteínas Nucleares Pequeñas/uso terapéutico , Replicación Viral/efectos de los fármacos
9.
Zhongguo Zhong Yao Za Zhi ; 43(17): 3477-3483, 2018 Sep.
Artículo en Zh | MEDLINE | ID: mdl-30347915

RESUMEN

The aim of this study was to investigated the biological diversity, antibacterial activites and the plant growth-promoting traits of endophytic fungi of sandal (Santalum album), and to assess their potential in the development of antibacterial substances and rapid cultivation of sandal. The results of isolation and taxa analysis of endophytic fungi from sandal showed that 325 strains of endophytic fungi belonging to 16 genera of endophytic fungi were isolated from sandal (of which 86 from roots, 105 from stems and 134 from leaves). The isolation rate and colonization rate of endophytic fungi in different sandal parts showed the same pattern of change: leave>stems>roots. The diversity index of endophytic fungi in sandal roots was significantly higher than that of stems and leaves. The dominant endophytic fungi of sandal roots, stems and leaves showed significant differences. The dominant endophytic fungi of roots were Fusarium (50.00%) and Alternaria (10.47%), Alternaria (58.11%) and Acremonium (20.00%) for stems, and Pantoea (74.63%) for leaves. The antibacterial activity of 40 representative strains of sandal endophytic fungi were analyzed and the results showed that 90% of endophytic fungi exhibited inhibitory activity against at least one of the tested bacteria strains, and the strains with inhibitory activity to Escherichia coli, Enterobacter aerogenes, Shigella dysenteriae, Salmonella typhimurium, Staphylococcus aureus, and Bacillus subtilis accounted for 45.0%, 30%, 47.5%, 55%, 72.5%, and 62.5%, respectively. The sandal fungal endophytes with plant growth-promoting characteristics were screened, and 5 strains of endophytic fungi with phosphorus-solubilizing activity, 8 strains of endophytic fungi producing IAA, and 4 strains of endophytic fungi producing siderophores were found. Among them, endophytic fungus Monilia sp TXRF45 clould produced IAA and siderophores, and also show phosphate-solubilizing activity. The results indicated that the endophytic fungi of Sandal were rich in species diversity and their distribution had a certain tissue specificity. Some strains showed good antibacterial activity and growth-promoting properties, which could potentially applicable for the development of antibacterial substances and rapid cultivation of sandal.


Asunto(s)
Antibiosis , Bacterias , Endófitos/química , Santalum/microbiología , Sideróforos/química , Biodiversidad , Endófitos/clasificación , Hongos/química , Hongos/clasificación , Hojas de la Planta/microbiología , Raíces de Plantas/microbiología , Tallos de la Planta/microbiología
10.
Cell Insight ; 3(4): 100178, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-39027058

RESUMEN

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide and presents a significant threat to human health. Despite its prevalence, the underlying regulatory mechanisms of HCC remain unclear. In this study, we integrated RNA-seq datasets, proteome dataset and survival analysis and unveiled Stratifin (SFN) as a potential prognostic biomarker for HCC. SFN knockdown inhibited HCC progression in cell cultures and mouse models. Conversely, ectopic expression of Sfn in primary mouse HCC model accelerated HCC progression. Mechanistically, SFN acted as an adaptor protein, activating AKT1 signaling by fostering the interaction between PDK1 and AKT1, with the R56 and R129 sites on SFN proving to be crucial for this binding. In the syngeneic implantation model, the R56A/R129A mutant of SFN inhibited Akt signaling activation and impeded HCC growth. Additionally, peptide inhibitors designed based on the binding motif of AKT1 to SFN significantly inhibited HCC progression. In summary, our findings establish that SFN promotes HCC progression by activating AKT signaling through the R56 and R129 binding sites. This discovery opens new avenues for a promising therapeutic strategy for the treatment of HCC.

11.
Virol Sin ; 38(3): 335-343, 2023 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-37141990

RESUMEN

Commensal microbiota is closely related to Hepatitis B virus (HBV) infection. Gut bacteria maturation accelerates HBV immune clearance in hydrodynamic injection (HDI) HBV mouse model. However, the effect of gut bacteria on HBV replication in recombinant adeno-associated virus (AAV)-HBV mouse model with immune tolerance remains obscure. We aim to investigate its role on HBV replication in AAV-HBV mouse model. C57BL/6 mice were administrated with broad-spectrum antibiotic mixtures (ABX) to deplete gut bacteria and intravenously injected with AAV-HBV to establish persistent HBV replication. Gut microbiota community was analyzed by fecal qPCR assay and 16S ribosomal RNA (rRNA) gene sequencing. HBV replication markers in blood and liver were determined by ELISA, qPCR assay and Western blot at indicated time points. Immune response in AAV-HBV mouse model was activated through HDI of HBV plasmid or poly(I:C) and then detected by quantifying the percentage of IFN-γ+/CD8+ T cells in the spleen via flow cytometry as well as the splenic IFN-γ mRNA level via qPCR assay. We found that antibiotic exposure remarkably decreased gut bacteria abundance and diversity. Antibiotic treatment failed to alter the levels of serological HBV antigens, intrahepatic HBV RNA transcripts and HBc protein in AAV-HBV mouse model, but contributed to HBsAg increase after breaking of immune tolerance. Overall, our data uncovered that antibiotic-induced gut bacteria depletion has no effect on HBV replication in immune tolerant AAV-HBV mouse model, providing new thoughts for elucidating the correlation between gut bacteria dysbiosis by antibiotic abuse and clinical chronic HBV infection.


Asunto(s)
Virus de la Hepatitis B , Hepatitis B , Ratones , Animales , Virus de la Hepatitis B/genética , Linfocitos T CD8-positivos , Ratones Endogámicos C57BL , Bacterias , Tolerancia Inmunológica , Replicación Viral , Modelos Animales de Enfermedad
12.
Nat Metab ; 5(5): 842-860, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-37188818

RESUMEN

Different organs undergo distinct transcriptional, epigenetic and physiological alterations that guarantee their functional maturation after birth. However, the roles of epitranscriptomic machineries in these processes have remained elusive. Here we demonstrate that expression of RNA methyltransferase enzymes Mettl3 and Mettl14 gradually declines during postnatal liver development in male mice. Liver-specific Mettl3 deficiency causes hepatocyte hypertrophy, liver injury and growth retardation. Transcriptomic and N6-methyl-adenosine (m6A) profiling identify the neutral sphingomyelinase, Smpd3, as a target of Mettl3. Decreased decay of Smpd3 transcripts due to Mettl3 deficiency results in sphingolipid metabolism rewiring, characterized by toxic ceramide accumulation and leading to mitochondrial damage and elevated endoplasmic reticulum stress. Pharmacological Smpd3 inhibition, Smpd3 knockdown or Sgms1 overexpression that counteracts Smpd3 can ameliorate the abnormality of Mettl3-deficent liver. Our findings demonstrate that Mettl3-N6-methyl-adenosine fine-tunes sphingolipid metabolism, highlighting the pivotal role of an epitranscriptomic machinery in coordination of organ growth and the timing of functional maturation during postnatal liver development.


Asunto(s)
Hígado , Metiltransferasas , Ratones , Masculino , Animales , Metiltransferasas/genética , Metiltransferasas/metabolismo , Hígado/metabolismo , Hepatocitos/metabolismo , Ceramidas , Estrés del Retículo Endoplásmico , Adenosina/metabolismo , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/metabolismo
13.
Cell Mol Gastroenterol Hepatol ; 13(4): 1001-1017, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-34896285

RESUMEN

BACKGROUND AND AIMS: The persistence of viral covalently closed circular DNA (cccDNA) is the major obstacle for antiviral treatment against hepatitis B virus (HBV). Basic and translational studies are largely hampered due to the lack of feasible small animal models to support HBV cccDNA formation. The aim of this study is to establish a novel mouse model harboring cccDNA. METHODS: An adeno-associated virus (AAV) vector carrying a replication-deficient HBV1.04-fold genome (AAV-HBV1.04) was constructed. The linear HBV genome starts from nucleotide 403 and ends at 538, which results in the splitting of HBV surface and polymerase genes. Different HBV replication markers were evaluated for AAV-HBV1.04 plasmid-transfected cells, the AAV-HBV1.04 viral vector-transduced cells, and mice injected with the AAV-HBV1.04 viral vector. RESULTS: Compared with the previously reported AAV-HBV1.2 construct, direct transfection of AAV-HBV1.04 plasmid failed to produce hepatitis B surface antigen and progeny virus. Interestingly, AAV-HBV1.04 viral vector transduction could result in the formation of cccDNA and the production of all HBV replication markers in vitro and in vivo. The formation of cccDNA could be blocked by ATR (ataxia-telangiectasia and Rad3-related protein) inhibitors but not HBV reverse transcription inhibitor or capsid inhibitors. The AAV-HBV1.04 mouse supported long-term HBV replication and responded to antiviral treatments. CONCLUSIONS: This AAV-HBV1.04 mouse model can support HBV cccDNA formation through ATR-mediated DNA damage response. The de novo formed cccDNA but not the parental AAV vector can lead to the production of hepatitis B surface antigen and HBV progeny. This model will provide a unique platform for studying HBV cccDNA and developing novel antivirals against HBV infection.


Asunto(s)
Antígenos de Superficie de la Hepatitis B , Virus de la Hepatitis B , Animales , Antivirales/metabolismo , Antivirales/farmacología , ADN Circular/genética , ADN Viral/genética , Dependovirus , Modelos Animales de Enfermedad , Antígenos de Superficie de la Hepatitis B/metabolismo , Antígenos de Superficie de la Hepatitis B/farmacología , Virus de la Hepatitis B/genética , Ratones , Replicación Viral
14.
mSphere ; 5(5)2020 10 07.
Artículo en Inglés | MEDLINE | ID: mdl-33028689

RESUMEN

Since the outbreak of coronavirus disease 2019 (COVID-19) in Wuhan, China, it has rapidly spread around the world. Persons with asymptomatic disease exhibit viral shedding, resulting in transmission, which presents disease control challenges. However, the clinical characteristics of these asymptomatic individuals remain elusive. We collected samples of 25 asymptomatic and 27 symptomatic COVID-19 patients. Viral titers of throat swabs were determined by quantitative reverse transcription-PCR (qRT-PCR). COVID-19 IgG and IgM were examined. Complete blood counts were determined, and serum biochemistry panels were performed. Cytokines, including gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin 2 (IL-2), IL-4, IL-6, and IL-10 were evaluated. T cell, B cell, and NK cell counts were measured using flow cytometry. Although similar viral loads were detected, asymptomatic patients had significantly faster virus turnover than symptomatic patients. Additionally, asymptomatic patients had higher counts of lymphocytes, T cells, B cells, and NK cells. While liver damage was observed in symptomatic patients, as indicated by elevated liver enzymes and decreased liver-synthesized proteins in the blood, asymptomatic patients showed normal liver measurements. Lactate dehydrogenase, a COVID-19 risk factor, was significantly lower in asymptomatic patients. These results suggest that asymptomatic COVID-19 patients had normal clinical indicators and faster viral clearance than symptomatic patients. Lymphocytes may play a role in their asymptomatic phenotype. Since asymptomatic patients may be a greater risk of virus transmission than symptomatic patients, public health interventions and a broader range of testing may be necessary for the control of COVID-19.IMPORTANCE Asymptomatic transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a potential problem for pandemic control through public health strategies. Our results demonstrate that asymptomatic COVID-19 patients have better outcomes than symptomatic patients. This may have been due to more active cellular immune responses and normal liver function. Since asymptomatic patients have no clinical symptoms which can easily prevent timely diagnosis and treatment, they may cause a greater risk of virus transmission than symptomatic patients, which poses a major challenge to infection control. Evidence suggests that nonpharmaceutical public health interventions, like social distancing and face mask ordinances, play important roles in the control of COVID-19. Looking forward, it may be necessary to proceed cautiously while reopening businesses in areas of epidemicity to prevent potential waves of COVID-19 in the future.


Asunto(s)
Infecciones Asintomáticas , Betacoronavirus , Infecciones por Coronavirus/diagnóstico , Neumonía Viral/diagnóstico , Adulto , Betacoronavirus/aislamiento & purificación , Biomarcadores/sangre , COVID-19 , Prueba de COVID-19 , Estudios de Casos y Controles , China , Técnicas de Laboratorio Clínico , Infecciones por Coronavirus/sangre , Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/transmisión , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pandemias/prevención & control , Neumonía Viral/sangre , Neumonía Viral/prevención & control , Neumonía Viral/transmisión , Estudios Retrospectivos , SARS-CoV-2 , Esparcimiento de Virus
15.
Probiotics Antimicrob Proteins ; 10(4): 748-754, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-29039076

RESUMEN

Bacterial species of Bacillus, Lactobacillus, and Bifidobacterium in the intestinal tract have been used as probiotics. Selections for probiotic candidates by the culture-based approaches are time-consuming and labor-consuming. The aim of this study was to develop a new method based on sequencing strategies to select the probiotic Bacillus, Lactobacillus, and Bifidobacterium. The Illumina-based sequencing strategies with different specific primers for Bacillus, Clostridium, and Bifidobacterium were applied to analyze diversity of the genera in goat feces. The average number of different Bacillus, Clostridium, and Bifidobacterium OTUs (operational taxonomic units) at the 97% similarity level ranged from 1922 to 63172. The coverage index values of Bacillus, Clostridium, and Bifidobacterium calculated from the bacterial OTUs were 0.89, 0.99, and 1.00, respectively. The most genera of Bacillus (37.9%), Clostridium (53%), and Bifidobacterium (99%) were detected in goat feces by the Illumina-based sequencing with the specific primers of the genera, respectively. Higher phylogenetic resolutions of the genera in goat feces were successfully established. The results suggest that the selection for probiotic Bacillus, Clostridium, and Bifidobacterium based on the Illumina sequencing with their specific primers is reliable and feasible, and the core Bacillus, Clostridium, and Bifidobacterium species of healthy goats possess the potentials as probiotic microbial consortia.


Asunto(s)
Bacillus/aislamiento & purificación , Bifidobacterium/aislamiento & purificación , Clostridium/aislamiento & purificación , Cabras/microbiología , Probióticos/aislamiento & purificación , Animales , Bacillus/clasificación , Bacillus/genética , Bifidobacterium/clasificación , Bifidobacterium/genética , Biodiversidad , Clostridium/clasificación , Clostridium/genética , Cartilla de ADN/genética , Heces/microbiología , Secuenciación de Nucleótidos de Alto Rendimiento , Filogenia , Probióticos/clasificación
16.
Environ Sci Pollut Res Int ; 24(1): 417-426, 2017 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-27726080

RESUMEN

Metal-resistant endophytic fungi from roots improved phytoremediation efficacy of host plants; however, the effects of endophytic fungi from plant aerial parts on host plants are unknown. The aim of this study was to develop a feasible method to screen fungal endophytes from stems and roots of Brassica napus and to investigate effects of the endophytic fungi on growth and phytoremediation efficiency of the plant. Endophytic Fusarium sp. CBRF44, Penicillium sp. CBRF65, and Alternaria sp. CBSF68 with different traits were isolated from roots and stems of rapes grown in a metal-contaminated soil. Fusarium sp. CBRF44 (resistant to 5 mM Cd and 15 mM Pb, isolated from roots) and Alternaria sp. CBSF68 (resistant to 1 mM Cd and 10 mM Pb, isolated from stems) could produce indole-3-acetic acid (IAA) and siderophore; Penicillium sp. CBRF65 (tolerate 2 mM Cd and 20 mM Pb, isolated from roots) could not produce IAA and siderophore but showed the highest phosphate-solubilizing activities. Fusarium sp. CBRF44 and Penicillium sp. CBRF65 significantly increased the rape biomass and promoted the extraction efficacy of Pb and Cd, while Alternaria sp. CBSF68 did not show similar results. Penicillium sp. CBRF65 and Fusarium sp. CBRF44 could be frequently recovered from inoculated rape roots, while Alternaria sp. CBSF68 was scarcely recovered. The results indicate that the colonizing capacity of endophytic fungi in roots is important to improve phytoremediation efficacy of host plants.


Asunto(s)
Alternaria , Brassica napus , Cadmio , Fusarium , Plomo , Penicillium , Contaminantes del Suelo , Alternaria/efectos de los fármacos , Alternaria/metabolismo , Biodegradación Ambiental , Biomasa , Brassica napus/crecimiento & desarrollo , Brassica napus/metabolismo , Brassica napus/microbiología , Cadmio/metabolismo , Cadmio/toxicidad , Fusarium/efectos de los fármacos , Fusarium/metabolismo , Ácidos Indolacéticos/metabolismo , Plomo/metabolismo , Plomo/toxicidad , Penicillium/efectos de los fármacos , Penicillium/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Raíces de Plantas/microbiología , Tallos de la Planta/crecimiento & desarrollo , Tallos de la Planta/metabolismo , Tallos de la Planta/microbiología , Microbiología del Suelo , Contaminantes del Suelo/metabolismo , Contaminantes del Suelo/toxicidad
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