Study on anti-HBV mechanism of Liuweiwuling Tablet (LWWL) based on transcriptomics
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PROVIDER: PRJNA761101 | ENA |
REPOSITORIES: ENA
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Project description:Transcriptomics were used to evaluate the possible mechanism and pathway of LWWL against HBV
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Project description:Hepatitis B virus (HBV) causes both acute and chronic liver inflammation. Approximately 600,000 CHB patients each year die of HBV-related diseases such as cirrhosis and liver cancer. Therefore, CHB remains a global health concern. Although there have been anti-HBV agents for treating CHB, they have some limitations including viral-drug resistance and adverse effects. Type III IFN or IFN-λ is promising to use as anti-HBV agents because of its anti-viral activities like type I IFNs. In addition, the expression of its receptor, IFNLR1, is limited only in epithelial cells including hepatocytes. Thus, treatment with IFN-lambda results in less side effects compared to IFN-alpha treatment. IFN-lambdas have been shown to inhibit the replication of several viruses including IAV, DENV, EMCV, HIV, HCV, and HBV; however, there have been no studies on the effects of IFN-λ3, the highest activity among other subtypes, on HBV replication. Therefore, this study aims to determine antiviral activities of IFN-λ3 against HBV replication and to investigate its molecular mechanism responsible for suppressing HBV propagation. The results showed that HBV transcripts and amount of intracellular HBV DNA were decreased in HepG2.2.15 cells, stable HBV-transfected hepatoblastoma cell line, treated with IFN-λ3 in a dose-dependent manner. This indicated that IFN-λ3 could inhibit HBV replication. Next, we performed quantitative proteomics to investigate the proteome changes in HepG2.2.15 treated with IFN-λ3. The proteins that changed their expressions were involved in several biological processes such as defense to viral infection, immune responses, cell-cell adhesion, transcription, translation, and metabolism. We further confirmed the proteomics results by immunoblotting assay. Consistent with MS data, it found that the expression of OAS3, SAMHD1 and STAT1 were increased as a result of IFN-λ3 stimulation. These results indicated that proteomics results were reproducible and reliable. Finally, we proposed 3 possible mechanisms involved in suppressing HBV replication including i.) IFN-λ3 induced anti-viral proteins affecting many steps in HBV life cycle ii.) IFN-λ3 promoted antigen processing and antigen presentation and iii.) IFN-λ3 rescued RIG-I signaling to promote both type I and type III IFN production.
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Project description:This study aimed to better characterize the repertoire of serum HBV RNAs during chronic HBV infection in humans, which remains understudied. Using RT-PCR, qPCR, RNA-sequencing and immuno-precipitation, we found that (i) >50% of serum samples bore different amounts of HBV replication-derived RNAs (rd-RNAs); (ii) a few samples contained RNAs transcribed from integrated HBV DNA including 5'-HBV-human-3' RNAs (integrant-derived RNAs or id-RNAs) and 5'-human-HBV-3' transcripts as a minority of serum HBV RNAs; (iii) spliced HBV RNAs were abundant in <50% of analyzed samples; (iv) most serum rd-RNAs were polyadenylated via conventional HBV polyadenylation signal; (v) pre-genomic RNA (pgRNA) was the major component of the pool of serum RNAs; (vi) area of HBV positions 1531-1739 had very high RNA reads coverage and thus should be used as a target for detecting serum HBV RNAs; (vii) vast majority of rd-RNAs and pgRNA were associated with HBV virions, but not with unenveloped capsids, exosomes, classic microvesicles or apoptotic vesicles and bodies; (viii) considerable rd-RNAs presence in the circulating immune complexes was found in a few samples; and (ix) serum rcDNA and rd-RNAs should be quantified simultaneously to evaluate HBV replication status and efficacy of anti-HBV therapy with nucleos(t)ide analogs. In summary, sera contain various HBV RNA types of different origin, which are likely secreted via different mechanisms. In addition, since we previously showed that id-RNAs were abundant or predominant HBV RNAs in many of liver and hepatocellular carcinoma tissues comparing to rd-RNAs, there is likely a mechanism favoring the egress of the replication-derived RNAs.
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Project description:Background and aims: The intrahepatic processes associated with chronic hepatitis B (CHB), especially in the context of HDV and HIV co-infection, require a better understanding. Spatial transcriptomics can provide new insights into the complex intrahepatic biological processes, guiding new personalized treatments. Our aim is to evaluate this method to characterize the hepatic transcriptional landscape, cellular composition and biological pathways in liver biopsy samples from patients infected with HBV and HDV or HIV co-infection. Method: The GeoMx nanostring Digital Spatial Profiling (DSP) platform was employed to assess expression of HBsAg and CD45 in formalin fixed paraffin embedded (FFPE) biopsies from three treatment-naïve patients with chronic HBV and HDV or HIV co-infection. The GeoMx whole transcriptome human atlas assay quantified the expression of genes enriched in specific regions of interest (ROIs). Cell type proportions within ROIs were deconvoluted using a training matrix from the human liver cell atlas. A weighted gene correlation network analysis (WGCNA) evaluated transcriptomic signatures across sampled regions. Results: We identified spatially discrete transcriptomic signatures and distinct biological pathways that associate with HBV infection/disease status and immune responses. Shared features including cytotoxicity and B cell receptor signaling were consistent across patients, suggesting common elements alongside individual traits. HDV/HBV co-infection exhibited upregulated genes linked to apoptosis and immune cell recruitment, whereas HIV/HBV co-infection featured genes related to interferon response regulation. Varied cellular characteristics and immune cell populations, with an abundance of T cells in the HDV/HBV sample, were observed within analyzed regions. Transcriptional differences in hepatocyte function suggest disrupted metabolic processes in HDV/HBV co-infection potentially impacting disease progression. Conclusion: This proof-of-principle study shows the value of this platform in investigating the complex immune landscape highlighting relevant host pathways to disease pathogenesis.
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Project description:: Hepatitis B virus (HBV) remains a major public health threat with more than 296 million people chronically infected worldwide at high risk to develop hepatocellular carcinoma. Current therapies are effective in suppressing HBV replication but rarely lead to cure. The fundamental limitation rests on the fact that current therapies do not affect the HBV covalently closed circular DNA (cccDNA), which serves as the template for viral transcription and replication and is highly stable in infected cells to ensure viral persistence. In this study, we aim to identify and elucidate the functional role of cccDNA-associated host factors using affinity purification and protein mass spectrometry in HBV-infected cells. HBV core protein (HBc) is associated with cccDNA. We used an anti-HBc antibody to pull down HBcAg-associated cccDNA complex in nuclear extract of HBV-infected HepG2-NTCP cells. By protein mass spectrometry, we identified many host proteins, including previously known proteins, that are associated with HBV cccDNA. Some of these proteins were functionally validated to play a role in HBV cccDNA activities.
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