Sera of individuals chronically infected with hepatitis B virus (HBV) contain diverse RNA types produced by HBV replication or derived from integrated HBV DNA
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ABSTRACT: 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.
Project description:Five matching sets of non-malignant liver tissues and HCCs from individuals chronically infected with hepatitis B virus (HBV) were examined. The HBV genomic sequences were determined using overlapping PCR amplicons covering the entire viral genome. Four pairs of tissues were infected with HBV of genotype C, while one pair - with genotype B. HBV replication markers were found in all tissues. In majority of HCC samples, the levels of pre-genomic/pre-core RNA (pgRNA) and covalently closed circular DNA (cccDNA) were lower than those of liver tissue counterparts. Regardless of the presence of HBV replication markers, (i) integrant-derived HBV RNAs (id-RNAs) were found using RT-PCR analysis in all tissues, and were considerably abundant or predominant in 6/10 tissue samples (2 livers and 4 HCCs); (ii) the RNAs that were polyadenylated using cryptic HBV polyadenylation signal and therefore could be produced by HBV replication or derived from integrated HBV DNA were found in 5/10 samples (3 livers and 2 HCCs), and were considerably abundant species in 3/10 tissues (2 livers and 1 HCC); and (iii) cccDNA-transcribed RNAs polyadenylated near position 1931 were not abundant in 7/10 tissues (2 livers and 5 HCCs), and were predominant only in two livers. Subsequent RNA sequencing analysis of selected liver/HCC samples also showed relative abundance of id-RNAs in most of examined tissues. Our findings suggesting that id-RNAs could represent a significant source of HBV envelope proteins, which is independent of viral replication, are discussed in the context of possible contribution of id-RNAs to the HBV life cycle.
Project description:Background and Aims Chronic infection with hepatitis B virus (HBV) has been known to cause liver cirrhosis and hepatocellular carcinoma. Although nucleos(t)ide analogs are mainly used for the treatment of HBV, they require long-term administration and may lead to the emergence of drug resistance. Therefore, to identify targets for the development of novel anti-HBV therapies, we screened HBV-suppressive host factors using RNA-bnding protein (RBP) expression plasmids library. Approach and Results We screened the RBP library by generating overexpressing RBP cell lines and observing anti-HBV effect. As a result, we identified NEDD4-binding protein 1 (N4BP1) as a candidate showing anti-HBV effect. In hepatocellular carcinoma cell lines, overexpression of N4BP1 decreased the relaxed circular DNA (rcDNA) levels, while suppression of N4BP1 expression increased rcDNA levels. Restoring N4BP1 expression in N4BP1 knockout cells regained the anti-HBV effect of N4BP1. Next, we constructed KH-like and RNase domain-deficient mutants of N4BP1 and examined their effects on HBV replication, and found that both the KH-like and RNase domains are required for its anti-HBV effect. N4BP1 suppresses the step where pregenomic RNA (pgRNA) is synthesized in the HBV life cycle by promoting degradation of pgRNA. Transcriptome analyses of primary human hepatocytes overexpressing N4BP1 suggested that N4BP1 may have anti-HBV activity independent of other host factors. Conclusions In summary, N4BP1 was found to be a novel anti-HBV factor. N4BP1 inhibited HBV replication by promoting pgRNA degradation.
Project description:The nuclear export of HBV RNAs allows the virus to synthesize its proteins through the translation machinery and replicate its genome through reverse transcription in the cytoplasm. However, the molecular mechanisms underlying this important process remain largely obscure. To illustrate this process, we took advantage of an unbiased HBV RNA-host protein interaction screen using a quantitative proteomics approach and identified embryonic lethal, abnormal vision, Drosophila-like 1 (ELAVL1) as a viral RNA binding partner. RNA scope and subcellular mRNA assays indicated that genetic and pharmaceutic inhibition of ELAVL1 inhibits HBV RNA nuclear export and suppresses viral replication in cell cultures. The observations of an HBV replication mouse model with ELAVL1 displayed similar results. RNA pulldown and RNA electrophoretic mobility shift assays revealed direct interaction between ELAVL1 and HBV pgRNA and confirmed AU-rich elements as the binding sites through site-directed mutagenesis of pgRNA. RNA-immunoprecipitation revealed that HBV RNAs associate with ELAVL1, which in turn binds to acidic leucine-rich nuclear phosphoprotein 32 family member A (ANP32A) and ANP32B. These interactions subsequently recruit CRM1. A nuclear RNase-targeted siRNA screen uncovered RNA exosome-mediated degradation of retardant HBV RNA after ELAVL1 or CRM1 knockdown. Further investigation revealed that ELAVL1 protects pgRNA from degradation. Notably, HBc deletion had no effect on pgRNA-CRM1 interaction and HBV RNA nuclear export. In summary, our work indicates that ELAVL1 functions both in HBV RNA stability and nucleocytoplasmic transport via the CRM1 nuclear export pathway.
Project description:N6-methyladenosine (m6A) RNA methylation is the most abundant epitranscriptomic modification of eukaryotic messenger RNAs (mRNAs). Previous reports have found m6A on both cellular and viral transcripts and defined its role in regulating numerous biological processes, including viral infection. Here, we show that m6A and its associated machinery regulate the life cycle of hepatitis B virus (HBV). HBV is a DNA virus that completes its life cycle via an RNA intermediate, termed pregenomic RNA (pgRNA). Silencing of enzymes that catalyze the addition of m6A to RNA resulted in increased HBV protein expression, but overall reduced reverse transcription of the pgRNA. We mapped the m6A site in the HBV RNA and found that a conserved m6A consensus motif situated within epsilon (stem loop structure is the site for m6A modification. The epsilon stem loop is located in the 3’ terminus of all HBV mRNAs and both termini (5’ and 3’) of the pgRNA. Mutational analysis of the identified m6A site in the 5’ epsilon stem loop of pgRNA revealed that m6A at this site is required for efficient reverse transcription of pgRNA, while m6A of the 3’ epsilon stem loop results in destabilization of all HBV transcripts, suggesting that m6A has dual regulatory functions in pgRNA. Overall, this study reveals how m6A regulates HBV gene expression and reverse transcription, leading to a new level of understanding of the HBV life cycle. .
Project description:We analyzed three clinical parameters with gene expression data from 122 liver tissues. Six healthy samples were used in validation. All hepatitis samples were HBV infected, which was validated by positive HBsAg or serum HBV-DNA. The samples with HCV infection or metabolic liver injury (e.g. fatty liver, chronic alcoholic hepatitis, etc.) were excluded. This dataset is part of the TransQST collection.
Project description:To determine host factors that regulate HBV, cells positive for and negative for HBV were compared. We identified three transcription regulators (HNF4A1, HNFA2, HNFA3) that support HBV replication
Project description:Purpose: Aberrantly methylated DNA are hallmarks for many cancers, HCC included. Tumor shed its DNA into circulation stream, and serum DNA methylation analysis is a less-invasive and accessable way to judge the primary tumor status. The goals of this study are to compare DNA methylation profiling in serum cell-free DNA from different stages of HCC progression including healthy control, chronic HBV carrier, HBV-related liver Cirrhosis and HCC, to establish HCC development-related aberrnat DNA methylation patterns. Methods: MBD methylCap/seq was carried out to screen differentially methylated CpG islands in serum cell-free DNA on four different stage of HBV-related HCC development. MSP and multiplex-BSP validation was performed using independent serum DNA or tumor and adjacent tissues. Results: Using a MBD methylCap/seq platform, we produced 33- to 37- million raw reads per sample and mapped them, in about half of the raw reads, to human genome(build h19) in the serum cf DNA of healthy control, HBV carrier, HBV cirrhosis and HCC. The mapped reads formed 180k to 260k peaks per sample, with 160 k common peaks shared by four samples. After subtraction of the common peaks, there left 51k, 107k and 78 k DMRs representing hypermethylations, in HBV carrier, HBV cirrhosis and HCC, respectively. We define those DMRs as early, middle and late when these DMRS occurred and maintained in HBV carrier, HBV cirrhosis and HCC, which including 27k, 24k and 19k DMRs, corresponding to 1,416, 1,337, 1,006 genes. GO analysis of them revealed gene categories and pathways associated with tumorogenenisis related process Conclusions: Our study represents the first detailed analysis of serum cf-DNA methylation profiling in the progression of HBV related HCC development. The processed data analysis here offers a comprehensive evaluation of DNA methylation in serum cf DNA. We conclude that MBD methylCap/seq based methylation profiling would benefit epigenetic research in HCC.
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.
Project description:RNA chemical modifications have been found to play important biological functions. Among which, the 5-methylcytosine (m5C) modification has been reported to participate in viral replication through affecting RNA processing, such as export, decay, translation and so on. In this study, we performed bisulfite sequencing (BS-seq) on HBV 1.1-mer-transfected huh7 cells to identify the m5C sites in HBV mRNA and their function in virus replication was verified. To investigate the mechanism by which m5C methyltransferase NSUN2 suppresses HBV replication, altered global m5C levels in host genes in HBV 1.1-mer-transfected cells were examined by BS-seq. We found that the m5C modification of genes associated with antiviral immunity changed significantly after viral infection. Our study provide new molecular insights into the mechanism of HBV-mediated IFN inhibition