Dataset Information

Label-free Proteomic Analysis of Exosomes Derived from Inducible Hepatitis B Virus-Replicating HepAD38 Cell Line

ABSTRACT: Hepatitis B virus (HBV) infection is a major health problem worldwide. Recent evidence suggests that various viruses can manipulate the infection process by secretion of specific viral and cellular components into exosomes, small nanometer-sized (30-150 nm) vesicles secreted from various cells. However, the impact of HBV replication on hepatocytes produced exosomes has not been fully delineated. In this work, an HBV-inducible cell line HepAD38 was used to directly compare changes in the protein content of exosomes secreted from HepAD38 cells with or without HBV replication. Exosomes were isolated from conditioned medium of HepAD38 cell cultures and the purity of exosomes were confirmed by transmission electron microscopy (TEM) and Western immunoblotting assays. Ion-intensity based label-free LC−MS/MS quantitation technologies were applied to analyze protein content of HBV-exosomes and HBV-free-exosomes. A total of 1412 exosomal proteins were identified, in which the abundance of 35 proteins were significantly altered. Strikingly, 5 subunit proteins from the 26S proteasome complex, including PSMC1, PSMC2, PSMD1, PSMD7 and PSMD14 were consistently enhanced in HBV-exosomes. Bioinformatic analysis of differential exosomal proteins revealed the significant enrichment of components involved in proteasomal catabolic process. Proteasome activity assays further suggested that HBV-exosomes had enhanced proteolytic activity compared to HBV-free-exosomes. Furthermore, Human peripheral monocytes incubated with HBV-exosomes induced a significant lower level of IL-6 secretion compared to HBV-free-exosomes. Irreversible inhibition of proteasomal activity within exosome restored the IL-6 induction in monocytes. These results suggest that transmission of proteasome subunit proteins by HBV-exosomes might modulate the innate sensing of pro-inflammatory molecules in the recipient monocytes. These results revealed the composition and potential function of exosomes produced during HBV replication, thus provide a new perspective on the role of exosomes in HBV-host interaction.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Xiaofang Jia

LAB HEAD: Zhenghong Yuan

PROVIDER: PXD004724 | Pride | 2017-03-01

REPOSITORIES: Pride

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Publications

Label-free Proteomic Analysis of Exosomes Derived from Inducible Hepatitis B Virus-Replicating HepAD38 Cell Line.

Jia Xiaofang X Chen Jieliang J Megger Dominik A DA Zhang Xiaonan X Kozlowski Maya M Zhang Lijun L Fang Zhong Z Li Jin J Chu Qiaofang Q Wu Min M Li Yaming Y Sitek Barbara B Yuan Zhenghong Z

Molecular & cellular proteomics : MCP 20170227 4 suppl 1

Hepatitis B virus (HBV) infection is a major health problem worldwide. Recent evidence suggests that some viruses can manipulate the infection process by packing specific viral and cellular components into exosomes, small nanometer-sized (30-150 nm) vesicles secreted from various cells. However, the impact of HBV replication on the content of exosomes produced by hepatocytes has not been fully delineated. In this work, an HBV-inducible cell line HepAD38 was used to directly compare changes in th ...[more]

PMID: 28242843

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Project description:Hepatitis B virus (HBV) infection is a major health problem worldwide. Recent evidence suggests that various viruses can manipulate the infection process by secretion of specific viral and cellular components into exosomes, small nanometer-sized (30-150 nm) vesicles secreted from various cells. However, the impact of HBV replication on hepatocytes produced exosomes has not been fully delineated. In this work, an HBV-inducible cell line HepAD38 was used to directly compare changes in the protein content of exosomes secreted from HepAD38 cells with or without HBV replication. Exosomes were isolated from conditioned medium of HepAD38 cell cultures and the purity of exosomes were confirmed by transmission electron microscopy (TEM) and Western immunoblotting assays. Ion-intensity based label-free LC?MS/MS quantitation technologies were applied to analyze protein content of HBV-exosomes and HBV-free-exosomes. A total of 1412 exosomal proteins were identified, in which the abundance of 35 proteins were significantly altered. Strikingly, 5 subunit proteins from the 26S proteasome complex, including PSMC1, PSMC2, PSMD1, PSMD7 and PSMD14 were consistently enhanced in HBV-exosomes. Bioinformatic analysis of differential exosomal proteins revealed the significant enrichment of components involved in proteasomal catabolic process. Proteasome activity assays further suggested that HBV-exosomes had enhanced proteolytic activity compared to HBV-free-exosomes. Furthermore, Human peripheral monocytes incubated with HBV-exosomes induced a significant lower level of IL-6 secretion compared to HBV-free-exosomes. Irreversible inhibition of proteasomal activity within exosome restored the IL-6 induction in monocytes. These results suggest that transmission of proteasome subunit proteins by HBV-exosomes might modulate the innate sensing of pro-inflammatory molecules in the recipient monocytes. These results revealed the composition and potential function of exosomes produced during HBV replication, thus provide a new perspective on the role of exosomes in HBV-host interaction.

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:The presentation of virus-derived peptides by HLA class I molecules on the surface of an infected cell and the recognition of these HLA-peptide complexes by, and subsequent activation of, CD8+ cytotoxic T cells provides an important mechanism for immune protection against viruses. Recent advances in proteogenomics have allowed researchers to discover a growing number of unique HLA-restricted viral peptides, resulting in a rapidly expanding repertoire of targets for immunotherapeutics (i.e. bispecific antibodies, engineered T-cell receptors (TCRs), chimeric antigen receptor T-cells (CAR-Ts)) to infected tissues. However, genomic variability between viral strains, such as Hepatitis-B virus (HBV), in combination with differences in patient HLA alleles, make it difficult to develop therapeutics against these targets. To address this challenge, we developed a novel proteogenomics approach for generating patient-specific databases that enable the identification of viral peptides based on the viral transcriptomes sequenced from individual patient liver samples. We also utilized DNA sequencing of patient samples to identify HLA genotypes and assist in target selection. Liver samples from 48 HBV infected patients, primarily from Asia, were examined to reconstruct patient-specific HBV genomes, identify regions within the human chromosomes targeted by HBV integrations and obtain a comprehensive view of HBV peptide epitopes using our HLA class-I (HLA-I) immunopeptidomics discovery platform. Two previously reported HLA associated HBV-derived peptides, HLA-A02 binder FLLTRILTI (S194-202) from the large surface antigen and HLA-A11 binder STLPETTVVRR (C141-151) from the capsid protein were validated by our discovery platform, but both were detected at a very low frequencies. In addition, we identified and validated, using heavy peptide analogues, novel strain-specific HBV-HLA associated peptides, such as GSLPQEHIVQK (P606-616) and variants. Overall, our novel approach can guide the development of bispecific antibody, TCR-T, or CAR-T based therapeutics for the treatment of HBV-related HCC and inform vaccine development.

Project description:Purpose: Chronic infection with hepatitis B virus is the leading global risk factor for the development of liver cancer. A large body of research has shown the many effects an HBV infection has on cellular physiology, particularly on pathways that may be involved in the development of HBV-associated diseases. Unfortunately, a significant portion of this research has been done in systems that may not mimic what is seen in a primary hepatocyte, and is not done on a transcriptome-wide scale. Because of this, we performed an RNA-seq analysis of primary rat hepatocytes either expressing HBV or not over a series of time points to determine the global changes HBV has on primary hepatocyte physiology. Methods: To do this RNA-seq analysis, triplicate samples of total RNA were collected from cultured primary rat hepatocytes (PRH) over the course of 72hr. PRH were collected immediately after isolation (0hr), or 24hr, 48hr, or 72hr after plating. In addition, PRH were infected 24hr after plating with adenovirus expressing GFP alone (AdGFP) or GFP along with a greater than unit length copy of the HBV genome (AdHBV) and collected at 48hr after plating (24hr after infection) or 72hr after plating (48hr after infection). cDNA libraries were sequenced using the Illumina NextSeq 500 platform to generate either 1x75bp reads. Reads were mapped using the STAR aligner, and output BAMs were further analyzed in R using the GenomicAlignments package, to quantify number of reads per transcript, and DESeq2, to determine differential expression. Reads per kilobase transcript per million total reads (RPKM) was calculated by dividing reads per transcript by the transcript length and then normalizing to the total number of reads in the sample. Results: Following this pipeline, we were able to identify a number of HBV-mediated differentially expressed transcripts at 48hr and 72hr. In addition, we noted considerable change to the hepatocyte transcriptome as a direct result of the isolation/plating procedure, regardless of the presence of HBV. Further pathway analysis of these differentially expressed transcripts identified many important cellular pathways, including those involved with cell cycle regulation and metabolism, as being differentially regulated by HBV in primary hepatocytes. mRNA profiles of cultured primary rat hepatocytes were generated, in triplicate, using the Illumina NextSeq 500 platform from freshly isolated cells (0hr), 24hr, 48hr, or 72hr after plating, and with or without expression of HBV 48hr or 72hr after plating.

Project description:Purpose: Chronic infection with hepatitis B virus is the leading global risk factor for the development of liver cancer. A large body of research has shown the many effects an HBV infection has on cellular physiology, particularly on pathways that may be involved in the development of HBV-associated diseases. Unfortunately, a significant portion of this research has been done in systems that may not mimic what is seen in a primary hepatocyte, and is not done on a transcriptome-wide scale. Because of this, we performed an RNA-seq analysis of primary rat hepatocytes expressing HBV to determine the global changes HBV has on primary hepatocyte physiology. Methods: To do this RNA-seq analysis, triplicate samples of total RNA were collected from cultured primary rat hepatocytes infected with adenovirus expressing GFP alone (AdGFP) or GFP along with a greater than unit length copy of the HBV genome (AdHBV). Samples were collected either 24h or 48h after infection. cDNA libraries were sequenced two times using the Illumina HiSeq or Illumina NextSeq platform to generate either 1x50bp or 1x75bp reads. Reads from each sequencing run were mapped using the STAR aligner, and output BAMs were merged into a single BAM for each sample. The merged BAM was further analyzed in R using the GenomicAlignments package to quantify number of reads per transcript and DESeq2 to determine differential expression. Reads per kilobase transcript per million total reads (RPKM) was calculated by dividing reads per transcript by the transcript length and then normalizing to the total number of reads in the sample. Results: Following this pipeline, we were able to identify a number of HBV-mediated differentially expressed transcripts at 24h and 48h post-infection. Further pathway analysis of these differentially expressed transcripts identified many important cellular pathways, including those involved with cell cycle regulation and metabolism, as being differentially regulated by HBV in primary hepatocytes. mRNA profiles of HBV-expressing and non-expressing primary rat hepatocytes were generated, in triplicate, 24h and 48h post-infection using Illumina HiSeq 2500 and NextSeq 500 instruments.

Dataset Information

Label-free Proteomic Analysis of Exosomes Derived from Inducible Hepatitis B Virus-Replicating HepAD38 Cell Line

Publications

Label-free Proteomic Analysis of Exosomes Derived from Inducible Hepatitis B Virus-Replicating HepAD38 Cell Line.

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