Project description:Hepatitis C virus (HCV) infection constitutes a global health problem with 71 million people currently chronically infected. Recent studies have reported that C19orf66 is expressed as an interferon (IFN)-stimulated gene; however, the intrinsic regulation of this gene within the liver as well as its antiviral effects against HCV remains elusive. In this study, we observed an upregulation of C19orf66 in vivo and ex vivo in response to HCV infection and to IFN therapy. Expression of C19orf66 restricted HCV infection, whereas CRIPSPR/Cas9 mediated knockout of C19orf66 attenuated IFN-mediated suppression of HCV replication. Co-immunoprecipitation followed by mass spectrometry identified a stress granule dominated interactome of C19orf66. Mechanistic studies revealed that C19orf66 expression impairs HCV-induced elevation of PI(4)P and alters the morphology of the viral replication organelle, designated membranous web, thus suppressing viral RNA replication. Collectively, our data suggest that C19orf66 contributes to the innate immune response against HCV in the liver.
Project description:MX1 is a well-characterized interferon-induced antiviral gene. MX1 is activated by viral infection due to interferon production in cells. We treated non-permissive Huh7 cells and permissive HRP4 cells with interferon. We compared the expression of genes induced by interferon to determine host factors affecting HCV replication.
Project description:MX1 is a well-characterized interferon-induced antiviral gene. MX1 is activated by viral infection due to interferon production in cells. We treated non-permissive Huh7 cells and permissive HRP4 cells with interferon. We compared the expression of genes induced by interferon to determine host factors affecting HCV replication. Huh7 cells and HRP4 cells were treated with 40U/ml interferon-α for 6h. RNA was extracted and hybridized on Affymetrix microarrays
Project description:Although treatment of chronic hepatitis C virus (HCV) infection with direct acting antivirals (DAAs) results in high rates of cure, liver fibrosis does not resolve immediately after HCV eradication. Resolution of fibrosis occurs in some, but not all patients, after HCV cure, and hepatic decompensation and hepatocellular carcinoma can still occur in patients with pre-existing cirrhosis. We hypothesized that evaluation of the host liver proteome in the context of HCV treatment would provide insight into how inflammatory and fibrinogenic pathways change upon HCV eradication. We evaluated the whole liver proteome and phosphoproteome using paired liver biopsies from 8 HCV-infected patients collected before or immediately after treatment with DAAs in clinical trials. We identify interferon stimulated proteins as the predominant pathways that decrease with HCV treatment, which is consistent with previous analyses of the liver transcriptome during DAA therapy. While there was no change in the proteome of pathways associated with liver fibrosis, we identified a decrease in the phosphoproteome signature for ERK1/ERK2 as a result of HCV treatment. Conclusion: There is a reduction in the endogenous interferon-mediated antiviral response and alterations in the phosphoproteome that may precede resolution of fibrosis in the liver immediately after treatment of HCV with DAAs.
Project description:The liver-specific microRNA, miR-122, is an essential host factor for replication of hepatitis C virus (HCV), an important infectious cause of chronic liver disease and hepatocellular carcinoma. miR-122 stabilizes the positive-strand HCV RNA genome and promotes viral RNA synthesis by binding two closely spaced sites (S1 and S2) near the 5’ end of the genome in association with Ago2. Ago2 is essential for both host factor activities, but whether other host proteins are involved is unknown. Using a quantitative proteomics approach, we identified TNRC6A (GW182) and its paralogs (TNRC6B and TNRC6C), as functionally important components of the miR-122/Ago2 host factor complex binding HCV RNA. Depletion of any two TNRC6 proteins reduced HCV replication in Huh-7.5 cells,but did not reduce viral RNA stability or translational activity, but rather dampened miR-122 stimulation of viral RNA synthesis. However, TNRC6 depletion had no effect on replication of HCV in which S2 was mutated so that miR-122 binds only S1, whereas it significantly enhanced replication when S1 was mutated and only S2 bound by miR-122. Consistent with this, we found that TNRC6 proteins preferentially associate with the S1 site, and that the association of Ago2 with S2 is increased in TNRC6-depleted cells. Collectively, these data suggest a model in which TNRC6 proteins, which are known to interact with Ago2, preferentially direct the miR-122/Ago2 complex to S1 while restricting its association with S2, thereby fine tuning the spatial organization of miR-122/Ago2 complexes bound to the viral RNA.
Project description:Chronic infection with HCV is manifested by dysregulation of innate immune responses and impaired T cell function at multiple levels. These changes may impact susceptibility to other infections, responsiveness to antiviral therapies, vaccine responsiveness, and development of complications such as hepatocellular carcinoma. Highly effective direct-acting antiviral (DAA) therapy has revolutionized the management of chronic HCV, with expected cure rates exceeding 95%. DAA treatment represents a unique opportunity to investigate to what extent elimination of viral replication and chronic antigen stimulation can restore immunologic phenotype. In this study we interrogated the global transcriptional profile of isolated peripheral blood T cells before, during and after IFN-free DAA therapy using single cell mRNA sequencing. Our results demonstrate that T cells mapped at single-cell resolution have dramatic transcriptomic changes early after initiation of DAA and many of these changes are sustained after completion of DAA therapy. Specifically, we see a significant reduction in transcripts associated with innate immune activation and interferon signaling such as ISG15, ISG20, IFIT3, OAS and MX1 in many different T cell subsets. Furthermore, we find an early upregulation of a gene involved in suppression of immune activation, DUSP1, in circulating T cells. Conclusion: This study provides the first in-depth transcriptomic analysis at the single-cell level of patients undergoing DAA therapy, demonstrating that IFN-free antiviral therapy in chronic HCV infection induces hitherto unrecognized shifts in innate immune and interferon signaling within T cell populations early, during, and long-term after treatment. The present study provides a rich data source to explore the effects of DAA treatment on bulk T cells.
Project description:Human hepatocyte chimeric mice were prepared and treated with hepatitis C virus (HCV) and/or interferon-alpha (IFN-α). To analyze the changes in gene expression, cDNA microarray analysis was performed with the collected human hepatocytes from the chimeric mouse livers. We consider that these results provide molecular insights into possible mechanisms used by HCV to evade innate immune responses, as well as novel therapeutic targets and a potential new indication for interferon therapy.
Project description:While hundreds of genes are induced by Type I interferons, their roles in restricting the influenza life cycle remain mostly unknown. Using a loss-of-function CRISPR screen in cells pre-stimulated with Type I interferon, we identified a small number of factors required for restricting influenza A virus replication. In addition to the known components of the interferon signaling pathway, we found a new factor, Replication Termination Factor 2 (RTF2). RTF2 restricts influenza, at least, at the nuclear stage of the viral life cycle based on several lines of evidence. First, a deficiency in RTF2 leads to higher levels of viral primary transcription, even in the presence of cycloheximide to block genome replication and secondary transcription. Second, cells that lack RTF2 have enhanced activity of a viral reporter that depends solely on four viral proteins that carry out replication and transcription in the nucleus. Third, when RTF2 protein is mislocalized outside the nucleus, it is not able to restrict replication. Furthermore, the absence of RTF2 not only led to enhanced viral transcription but also to reduced expression of anti-viral factors in response to interferon. RTF2 thus inhibits primary influenza transcription, likely acts in the nucleus, and contributes to upregulation of antiviral effectors in response to Type I interferons
Project description:Transcriptional profiling provides global snapshots of virus-mediated cellular reprogramming, which can simultaneously encompass pro- and antiviral components. To determine early transcriptional signatures associated with HCV infection of authentic target cells, we performed ex vivo infections of adult primary human hepatocytes (PHHs) from seven donors. Coordinated sampling identified minimal gene dysregulation at six hours post infection (hpi) in PHHs. In contrast, at 72 hpi, massive increases in the breadth and magnitude of HCV-induced gene dysregulation were apparent, affecting gene classes associated with diverse biological processes. Comparison with HCV-induced transcriptional dysregulation in Huh-7.5 cells identified limited overlap between the two systems. Of note, in PHHs, HCV infection initiated broad upregulation of canonical interferon (IFN)-mediated defense programs, limiting viral RNA replication and abrogating virion release. In addition, we confirm that constitutive expression of IRF1 in PHHs maintains a steady-state antiviral program in the absence of infection which can further reduce HCV RNA replication. We also detected infection-induced signatures of translational shutoff in PHHs - downregulation of ~90 genes encoding components of the EIF2 translation initiation complex and ribosomal subunits. As HCV polyprotein translation occurs independently of the EIF2 complex, this process is pro-viral: only translation initiation of host transcripts is arrested. The combination of antiviral intrinsic and inducible immunity, balanced against pro-viral programs, including translational arrest, maintains HCV replication at a low-level in PHHs. This may ultimately keep HCV under the radar of extra-hepatocyte immune surveillance while initial infection is established, promoting tolerance, preventing clearance and facilitating progression to chronicity.
Project description:This study characterizes the effects of chronic Hepatitis C virus (HCV) infection on gene expression by analyzing blood samples from 10 treatment-naive HCV patients and 6 healthy volunteers. Differential expression analysis of microarray data from peripheral blood mononuclear cells (PBMCs) identified a 136 gene signature, including 66 genes elevated in infected individuals. Most of the up-regulated genes were associated with interferon (IFN) activity (including members of the OAS and MX families, ISG15 and IRF7), suggesting an ongoing immune response. This HCV signature was also found to be consistently enriched in many other viral infection and vaccination datasets. Validation of these genes was carried out using a second cohort composed of 5 HCV patients and 5 healthy volunteers, confirming the up-regulation of the IFN signature. In summary, this is the first study to directly compare blood transcriptional profiles from HCV patients with healthy controls. The results show that chronic HCV infection has a pronounced effect on gene expression in PBMCs of infected individuals, and significantly elevates the expression of a subset of interferon-stimulated genes.