Promotion of hepatocellular carcinoma by hepatitis C virus

Persistent infection with the hepatitis C virus (HCV) is a major global health problem. Around 2-3% of the world’s population are chronically infected, and infected individuals are at high risk of developing steatosis, fibrosis, and liver cirrhosis. The latter is a major predisposing factor for the development of hepatocellular carcinoma (HCC). It is generally accepted that an inflammatory response triggered by persistent HCV infection leads to increased cell proliferation and fibrogenesis that in turn promotes cirrhosis and ultimately HCC development. This indirect mechanism of tumor induction would explain the long incubation period from primary HCV infection to HCC and the requirement for additional cofactors such as toxins or drugs (most notably alcohol), metabolic liver diseases, steatosis, nonalcoholic liver disease, or diabetes. With the advent of adequate cell culture systems for HCV it is, however, becoming increasingly clear that the virus also contributes directly to HCC formation. Examples are the continuous induction of stress response or the massive accumulation of intracellular lipids. Moreover, viral proteins can bind to and sequester cell cycle control factors such as the retinoblastoma protein or the tumor suppressor DDX3. Thus, HCV-associated liver cancer is most likely promoted by the combined action of long-term chronic inflammation and targeted perturbations of cellular key pathways involved in metabolic homeostasis as well as cell cycle control. Copyright (c) 2012 S. Karger AG, Base

Hepatitis C Virus p7 Protein Is Crucial for Assembly and Release of Infectious Virions

Hepatitis C virus (HCV) infection is associated with chronic liver disease and currently affects about 3% of the world population. Although much has been learned about the function of individual viral proteins, the role of the HCV p7 protein in virus replication is not known. Recent data, however, suggest that it forms ion channels that may be targeted by antiviral compounds. Moreover, this protein was shown to be essential for infectivity in chimpanzee. Employing the novel HCV infection system and using a genetic approach to investigate the function of p7 in the viral replication cycle, we find that this protein is essential for efficient assembly and release of infectious virions across divergent virus strains. We show that p7 promotes virus particle production in a genotype-specific manner most likely due to interactions with other viral factors. Virus entry, on the other hand, is largely independent of p7, as the specific infectivity of released virions with a defect in p7 was not affected. Together, these observations indicate that p7 is primarily involved in the late phase of the HCV replication cycle. Finally, we note that p7 variants from different isolates deviate substantially in their capacity to promote virus production, suggesting that p7 is an important virulence factor that may modulate fitness and in turn virus persistence and pathogenesis

A Coupled Mathematical Model of the Intracellular Replication of Dengue Virus and the Host Cell Immune Response to Infection

Dengue virus (DV) is a positive-strand RNA virus of the Flavivirus genus. It is one of the most prevalent mosquito-borne viruses, infecting globally 390 million individuals per year. The clinical spectrum of DV infection ranges from an asymptomatic course to severe complications such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), the latter because of severe plasma leakage. Given that the outcome of infection is likely determined by the kinetics of viral replication and the antiviral host cell immune response (HIR) it is of importance to understand the interaction between these two parameters. In this study, we use mathematical modeling to characterize and understand the complex interplay between intracellular DV replication and the host cells' defense mechanisms. We first measured viral RNA, viral protein, and virus particle production in Huh7 cells, which exhibit a notoriously weak intrinsic antiviral response. Based on these measurements, we developed a detailed intracellular DV replication model. We then measured replication in IFN competent A549 cells and used this data to couple the replication model with a model describing IFN activation and production of IFN stimulated genes (ISGs), as well as their interplay with DV replication. By comparing the cell line specific DV replication, we found that host factors involved in replication complex formation and virus particle production are crucial for replication efficiency. Regarding possible modes of action of the HIR, our model fits suggest that the HIR mainly affects DV RNA translation initiation, cytosolic DV RNA degradation, and naïve cell infection. We further analyzed the potential of direct acting antiviral drugs targeting different processes of the DV lifecycle in silico and found that targeting RNA synthesis and virus assembly and release are the most promising anti-DV drug targets

SARS-CoV-2 nsp3 and nsp4 are minimal constituents of a pore spanning replication organelle

Funding Information: We thank the Infectious Diseases Imaging Platform (IDIP) at the Center for Integrative Infectious Disease Research Heidelberg, the cryo-EM network at the Heidelberg University (HD-cryoNET) and Heidelberg University Electron Microscopy Core Facility for support and assistance. The authors gratefully acknowledge the data storage service SDS@hd supported by the Ministry of Science, Research, and the Arts Baden-Württemberg (MWK), the German Research Foundation (DFG) through grant INST 35/1314-1 FUGG and INST 35/1503-1 FUGG. This work was supported by a research grant from the Chica and Heinz Schaller Foundation (Schaller Research Group Leader Program). Work of P.C. and R.B. is supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) project no. 240245660–SFB1129 and project number 437060729 (PC and MWM). In addition, work of R.B. is supported by DFG project no. 272983813–TRR 179) and by the project “Virological and immunological determinants of COVID-19 pathogenesis – lessons to get prepared for future pandemics (KA1-Co-02 “COVIPA”)”, a grant from the Helmholtz Association’s Initiative and Networking Fund. V.P. is supported by a European Molecular Biology Organization (EMBO) long-term fellowship (ALTF454-2020). LZ and JM are supported by CoVLP project of the Flagship Initiative Engineering Molecular Systems. ZH received support for this work from FCT - Fundação para a Ciência e a Tecnologia, I.P., through MOSTMICRO-ITQB R&D Unit (UIDB/04612/2020, 510 UIDP/04612/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020) and from a joint research agreement with the Okinawa Institute of Science and Technology. Funding Information: We thank the Infectious Diseases Imaging Platform (IDIP) at the Center for Integrative Infectious Disease Research Heidelberg, the cryo-EM network at the Heidelberg University (HD-cryoNET) and Heidelberg University Electron Microscopy Core Facility for support and assistance. The authors gratefully acknowledge the data storage service SDS@hd supported by the Ministry of Science, Research, and the Arts Baden-Württemberg (MWK), the German Research Foundation (DFG) through grant INST 35/1314-1 FUGG and INST 35/1503-1 FUGG. This work was supported by a research grant from the Chica and Heinz Schaller Foundation (Schaller Research Group Leader Program). Work of P.C. and R.B. is supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) project no. 240245660–SFB1129 and project number 437060729 (PC and MWM). In addition, work of R.B. is supported by DFG project no. 272983813–TRR 179) and by the project “Virological and immunological determinants of COVID-19 pathogenesis – lessons to get prepared for future pandemics (KA1-Co-02 “COVIPA”)”, a grant from the Helmholtz Association’s Initiative and Networking Fund. V.P. is supported by a European Molecular Biology Organization (EMBO) long-term fellowship (ALTF454-2020). LZ and JM are supported by CoVLP project of the Flagship Initiative Engineering Molecular Systems. ZH received support for this work from FCT - Fundação para a Ciência e a Tecnologia, I.P., through MOSTMICRO-ITQB R&D Unit (UIDB/04612/2020, 510 UIDP/04612/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020) and from a joint research agreement with the Okinawa Institute of Science and Technology. Publisher Copyright: © 2023, The Author(s).Coronavirus replication is associated with the remodeling of cellular membranes, resulting in the formation of double-membrane vesicles (DMVs). A DMV-spanning pore was identified as a putative portal for viral RNA. However, the exact components and the structure of the SARS-CoV-2 DMV pore remain to be determined. Here, we investigate the structure of the DMV pore by in situ cryo-electron tomography combined with subtomogram averaging. We identify non-structural protein (nsp) 3 and 4 as minimal components required for the formation of a DMV-spanning pore, which is dependent on nsp3-4 proteolytic cleavage. In addition, we show that Mac2-Mac3-DPUP-Ubl2 domains are critical for nsp3 oligomerization and crown integrity which influences membrane curvature required for biogenesis of DMVs. Altogether, SARS-CoV-2 nsp3-4 have a dual role by driving the biogenesis of replication organelles and assembly of DMV-spanning pores which we propose here to term replicopores.publishersversionpublishe

Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication

All positive strand RNA viruses are known to replicate their genomes in close association with intracellular membranes. In case of the hepatitis C virus (HCV), a member of the family Flaviviridae, infected cells contain accumulations of vesicles forming a membranous web (MW) that is thought to be the site of viral RNA replication. However, little is known about the biogenesis and three-dimensional structure of the MW. In this study we used a combination of immunofluorescence- and electron microscopy (EM)-based methods to analyze the membranous structures induced by HCV in infected cells. We found that the MW is derived primarily from the endoplasmic reticulum (ER) and contains markers of rough ER as well as markers of early and late endosomes, COP vesicles, mitochondria and lipid droplets (LDs). The main constituents of the MW are single and double membrane vesicles (DMVs). The latter predominate and the kinetic of their appearance correlates with kinetics of viral RNA replication. DMVs are induced primarily by NS5A whereas NS4B induces single membrane vesicles arguing that MW formation requires the concerted action of several HCV replicase proteins. Three-dimensional reconstructions identify DMVs as protrusions from the ER membrane into the cytosol, frequently connected to the ER membrane via a neck-like structure. In addition, late in infection multi-membrane vesicles become evident, presumably as a result of a stress-induced reaction. Thus, the morphology of the membranous rearrangements induced in HCV-infected cells resemble those of the unrelated picorna-, corona- and arteriviruses, but are clearly distinct from those of the closely related flaviviruses. These results reveal unexpected similarities between HCV and distantly related positive-strand RNA viruses presumably reflecting similarities in cellular pathways exploited by these viruses to establish their membranous replication factories

Type I and type II interferon responses in two human liver cell lines (Huh-7 and HuH6)

AbstractMost studies investigating the biology of Hepatitis C virus (HCV) have used the human hepatoma cell line Huh-7 or subclones thereof, as these are the most permissive cell lines for HCV infection and replication. Other cell lines also support replication of HCV, most notably the human hepatoblastoma cell line HuH6. HCV replication in cell culture is generally highly sensitive to interferons (IFNs) and differences in the IFN-mediated inhibition of virus replication may reflect alterations in the IFN-induced antiviral response inherent to different host cells. For example, HCV replication is highly sensitive to IFN-γ treatment in Huh-7, but not in HuH6 cells. In this study, we used microarray-based gene expression profiling to compare the response of Huh-7 and HuH6 cells to stimulation with IFN-α and IFN-γ. Furthermore, we determined whether the resistance of HCV replication in HuH6 cells can be linked to differences in the expression profile of IFN-regulated genes. Although both cells lines responded to IFNs with rapid changes in gene expression, thereby demonstrating functional type I and type II signaling pathways, differences were observed for a number of genes. Raw and normalized expression data have been deposited in GEO under accession number GSE68927

Dynamics of torque teno virus load in kidney transplant recipients with indication biopsy and therapeutic modifications of immunosuppression

Following kidney transplantation, lifelong immunosuppressive therapy is essential to prevent graft rejection. On the downside, immunosuppression increases the risk of severe infections, a major cause of death among kidney transplant recipients (KTRs). To improve post-transplant outcomes, adequate immunosuppressive therapy is therefore a challenging but vital aspect of clinical practice. Torque teno virus load (TTVL) was shown to reflect immune competence in KTRs, with low TTVL linked to an elevated risk for rejections and high TTVL associated with infections in the first year post-transplantation. Yet, little is known about the dynamics of TTVL after the first year following transplantation and how TTVL changes with respect to short-term modifications in immunosuppressive therapy. Therefore, we quantified TTVL in 106 KTRs with 108 clinically indicated biopsies, including 65 biopsies performed >12 months post-transplantation, and correlated TTVL to histopathology. In addition, TTVL was quantified at 7, 30, and 90 days post-biopsy to evaluate how TTVL was affected by changes in immunosuppression resulting from interventions based on histopathological reporting. TTVL was highest in patients biopsied between 1 and 12 months post-transplantation (N = 23, median 2.98 × 107 c/mL) compared with those biopsied within 30 days (N = 20, median 7.35 × 103 c/mL) and > 1 year post-transplantation (N = 65, median 1.41 × 104 c/mL; p < 0.001 for both). Patients with BK virus-associated nephropathy (BKVAN) had significantly higher TTVL than patients with rejection (p < 0.01) or other pathologies (p < 0.001). When converted from mycophenolic acid to a mTOR inhibitor following the diagnosis of BKVAN, TTVL decreased significantly between biopsy and 30 and 90 days post-biopsy (p < 0.01 for both). In KTR with high-dose corticosteroid pulse therapy for rejection, TTVL increased significantly between biopsy and 30 and 90 days post-biopsy (p < 0.05 and p < 0.01, respectively). Of note, no significant changes were seen in TTVL within 7 days of changes in immunosuppressive therapy. Additionally, TTVL varied considerably with time since transplantation and among individuals, with a significant influence of age and BMI on TTVL (p < 0.05 for all). In conclusion, our findings indicate that TTVL reflects changes in immunosuppressive therapy, even in the later stages of post-transplantation. To guide immunosuppressive therapy based on TTVL, one should consider inter- and intraindividual variations, as well as potential confounding factors

Generation of T-cell receptors targeting a genetically stable and immunodominant cytotoxic T-lymphocyte epitope within hepatitis C virus non-structural protein 3

Hepatitis C virus (HCV) is a major cause of severe liver disease, and one major contributing factor is thought to involve a dysfunction of virus-specific T-cells. T-cell receptor (TCR) gene therapy with HCV-specific TCRs would increase the number of effector T-cells to promote virus clearance. We therefore took advantage of HLA-A2 transgenic mice to generate multiple TCR candidates against HCV using DNA vaccination followed by generation of stable T-cell–BW (T-BW) tumour hybrid cells. Using this approach, large numbers of non-structural protein 3 (NS3)-specific functional T-BW hybrids can be generated efficiently. These predominantly target the genetically stable HCV genotype 1 NS31073–1081 CTL epitope, frequently associated with clearance of HCV in humans. These T-BW hybrid clones recognized the NS31073 peptide with a high avidity. The hybridoma effectively recognized virus variants and targeted cells with low HLA-A2 expression, which has not been reported previously. Importantly, high-avidity murine TCRs effectively redirected human non-HCV-specific T-lymphocytes to recognize human hepatoma cells with HCV RNA replication driven by a subgenomic HCV replicon. Taken together, TCR candidates with a range of functional avidities, which can be used to study immune recognition of HCV-positive targets, have been generated. This has implications for TCR-related immunotherapy against HCV

Normalizing for individual cell population context in the analysis of high-content cellular screens

<p>Abstract</p> <p>Background</p> <p>High-content, high-throughput RNA interference (RNAi) offers unprecedented possibilities to elucidate gene function and involvement in biological processes. Microscopy based screening allows phenotypic observations at the level of individual cells. It was recently shown that a cell's population context significantly influences results. However, standard analysis methods for cellular screens do not currently take individual cell data into account unless this is important for the phenotype of interest, i.e. when studying cell morphology.</p> <p>Results</p> <p>We present a method that normalizes and statistically scores microscopy based RNAi screens, exploiting individual cell information of hundreds of cells per knockdown. Each cell's individual population context is employed in normalization. We present results on two infection screens for hepatitis C and dengue virus, both showing considerable effects on observed phenotypes due to population context. In addition, we show on a non-virus screen that these effects can be found also in RNAi data in the absence of any virus. Using our approach to normalize against these effects we achieve improved performance in comparison to an analysis without this normalization and hit scoring strategy. Furthermore, our approach results in the identification of considerably more significantly enriched pathways in hepatitis C virus replication than using a standard analysis approach.</p> <p>Conclusions</p> <p>Using a cell-based analysis and normalization for population context, we achieve improved sensitivity and specificity not only on a individual protein level, but especially also on a pathway level. This leads to the identification of new host dependency factors of the hepatitis C and dengue viruses and higher reproducibility of results.</p