Monitoring the Antiviral Effect of Alpha Interferon on Individual Cells

Hepatitis C virus (HCV) particles are described as lipoviroparticles which are released similarly to very-low-density lipoproteins (VLDLs). However, the release mechanism is still poorly understood; the canonical endoplasmic reticulum-Golgi intermediate compartment (ERGIC) pathway as well as endosome-dependent release has been proposed. Recently, the role of exosomes in the transmission of HCV has been reported. Only a minor fraction of the de novo-synthesized lipoviroparticles is released by the infected cell. To investigate the relevance of multivesicular bodies (MVBs) for viral morphogenesis and release, the MVB inhibitor U18666A was used. Intracellular trafficking was analyzed by confocal microscopy and electron microscopy. Moreover, an mCherry-tagged HCV variant was used. Conditions were established that enable U18666A-dependent inhibition of MVBs without affecting viral replication. Under these conditions, significant inhibition of the HCV release was observed. The assembly of viral particles is not affected. In U18666A-treated cells, intact infectious viral particles accumulate in CD63-positive exosomal structures and large dysfunctional lysosomal structures (multilamellar bodies). These retained particles possess a lower density, reflecting a misloading with lipids. Our data indicate that at least a fraction of HCV particles leaves the cell via the endosomal pathway. Endosomes facilitate the sorting of HCV particles for release or degradation. IMPORTANCEThere are still a variety of open questions regarding morphogenesis and release of hepatitis C virus. The HCV-infected cell produces significant more viral particles that are released, raising the question about the fate of the nonreleased particles. Moreover, the relevance of the endosomal pathway for the release of HCV is under debate. Use of the MVB (multivesicular body) inhibitor U18666A enabled a detailed analysis of the impact of MVBs for viral morphogenesis and release. It was revealed that infectious, fully assembled HCV particles are either MVB-dependently released or intracellularly degraded by the lysosome. Our data indicate that at least a fraction of HCV particles leaves the cell via the endosomal pathway independent from the constitutive secretory pathway. Our study describes a so-far-unprecedented cross talk between two pathways regulating on the one hand the release of infectious viral particles and on the other hand the intracellular degradation of nonreleased particles. Hepatitis C virus (HCV) is still a major health problem, as approximately 170 million people are chronically infected with HCV worldwide (1). Chronic infection can cause liver cirrhosis and hepatocellular carcinoma, finally leading to death (2).HCV belongs to the Flaviviridae family. Flaviviridae have a single-stranded positive-sense RNA genome (3). The HCV RNA encompasses about 9,600 bases and is translated into a single polyprotein of about 3,100 amino acids, which is processed by viral and host proteases into the mature viral proteins (4). The members of the st...

Section: Time-and Dose-dependent Inhibition Of the Viral Replication mentioning

confidence: 99%

The Intracellular Cholesterol Transport Inhibitor U18666A Inhibits the Exosome-Dependent Release of Mature Hepatitis C Virus

Elgner

Ren

Medvedev

et al. 2016

“…The localization of NS5A and other viral components, especially core protein, on LDs is a prerequisite for the production of infectious HCV particles (38,41,42). Studies concerning the subcellular sites of viral RNA replication and virion morphogenesis have been reinforced by live cell imaging employing HCV genomes which express a functional NS5A-GFP fusion protein (34,(43)(44)(45)(46).…”

mentioning

confidence: 99%

Functional Characterization of Bovine Viral Diarrhea Virus Nonstructural Protein 5A by Reverse Genetic Analysis and Live Cell Imaging

Isken

Langerwisch

Schönherr

et al. 2014

Nonstructural protein 5A (NS5A) of bovine viral diarrhea virus (BVDV) is a hydrophilic phosphoprotein with RNA binding activity and a critical component of the viral replicase. In silico analysis suggests that NS5A encompasses three domains interconnected by two low-complexity sequences (LCSs). While domain I harbors two functional determinants, an N-terminal amphipathic helix important for membrane association, and a Zn-binding site essential for RNA replication, the structure and function of the C-terminal half of NS5A are still ill defined. In this study, we introduced a panel of 10 amino acid deletions covering the C-terminal half of NS5A. In the context of a highly efficient monocistronic replicon, deletions in LCS I and the N-terminal part of domain II, as well as in domain III, were tolerated with regard to RNA replication. When introduced into a bicistronic replicon, only deletions in LCS I and the N-terminal part of domain II were tolerated. In the context of the viral full-length genome, these mutations allowed residual virion morphogenesis. Based on these data, a functional monocistronic BVDV replicon coding for an NS5A variant with an insertion of the fluorescent protein mCherry was constructed. Live cell imaging demonstrated that a fraction of NS5A-mCherry localizes to the surface of lipid droplets. Taken together, this study provides novel insights into the functions of BVDV NS5A. Moreover, we established the first pestiviral replicon expressing fluorescent NS5A-mCherry to directly visualize functional viral replication complexes by live cell imaging.

“…First, the effect of an anti-HMGB1 antibody on HCV infection was monitored by adding the antibody to culture medium after HCV (JFH/5aRluc) infection (22). This virus contains the Renilla luciferase gene inserted into the NS5A region and allows the quantitative measurement of HCV infectivity (22). We measured luciferase activity in Huh7.5.1 cells at 3 dpi, a time that corresponds to the first and second rounds of HCV infection.…”

Section: Hcv Infection Promotes Nuclear-to-cytoplasmic Translocation mentioning

confidence: 99%

“…Because various cytokine functions of HMGB1 have been reported, we next investigated the effect of secreted HMGB1 on HCV infection. First, the effect of an anti-HMGB1 antibody on HCV infection was monitored by adding the antibody to culture medium after HCV (JFH/5aRluc) infection (22). This virus contains the Renilla luciferase gene inserted into the NS5A region and allows the quantitative measurement of HCV infectivity (22).…”

Section: Hcv Infection Promotes Nuclear-to-cytoplasmic Translocation mentioning

confidence: 99%

See 1 more Smart Citation

Hepatitis C Virus Infection Is Blocked by HMGB1 Released from Virus-Infected Cells

Jung¹,

Park²,

Jee³

et al. 2011

Self Cite

High-mobility group box 1 (HMGB1), an abundant nuclear protein that triggers host immune responses, is an endogenous danger signal involved in the pathogenesis of various infectious agents. However, its role in hepatitis C virus (HCV) infection is not known. Here, we show that HMGB1 protein is translocated from the nucleus to cytoplasm and subsequently is released into the extracellular milieu by HCV infection. Secreted HMGB1 triggers antiviral responses and blocks HCV infection, a mechanism that may limit HCV propagation in HCV patients. Secreted HMGB1 also may have a role in liver cirrhosis, which is a common comorbidity in HCV patients. Further investigations into the roles of HMGB1 in the diseases caused by HCV infection will shed light on and potentially help prevent these serious and prevalent HCV-related diseases.Hepatitis C virus (HCV) is one of the major causative agents of hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC) (17, 30). More than 170 million people are estimated to suffer from HCV infection worldwide (17). Chronic and persistent infection is a characteristic feature of HCV pathogenesis (30). During chronic infection, the production of virus particles is limited, and a restricted number of liver cells are infected. As a result, the viral dose in patients' blood generally is lower than that of other hepatitis-causing viruses, such as hepatitis B virus (HBV) (4). Moreover, a large portion of hepatocytes often remains uninfected by the virus even after long-term infection (28). These phenomena indicate the existence of balance between the HCV infection process and host mechanisms that protect against HCV infection. We speculate that innate and adaptive immunities contribute to the balance between infection and protection.High-mobility group box 1 (HMGB1) protein is a highly conserved nuclear protein that participates in DNA organization and the regulation of transcription. In addition to its nuclear function, HMGB1 plays an important role as a cytokine, mediating the responses to infection, injury, and inflammation (1, 2, 29, 42). HMGB1 is released passively from necrotic cells and is actively secreted from activated immune cells, such as macrophages, natural killer cells, and mature dendritic cells (2). The functionality of actively secreted HMGB1 is known to be modulated by posttranslational modifications, such as oxidation (2, 36). Extracellular HMGB1 can function by itself and/or in association with other molecules, including CpG DNA, lipopolysaccharide (LPS), and interleukin-1 (IL-1) (5). HMGB1 induces a variety of cellular responses that contribute to innate immunity, tissue repair, and cell migration through interactions with various receptors that activate multiple signal transduction responses. The Toll-like receptors (e.g., TLR2, TLR4, and TLR9) and the receptor for advanced glycation end products (RAGE) are known receptors for the cytokine functions of HMGB1 (2). TLR4, the major component of the LPS recognition receptor complex, engages in downstream signaling through My...