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...

HCV (hepatitis C virus) is a member of the Flaviviridae family that contains a single-stranded positive-sense RNA genome of approximately 9600 bases. HCV is a major causative agent for chronic liver diseases such as steatosis, fibrosis, cirrhosis, and hepatocellular carcinoma which are caused by multifactorial processes. Elevated levels of reactive oxygen species (ROS) are considered as a major factor contributing to HCV-associated pathogenesis. This review summarizes the mechanisms involved in formation of ROS in HCV replicating cells and describes the interference of HCV with ROS detoxifying systems. The relevance of ROS for HCV-associated pathogenesis is reviewed with a focus on the interference of elevated ROS levels with processes controlling liver regeneration. The overview about the impact of ROS for the viral life cycle is focused on the relevance of autophagy for the HCV life cycle and the crosstalk between HCV, elevated ROS levels, and the induction of autophagy.

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1Seventy million people worldwide are chronically infected with hepatitis C virus (HCV). Chronic HCV infection is associated with an elevated risk of developing liver cirrhosis and hepatocellular carcinoma (1). HCV is a single-stranded, positivesense RNA virus that belongs to the Flaviviridae family. The viral RNA encodes a single polyprotein of about 3,100 amino acids, which is cleaved co-and posttranslationally by cellular and viral proteases into 10 viral proteins: the structural proteins (core, E1, E2), which build up the viral particles; the p7 polypeptide, which forms an ion channel; and the nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B), which support viral replication and assembly processes (2-5).NS5A is known to act as a regulator of intracellular signal transduction cascades. The kinase c-Raf was identified to bind to the C-terminal domain of NS5A. Thereby, c-Raf is located at the HCV replication complex (6, 7). The interaction of NS5A with c-Raf leads to the activation of c-Raf in association with the phosphorylation of c-Raf at serine 338 (6, 7). Moreover, it was found that inhibition of c-Raf blocks HCV replication (6,8). However, due to the delocalization of c-Raf to the replicon complex/endoplasmic reticulum (ER) membrane, c-Raf in HCV-replicating cells is withdrawn from the classic MEK/extracellular signal-regulated kinase (ERK) signaling pathway (6). Therefore, although c-Raf is activated in HCV-replicating cells, signal transduction to the MEK/ERK pathway is impaired.The HCV infection cycle is tightly associated with lipid metabolism. HCV replication occurs on the cytoplasmic face of the ER in the replication complexes (RCs) formed by nonstructural proteins. HCV replication and morphogenesis take place at specialized rearranged intracellular ER membranes, the socalled membranous web, that are enriched in proteins involved

Autophagy is a highly conserved and regulated intracellular lysosomal degradation pathway that is essential for cell survival. Dysregulation has been linked to the development of various human diseases, including neurodegeneration and tumorigenesis, infection, and aging. Besides, many viruses hijack the autophagosomal pathway to support their life cycle. The hepatitis C virus (HCV), a major cause of chronic liver diseases worldwide, has been described to induce autophagy. The autophagosomal pathway can be further activated in response to elevated levels of reactive oxygen species (ROS). HCV impairs the Nrf2/ARE-dependent induction of ROS-detoxifying enzymes by a so far unprecedented mechanism. In line with this, this review aims to discuss the relevance of HCV-dependent elevated ROS levels for the induction of autophagy as a result of the impaired Nrf2 signaling and the described crosstalk between p62 and the Nrf2/Keap1 signaling pathway. Moreover, autophagy is functionally connected to the endocytic pathway as components of the endosomal trafficking are involved in the maturation of autophagosomes. The release of HCV particles is still not fully understood. Recent studies suggest an involvement of exosomes that originate from the endosomal pathway in viral release. In line with this, it is tempting to speculate whether HCV-dependent elevated ROS levels induce autophagy to support exosome-mediated release of viral particles. Based on recent findings, in this review, we will further highlight the impact of HCV-induced autophagy and its interplay with the endosomal pathway as a novel mechanism for the release of HCV particles.