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

Abstract

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...

Hepatitis C Virus Lipoviroparticles Assemble in the Endoplasmic Reticulum (ER) and Bud off from the ER to the Golgi Compartment in COPII Vesicles

“…This reasoning also applies to a recent study by Mankouri et al, in which they conclude that the inhibition of Rab GTPase did not affect VLDL secretion and therefore that VLDLs are not associated with HCV secretion (28). Some studies implicated noncanonical secretion in HCV egress involving the endosomal compartments, autophagosomes, endocytic machinery, and ESCRT proteins (35)(36)(37)49). The presence of complex glycans on virus-associated glycoproteins substantiates that HCV virions traverse through the Golgi apparatus (43).…”

“…Intriguingly, the endocytic and endosomal sorting complex required for transport (ESCRT) machineries have also been implicated in HCV secretion (35,36). The intracellular cholesterol transport inhibitor U18666A promotes the accumulation of HCV particles in autophagosomal and exosomal structures, reflecting the involvement of exosomes in HCV secretion (34,37).…”

Cellular Membrane Trafficking Machineries Used by the Hepatitis Viruses

“…Recent findings have suggested that exosomes could contain HCV particles or its genome to support the infective transmission between hepatocytes . Further, a recent study using an MVB inhibitor found that at least a fraction of mature HCV particles are released by MVBs . In that study, it was suggested that HCV particles are assembled in a membranous web of the ER that could be transported to an MVB compartment.…”

“…), it remains undetermined how the viral envelope proteins are transported onto MVBs. Figure depicts published images of confocal microscopy or TEM, showing that proteins or particles of hepatitis viruses are found in MVBs . The multisubunit membrane remodeling complex termed endosomal sorting complexes required for transport machinery (ESCRT) catalyzes the biogenesis of ILVs.…”

“…The graph shows the intensities of 4′,6‐diamidino‐2‐phenylindole, core, and CD63 along the white arrow. Republished with permission from an article . (D) PLC/PRF/5 cells that were infected with HEV were stained with antibodies to CD63, HEV ORF2 protein, and HEV ORF3 protein.…”