Hepatitis C Virus Is Released via a Noncanonical Secretory Route

We analyzed hepatitis C virus (HCV) morphogenesis using viral genomes encoding a mCherry-tagged E1 glycoprotein. HCV-E1mCherry polyprotein expression, intracellular localization, and replication kinetics were comparable to those of untagged HCV, and E1-mCherry-tagged viral particles were assembled and released into cell culture supernatants. Expression and localization of structural E1 and nonstructural NS5A followed a temporospatial pattern with a succinct decrease in the number of replication complexes and the appearance of E1-mCherry punctae. Interaction of the structural proteins E1, Core, and E2 increased at E1mCherry punctae in a time-dependent manner, indicating that E1-mCherry punctae represent assembled or assembling virions. E1-mCherry did not colocalize with Golgi markers. Furthermore, the bulk of viral glycoproteins within released particles revealed an EndoH-sensitive glycosylation pattern, indicating an absence of viral glycoprotein processing by the Golgi apparatus. In contrast, HCV-E1-mCherry trafficked with Rab9-positive compartments and inhibition of endosomes specifically suppressed HCV release. Our data suggest that assembled HCV particles are released via a noncanonical secretory route involving the endosomal compartment. IMPORTANCE The goal of this study was to shed light on the poorly understood trafficking and release routes of hepatitis C virus (HCV). For this, we generated novel HCV genomes which resulted in the production of fluorescently labeled viral particles. We used live-cell microscopy and other imaging techniques to follow up on the temporal dynamics of virus particle formation and trafficking in HCV-expressing liver cells. While viral particles and viral structural protein were found in endosomal compartments, no overlap of Golgi structures could be observed. Furthermore, biochemical and inhibitor-based experiments support a HCV release route which is distinguishable from canonical Golgi-mediated secretion. Since viruses hijack cellular pathways to generate viral progeny, our results point toward the possible existence of a not-yet-described cellular secretion route.