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-E1-mCherry 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 E1-mCherry 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.

Surface glycoprotein of Borna disease virus mediates virus spread from cell to cell.

Borna disease virus (BDV) is a non-segmented negative-stranded RNA virus that maintains a strictly neurotropic and persistent infection in affected end hosts. The primary target cells for BDV infection are brain cells, e.g. neurons and astrocytes. The exact mechanism of how infection is propagated between these cells and especially the role of the viral glycoprotein (GP) for cell-cell transmission, however, are still incompletely understood. Here, we use different cell culture systems, including rat primary astrocytes and mixed cultures of rat brain cells, to show that BDV primarily spreads through cell-cell contacts. We employ a highly stable and efficient peptidomimetic inhibitor to inhibit the furin-mediated processing of GP and demonstrate that cleaved and fusion-active GP is strictly necessary for the cell-to-cell spread of BDV. Together, our quantitative observations clarify the role of Borna disease virus-glycoprotein for viral dissemination and highlight the regulation of GP expression as a potential mechanism to limit viral spread and maintain persistence. These findings furthermore indicate that targeting host cell proteases might be a promising approach to inhibit viral GP activation and spread of infection.

The intraviral protein interaction network of hepatitis C virus.

Hepatitis C virus (HCV) is a global health problem and one of the main reasons for chronic liver diseases such as cirrhosis and hepatocellular carcinoma. The HCV genome is translated into a polyprotein which is proteolytically processed into 10 viral proteins. The interactome of the HCV proteins with the host cell has been worked out; however, it remains unclear how viral proteins interact with each other. We aimed to generate the interaction network of these 10 HCV proteins using a flow-cytometry-based FRET assay established in our laboratory (Banning, C., Votteler, J., Hoffmann, D., Koppensteiner, H., Warmer, M., Reimer, R., Kirchhoff, F., Schubert, U., Hauber, J., and Schindler, M. (2010) A flow cytometry-based FRET assay to identify and analyse protein-protein interactions in living cells. PLoS One 5, e9344). HCV proteins were constructed as fusions with the chromophores CFP and YFP. All HCV fusions were expressed and localized to specific subcellular compartments, indicating that they were functional. FACS-FRET measurements identified a total of 20 interactions; 13 of these were previously described and have now been confirmed in living cells via our method. Among the seven novel protein binding pairs, HCV p7 plays a pivotal role. It binds to the HCV capsid protein Core and the two glycoproteins E1 and E2. These interplays were further demonstrated in the relevant context of Huh7.5 liver cells expressing infectious HCV. Our work demonstrates the feasibility of rapidly generating small interaction networks via FACS-FRET and defines the network of intra-HCV protein interactions. Furthermore, our data support an important role of p7 in HCV assembly.