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-cytometrybased 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 Hepatitis C virus (HCV)1 belongs to the family of Flaviviridae and is the only member of the genus Hepacivirus. The ϳ9.5-kB positive-strand RNA genome is directly translated via an internal ribosomal entry site into a polyprotein. This is proteolytically processed by cellular and viral proteases into structural (Core, E1, E2) and nonstructural (p7, NS2, NS3, NS4A/B, and NS5A/B) proteins (1). In recent decades, light was shed on the importance and biological relevance of most HCV proteins, which ultimately led to the development of the first specific antiviral therapy involving inhibition of the NS3 serine protease (2). However, because HCV is highly variable and because of the rapid emergence of drug resistance, additional therapeutic approaches are urgently needed (2). An impressive body of data was derived from protein interaction or siRNA screens investigating the interplay of HCV proteins with cellular factors (3-5). Although these screens are essential in order for researchers to understand how HCV manipulates the host cell, their potential benefit for novel therapeutic approaches could be limited. HCV is a chronic viral infection, and targeting host factors might result in drugs with severe adverse effects. Thus, a promising strategy would be to specifically inhibit interactions among viral proteins. Surprisingly, until now, a comprehensive analysis of the putative interactions and the interplay of HCV proteins with each other in living human cells has been lacking.In the present work, we did an extensive and thorough analysis of intra-HCV protein interactions. We used our novel flow-cytometry-based FRET assay that allows rapid assessment of the interplay between proteins in thousands of living cells (6). Therefore, this experimental approach enables quantification and statistical evaluation of all results. From the total of 20 interactions established by FACS-FRET, we chose to further investigate three that were not yet described in the literature. The putative HCV viroporin p7 binds to the structural proteins, and this was verified via biochemical methods in cells expressing fully infectious HCV.The established network of intra-HCV protein interactions in living mammalian cells provides new insights into the biology of this important human pathogen. Furthermore, we identified several HCV protein interactions that could be targeted for an...

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. IMPORTANCEThe 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. Hepatitis C virus (HCV) belongs to the Flavivirus genus and has a positive-strand RNA genome. This encodes a polyprotein which is posttranslationally cleaved into six nonstructural (NS) proteins, the ion channel p7 protein, and the structural proteins Core, E1, and E2 (1). The NS proteins reside at the outer leaflet of the endoplasmic reticulum (ER) membrane where NS4B and NS5A in particular induce membrane alterations resulting in the formation of the membranous web, which is the major site for HCV replication (2-5). Core is targeted to adjacent lipid droplets (LDs) (6, 7), which represent intracellular lipid deposits and are considered important for production of infectious particles (1,7,8). The E1 and E2 envelope proteins are incorporated into ER membranes with ectodomains facing the ER lumen (9, 10). Later, they are recruited to assembly sites via the NS2 complex (11,12). Upon recruitment of all requi...

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.

No abstract

No abstract