The Intraviral Protein Interaction Network of Hepatitis C Virus

Abstract

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...

HIV-1 Nef and Vpu Interfere with L-Selectin (CD62L) Cell Surface Expression To Inhibit Adhesion and Signaling in Infected CD4+T Lymphocytes

“…Importantly, the method allows the measurement of protein interactions in living cells, and independently and multiple times, we confirmed FRET-measured interactions by biochemical assays and in virus-infected cells, demonstrating that FACS-FRET is a robust assay to assess protein interactions (21,26,27). Briefly, 150,000 293T cells were seeded in a 12-well plate and transfected 12 h later.…”

Hepatitis C Virus Is Released via a Noncanonical Secretory Route

“…Recently, it has been proposed that the cellular Ewing sarcoma breakpoint region 1 (EWSR1) protein is important for regulation of the switch from translation to replication by binding to the cis-acting replication element of HCV (14). Furthermore, transport of HCV Core toward LDs by the enzyme diacylglycerol acyltransferase-1 (DGAT1) is crucial for production of newly formed virions (15, 16), and the NS2 protein, together with p7, might be a major player in coordinating assembly (11,17,18).How HCV is released from infected cells is still under debate.HCV was found to associate with constituents of very-low-density lipoproteins (VLDL), such as , and proteins of the VLDL secretory pathway, including the transcription factor hepatocyte nuclear factor 4 (HNF4) (22). Thus, it has been assumed that HCV budding, maturation, and release might intersect the VLDL secretion pathway (13), but a precise model is still lacking and a recent study suggested that HCV release is independent of the VLDL route (23).…”

“…Recently, it has been proposed that the cellular Ewing sarcoma breakpoint region 1 (EWSR1) protein is important for regulation of the switch from translation to replication by binding to the cis-acting replication element of HCV (14). Furthermore, transport of HCV Core toward LDs by the enzyme diacylglycerol acyltransferase-1 (DGAT1) is crucial for production of newly formed virions (15, 16), and the NS2 protein, together with p7, might be a major player in coordinating assembly (11,17,18).…”

“…Immunodetection was done with primary antibodies from rabbit directed against mCherry (BioVision) and mouse anti-E2 (AP-33), anti-Core (C7-50; Abcam), anti-NS3 (clone F3A6B2C3 against epitopes 1322 to 1662 of JFH-1 NS3), or anti-NS5A (2F6/G11; IBT) diluted 1:100 in 1% BSA-PBS for 2 h at room temperature. Afterwards, samples were prepared according to the protocol of the manufacturer (Duolink; Sigma-Aldrich) and as described previously (18). Spots of the fluorescent substrate were detected by confocal spinning-disc microscopy (Nikon Ti Eclipse microscope with an UltraView VoX system; PerkinElmer).…”

AP-2 Is the Crucial Clathrin Adaptor Protein for CD4 Downmodulation by HIV-1 Nef in Infected Primary CD4 + T Cells

“…While most previous studies have been conducted in the wellcharacterized HeLa cell model, providing a good rational for molecular and cell biological experiments, our goal was to clarify the roles of the different cellular proteins in cells relevant to HIV-1 infection, such as the CD4 ϩ T cell line SupT1 and primary CD4 ϩ T cells. Furthermore, we aimed to investigate binding of host cell factors with Nef by our established fluorescence-activated cell sorter (FACS)-Förster resonance energy transfer (FRET) assay, allowing detection of transient protein interactions in living cells (16,17). We utilized cyan fluorescent protein (CFP)-tagged variants of the adaptor protein subunits AP-1, AP-2, AP-2, and AP-3; the thioesterase ACOT8; ␤-COP; the ATPase V1H; and CD4 (all ligated into Clontech pECFP/EYFP vectors as described previously [16]).…”