Subcellular Localization and Function of an Epitope-Tagged p7 Viroporin in Hepatitis C Virus-Producing Cells

Vièyres, Gabrielle; Brohm, Christiane; Friesland, Martina; Gentzsch, Juliane; Wölk, Benno; Roingeard, Philippe; Steinmann, Eike; Pietschmann, Thomas

doi:10.1128/jvi.02782-12

Cited by 40 publications

(65 citation statements)

References 70 publications

(149 reference statements)

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“…1B, compare lanes 1 and 3). These results confirmed the recent report by Vieyres and colleagues who showed that introducing an N-terminal double HA tag to the p7 of the gt2a/2a chimera Jc1 allowed the production of virus, albeit at a reduced level compared to that from Jc1 (42). Since p7 in HJ3-5 is derived from gt1a, these results also indicate that p7 with an N-terminal epitope tag was functional in virus production in both gt1a and gt2a genetic backgrounds.…”

Section: Resultssupporting

confidence: 81%

Efficiency of E2-p7 Processing Modulates Production of Infectious Hepatitis C Virus

Shanmugam

2013

J Virol

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Previous studies indicate that the processing of hepatitis C virus (HCV) E2-p7-NS2 precursor mediated by host signal peptidase is relatively inefficient, resulting in the accumulation of E2-p7-NS2 and E2-p7 precursors in addition to E2 in mammalian cells. In this study, we discovered that a significant inhibition of the processing at an E2-p7 junction site is detrimental for HCV production, whether it was caused by the mutations in p7 or by the strategic introduction of a mutation at a terminal residue of E2 to block the signal peptidase-mediated cleavage of this junction site. However, complete separation of E2 and p7 by inserting an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) between these two proteins also moderately inhibited virus production. These results indicate that optimal processing of the E2-p7 junction site is critical for efficient HCV production. We further demonstrated that disrupting E2-p7 processing inhibits both NS2 localization to the putative virus assembly sites near lipid droplets (LD) and NS2 interaction with NS3 and E2. However, the impact, if any, of the p7-NS2 processing efficiency on HCV production seems relatively minor. In conclusion, these results imply that effective release of E2 and p7 from the precursor E2-p7 promotes HCV production by enhancing NS2-associated virus assembly complex formation near LD. Hepatitis C virus (HCV) is one of the major agents responsible for causing severe liver diseases, including chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (1, 2). It is an enveloped, positive-stranded RNA virus belonging to the Hepacivirus genus in the Flaviviridae family of viruses (3, 4). HCV encodes a single, large open reading frame encoding an ϳ3,000-amino-acid polyprotein flanked by 5= and 3= noncoding sequences. The internal ribosome entry site (IRES) located at the 5= noncoding region mediates translation of a polyprotein that is subsequently processed by both host-and virus-encoded proteases to yield 10 different proteins. The N-terminal one-third of the polyprotein encoding the structural proteins, including core and envelope proteins (E1 and E2), followed by p7 and NS2, is processed by host signal peptidase (5-7). Core protein is further processed by signal peptide peptidase into a mature form (8). The junction site between NS2 and NS3 is cleaved by an NS2-NS3 autoprotease (9, 10), and the remaining nonstructural proteins (NS3, NS4A, NS4B, NS5A, and NS5B) are processed by NS3 protease with its cofactor NS4A (11-13). Proteins NS3 to NS5B, along with the 5=-and 3=-terminal noncoding regions, were shown to be sufficient for the autonomous replication of HCV RNA (14).Recent advances in the infectious HCV cell culture model allowed the investigation of HCV virus particle assembly processes (15-18). We have learned that the association of HCV core protein with lipid droplets (LD) is critical for virus assembly since disrupting the core protein localization to LD inhibited infectious virus production (19,20). We also learned that the...

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Section: Resultssupporting

confidence: 81%

Efficiency of E2-p7 Processing Modulates Production of Infectious Hepatitis C Virus

Shanmugam

2013

J Virol

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“…p7 has been shown by overexpression studies and in full-length HCV to predominantly localize to ER membranes (Carrère-Kremer et al, 2002;Haqshenas et al, 2007;Wozniak et al, 2010), including those associated with mitochondria (Griffin et al, 2005). Cell surface expression has also been noted (Carrère-Kremer et al, 2002) and recent studies of HA-tagged or native proteins in full-length virus have observed associations with HCV core, E2 and NS5A proteins (Bentham et al, 2013;Vieyres et al, 2013).…”

Section: Hcv P7mentioning

confidence: 99%

“…Viable full-length HCV containing IRES elements inserted between E2 and p7 or p7 and NS2 argued against a functional role for p7 precursors (Jones et al, 2007). Both early-and late-acting defects in virion production have been described where p7 was (partially) deleted, mutated at specific residues or treated with inhibitors (Bentham et al, 2013;Foster et al, 2011Foster et al, , 2014Jones et al, 2007;Steinmann et al, 2007a;Vieyres et al, 2013;Wozniak et al, 2010). This is now known to result from p7 performing multiple functions within infected cells, comprising distinct protein-protein interactions as well as its channel-forming activity.…”

Section: Hcv P7mentioning

confidence: 99%

Viroporins: structure, function and potential as antiviral targets

Scott

Griffin

2015

Journal of General Virology

119

146

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The channel-forming activity of a family of small, hydrophobic integral membrane proteins termed 'viroporins' is essential to the life cycles of an increasingly diverse range of RNA and DNA viruses, generating significant interest in targeting these proteins for antiviral development. Viroporins vary greatly in terms of their atomic structure and can perform multiple functions during the virus life cycle, including those distinct from their role as oligomeric membrane channels. Recent progress has seen an explosion in both the identification and understanding of many such proteins encoded by highly significant pathogens, yet the prototypic M2 proton channel of influenza A virus remains the only example of a viroporin with provenance as an antiviral drug target. This review attempts to summarize our current understanding of the channel-forming functions for key members of this growing family, including recent progress in structural studies and drug discovery research, as well as novel insights into the life cycles of many viruses revealed by a requirement for viroporin activity. Ultimately, given the successes of drugs targeting ion channels in other areas of medicine, unlocking the therapeutic potential of viroporins represents a valuable goal for many of the most significant viral challenges to human and animal health.

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“…These findings could give a functional rationale for previously published data. Furthermore, it was suggested that E2 and p7 might interact (9,44,45), and Gentzsch and colleagues postulated that the p7-RR/QQ mutant has a defect at the step of capsid envelopment. Strikingly, our data suggest that mutation of the dibasic motif in p7 selectively impairs the interaction with E2 (44).…”

Section: Hcv Protein Interactions Determined Via Facs-fret-tomentioning

confidence: 99%

“…Then immunofluorescence staining with specific antibodies was performed to detect areas of protein co-localization by means of confocal microscopy. We quantified co-localization using Costes Pearson's correlation (84) major functional impairments as a consequence of the tag (9,10,16,(23)(24)(25)(26)(27)(28)(29).…”

Section: Hcv Protein Interactions Determined Via Facs-fret-tomentioning

confidence: 99%

The Intraviral Protein Interaction Network of Hepatitis C Virus

Hagen

Bayer

Rösch

et al. 2014

Molecular & Cellular Proteomics

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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...

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Subcellular Localization and Function of an Epitope-Tagged p7 Viroporin in Hepatitis C Virus-Producing Cells

Cited by 40 publications

References 70 publications

Efficiency of E2-p7 Processing Modulates Production of Infectious Hepatitis C Virus

Efficiency of E2-p7 Processing Modulates Production of Infectious Hepatitis C Virus

Viroporins: structure, function and potential as antiviral targets

The Intraviral Protein Interaction Network of Hepatitis C Virus

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