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Identification of HNRNPK as Regulator of Hepatitis C Virus Particle Production

Marion Poenisch, Philippe Metz, Hagen Blankenburg, Alessia Ruggieri, Ji-Young Lee, Daniel Rupp, Ilka Rebhan, Kathrin Diederich, Lars Kaderali, Francisco S. Domingues, Mario Albrecht, Volker Lohmann (+3 others)
2015 PLoS Pathogens  
Hepatitis C virus (HCV) is a major cause of chronic liver disease affecting around 130 million people worldwide. While great progress has been made to define the principle steps of the viral life cycle, detailed knowledge how HCV interacts with its host cells is still limited. To overcome this limitation we conducted a comprehensive whole-virus RNA interference-based screen and identified 40 host dependency and 16 host restriction factors involved in HCV entry/replication or assembly/ release.
more » ... f these factors, heterogeneous nuclear ribonucleoprotein K (HNRNPK) was found to suppress HCV particle production without affecting viral RNA replication. This suppression of virus production was specific to HCV, independent from assembly competence and genotype, and not found with the related Dengue virus. By using a knock-down rescue approach we identified the domains within HNRNPK required for suppression of HCV particle production. Importantly, HNRNPK was found to interact specifically with HCV RNA and this interaction was impaired by mutations that also reduced the ability to suppress HCV particle production. Finally, we found that in HCV-infected cells, subcellular distribution of HNRNPK was altered; the protein was recruited to sites in close proximity of lipid droplets and colocalized with core protein as well as HCV plus-strand RNA, which was not the case with HNRNPK variants unable to suppress HCV virion formation. These results suggest that HNRNPK might determine efficiency of HCV particle production by limiting the availability of viral RNA for incorporation into virions. This study adds a new function to HNRNPK that acts as central hub in the replication cycle of multiple other viruses.
doi:10.1371/journal.ppat.1004573 pmid:25569684 pmcid:PMC4287573 fatcat:mjwc2snulbcmhn7ebt6whiilme

Normalizing for individual cell population context in the analysis of high-content cellular screens

Bettina Knapp, Ilka Rebhan, Anil Kumar, Petr Matula, Narsis A Kiani, Marco Binder, Holger Erfle, Karl Rohr, Roland Eils, Ralf Bartenschlager, Lars Kaderali
2011 BMC Bioinformatics  
High-content, high-throughput RNA interference (RNAi) offers unprecedented possibilities to elucidate gene function and involvement in biological processes. Microscopy based screening allows phenotypic observations at the level of individual cells. It was recently shown that a cell's population context significantly influences results. However, standard analysis methods for cellular screens do not currently take individual cell data into account unless this is important for the phenotype of
more » ... rest, i.e. when studying cell morphology. Results: We present a method that normalizes and statistically scores microscopy based RNAi screens, exploiting individual cell information of hundreds of cells per knockdown. Each cell's individual population context is employed in normalization. We present results on two infection screens for hepatitis C and dengue virus, both showing considerable effects on observed phenotypes due to population context. In addition, we show on a nonvirus screen that these effects can be found also in RNAi data in the absence of any virus. Using our approach to normalize against these effects we achieve improved performance in comparison to an analysis without this normalization and hit scoring strategy. Furthermore, our approach results in the identification of considerably more significantly enriched pathways in hepatitis C virus replication than using a standard analysis approach. Conclusions: Using a cell-based analysis and normalization for population context, we achieve improved sensitivity and specificity not only on a individual protein level, but especially also on a pathway level. This leads to the identification of new host dependency factors of the hepatitis C and dengue viruses and higher reproducibility of results.
doi:10.1186/1471-2105-12-485 pmid:22185194 pmcid:PMC3259109 fatcat:onfdgkkgxjh6xay3d6cgtyahxq

The Lipid Kinase Phosphatidylinositol-4 Kinase III Alpha Regulates the Phosphorylation Status of Hepatitis C Virus NS5A

Simon Reiss, Christian Harak, Inés Romero-Brey, Danijela Radujkovic, Rahel Klein, Alessia Ruggieri, Ilka Rebhan, Ralf Bartenschlager, Volker Lohmann, Michael Gale
2013 PLoS Pathogens  
The lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KIIIa) is an essential host factor of hepatitis C virus (HCV) replication. PI4KIIIa catalyzes the synthesis of phosphatidylinositol 4-phosphate (PI4P) accumulating in HCV replicating cells due to enzyme activation resulting from its interaction with nonstructural protein 5A (NS5A). This study describes the interaction between PI4KIIIa and NS5A and its mechanistic role in viral RNA replication. We mapped the NS5A sequence involved in
more » ... 4KIIIa interaction to the carboxyterminal end of domain 1 and identified a highly conserved PI4KIIIa functional interaction site (PFIS) encompassing seven amino acids, which are essential for viral RNA replication. Mutations within this region were also impaired in NS5A-PI4KIIIa binding, reduced PI4P levels and altered the morphology of viral replication sites, reminiscent to the phenotype observed by silencing of PI4KIIIa. Interestingly, abrogation of RNA replication caused by mutations in the PFIS correlated with increased levels of hyperphosphorylated NS5A (p58), indicating that PI4KIIIa affects the phosphorylation status of NS5A. RNAi-mediated knockdown of PI4KIIIa or pharmacological ablation of kinase activity led to a relative increase of p58. In contrast, overexpression of enzymatically active PI4KIIIa increased relative abundance of basally phosphorylated NS5A (p56). PI4KIIIa therefore regulates the phosphorylation status of NS5A and viral RNA replication by favoring p56 or repressing p58 synthesis. Replication deficiencies of PFIS mutants in NS5A could not be rescued by increasing PI4P levels, but by supplying functional NS5A, supporting an essential role of PI4KIIIa in HCV replication regulating NS5A phosphorylation, thereby modulating the morphology of viral replication sites. In conclusion, we demonstrate that PI4KIIIa activity affects the NS5A phosphorylation status. Our results highlight the importance of PI4KIIIa in the morphogenesis of viral replication sites and its regulation by facilitating p56 synthesis.
doi:10.1371/journal.ppat.1003359 pmid:23675303 pmcid:PMC3649985 fatcat:6eapunzfindyxft225vsbpaoei

Detecting host factors involved in virus infection by observing the clustering of infected cells in siRNA screening images

Apichat Suratanee, Ilka Rebhan, Petr Matula, Anil Kumar, Lars Kaderali, Karl Rohr, Ralf Bartenschlager, Roland Eils, Rainer König
2010 Computer applications in the biosciences : CABIOS  
Motivation: Detecting human proteins that are involved in virus entry and replication is facilitated by modern high-throughput RNAi screening technology. However, hit lists from different laboratories have shown only little consistency. This may be caused by not only experimental discrepancies, but also not fully explored possibilities of the data analysis. We wanted to improve reliability of such screens by combining a population analysis of infected cells with an established dye intensity
more » ... out. Results: Viral infection is mainly spread by cell-cell contacts and clustering of infected cells can be observed during spreading of the infection in situ and in vivo. We employed this clustering feature to define knockdowns which harm viral infection efficiency of human Hepatitis C Virus. Images of knocked down cells for 719 human kinase genes were analyzed with an established point pattern analysis method (Ripley's K-function) to detect knockdowns in which virally infected cells did not show any clustering and therefore were hindered to spread their infection to their neighboring cells. The results were compared with a statistical analysis using a common intensity readout of the GFP-expressing viruses and a luciferasebased secondary screen yielding five promising host factors which may suit as potential targets for drug therapy. Conclusion: We report of an alternative method for high-throughput imaging methods to detect host factors being relevant for the infection efficiency of viruses. The method is generic and has the potential to be used for a large variety of different viruses and treatments being screened by imaging techniques. Contact
doi:10.1093/bioinformatics/btq398 pmid:20823335 pmcid:PMC2935410 fatcat:zk4dd3czj5hz3ondguq53m23ou

Recruitment and Activation of a Lipid Kinase by Hepatitis C Virus NS5A Is Essential for Integrity of the Membranous Replication Compartment

Simon Reiss, Ilka Rebhan, Perdita Backes, Ines Romero-Brey, Holger Erfle, Petr Matula, Lars Kaderali, Marion Poenisch, Hagen Blankenburg, Marie-Sophie Hiet, Thomas Longerich, Sarah Diehl (+12 others)
2011 Cell Host and Microbe  
Hepatitis C virus (HCV) is a major causative agent of chronic liver disease in humans. To gain insight into host factor requirements for HCV replication, we performed a siRNA screen of the human kinome and identified 13 different kinases, including phosphatidylinositol-4 kinase III alpha (PI4KIIIa), as being required for HCV replication. Consistent with elevated levels of the PI4KIIIa product phosphatidylinositol-4-phosphate (PI4P) detected in HCV-infected cultured hepatocytes and liver tissue
more » ... rom chronic hepatitis C patients, the enzymatic activity of PI4KIIIa was critical for HCV replication. Viral nonstructural protein 5A (NS5A) was found to interact with PI4KIIIa and stimulate its kinase activity. The absence of PI4KIIIa activity induced a dramatic change in the ultrastructural morphology of the membranous HCV replication complex. Our analysis suggests that the direct activation of a lipid kinase by HCV NS5A contributes critically to the integrity of the membranous viral replication complex.
doi:10.1016/j.chom.2010.12.002 pmid:21238945 pmcid:PMC3433060 fatcat:lz4vnk5pmnacfozwfb2st2yhtu

Computational Analysis of RNAi Screening Data to Identify Host Factors Involved in Viral Infection and to Characterize Protein-Protein Interactions

Apichat Suratanee
Data source The experimental data were generated by our collaborator, Ilka Rebhan at the Department of Molecular Virology, UniversitätsKlinikum Heidelberg.  ...  Rebhan, P. Matula, A. Kumar, L. Kaderali, K. Rohr, R. • M. Gipp, G. Marcus, N. Harder, A. Suratanee, K. Rohr, R. König and R. Männer.  ... 
doi:10.11588/heidok.00013845 fatcat:abgmicfnobdv3plze2lrlqintm

Analyse des Reepithelialisierungsmechanismus auf zellulärer Ebene mittels organotypischer in vitro Wundheilungsmodelle aus systembiologischer Perspektive

Kai Safferling
Thomas Sütterlin, Bernd Lahrmann, Ilka Rebhan und Jessica Kaufmann danke ich für die stets amüsanten Mittagspausen und die geistreichen Diskussionen abseits meiner wissenschaftlichen Fragestellung.  ... 
doi:10.11588/heidok.00014872 fatcat:5quhgjyvmjhvvflix6fnwvcv6a

RNA Interference Data: from a Statistical Analysis to Network Inference

Bettina Knapp
The first screen has been done by Ilka Rebhan and aims at the identification of host cell factors involved in Hepatitis C virus replication.  ... 
doi:10.11588/heidok.00013322 fatcat:35k3zclc75ghrcdj5nm2xmy5ny