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author:("rebhun, ilk")
1.  Identification of HNRNPK as Regulator of Hepatitis C Virus Particle Production 
PLoS Pathogens  2015;11(1):e1004573.
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. Of 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.
Author Summary
As obligate intracellular parasites with limited gene coding capacity viruses exploit host cell machineries for the sake of efficient replication and spread. Thus, identification of these cellular machineries and factors is necessary to understand how a given virus achieves efficient replication and eventually causes host cell damage. Hepatitis C virus (HCV) is an RNA virus replicating in the cytoplasm of hepatocytes. While viral proteins have been studied in great detail, our knowledge about how host cell factors are used by HCV for efficient replication and spread is still scarce. In the present study we conducted a comprehensive RNA-interference-based screen and identified 40 genes that promote the HCV lifecycle and 16 genes that suppress it. Follow-up studies revealed that one of these genes, the heterogeneous nuclear ribonucleoprotein K (HNRNPK), selectively suppresses production of infectious HCV particles. We mapped the domains of HNRNPK required for this suppression and demonstrate that this protein selectively binds to the HCV RNA genome. Based on the correlation between suppression of virus production, HCV RNA binding and recruitment to lipid droplets, we propose that HNRNPK might limit the amount of viral RNA genomes available for incorporation into virus particles. This study provides novel insights into the complexity of reactions that are involved in the formation of HCV virions.
PMCID: PMC4287573  PMID: 25569684
2.  The Lipid Kinase Phosphatidylinositol-4 Kinase III Alpha Regulates the Phosphorylation Status of Hepatitis C Virus NS5A 
PLoS Pathogens  2013;9(5):e1003359.
The lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KIIIα) is an essential host factor of hepatitis C virus (HCV) replication. PI4KIIIα 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 PI4KIIIα and NS5A and its mechanistic role in viral RNA replication. We mapped the NS5A sequence involved in PI4KIIIα interaction to the carboxyterminal end of domain 1 and identified a highly conserved PI4KIIIα functional interaction site (PFIS) encompassing seven amino acids, which are essential for viral RNA replication. Mutations within this region were also impaired in NS5A-PI4KIIIα binding, reduced PI4P levels and altered the morphology of viral replication sites, reminiscent to the phenotype observed by silencing of PI4KIIIα. Interestingly, abrogation of RNA replication caused by mutations in the PFIS correlated with increased levels of hyperphosphorylated NS5A (p58), indicating that PI4KIIIα affects the phosphorylation status of NS5A. RNAi-mediated knockdown of PI4KIIIα or pharmacological ablation of kinase activity led to a relative increase of p58. In contrast, overexpression of enzymatically active PI4KIIIα increased relative abundance of basally phosphorylated NS5A (p56). PI4KIIIα 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 PI4KIIIα in HCV replication regulating NS5A phosphorylation, thereby modulating the morphology of viral replication sites. In conclusion, we demonstrate that PI4KIIIα activity affects the NS5A phosphorylation status. Our results highlight the importance of PI4KIIIα in the morphogenesis of viral replication sites and its regulation by facilitating p56 synthesis.
Author Summary
Hepatitis C virus (HCV) infections affect about 170 million people worldwide and often result in severe chronic liver disease. HCV is a positive-strand RNA virus inducing massive rearrangements of intracellular membranes to generate the sites of genome replication, designated the membranous web. The complex biogenesis of the membranous web is still poorly understood, but requires the concerted action of several viral nonstructural proteins and cellular factors. Recently, we and others identified the lipid kinase phosphatidylinositol-4 kinase III alpha (PI4KIIIα), catalyzing the synthesis of phosphatidylinositol 4-phosphate (PI4P), as an essential host factor involved in the formation of the membranous web. In this study, we characterized the virus-host interaction in greater detail using a genetic approach. We identified a highly conserved region in the viral phosphoprotein NS5A crucial for the interaction with PI4KIIIα. Surprisingly, we found that PI4KIIIα, despite being a lipid kinase, appeared to regulate the phosphorylation status of NS5A, thus contributing to viral replication. Our results furthermore suggest that the morphology of the membranous web is regulated by NS5A phosphorylation, providing novel insights into the complex regulation of viral RNA replication.
PMCID: PMC3649985  PMID: 23675303
3.  Recruitment and activation of a lipid kinase by hepatitis C virus NS5A is essential for integrity of the membranous replication compartment 
Cell host & microbe  2011;9(1):32-45.
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 (PI4KIIIα) as required for HCV replication. Consistent with elevated levels of the PI4KIIIα product phosphatidylinositol-4-phosphate (PI4P) detected in HCV infected cultured hepatocytes and liver tissue from chronic hepatitis C patients, the enzymatic activity of PI4KIIIα was critical for HCV replication. Viral nonstructural protein 5A (NS5A) was found to interact with PI4KIIIα and stimulate its kinase activity. The absence of PI4KIIIα 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.
PMCID: PMC3433060  PMID: 21238945
4.  Normalizing for individual cell population context in the analysis of high-content cellular screens 
BMC Bioinformatics  2011;12:485.
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 interest, i.e. when studying cell morphology.
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 non-virus 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.
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.
PMCID: PMC3259109  PMID: 22185194
5.  Detecting host factors involved in virus infection by observing the clustering of infected cells in siRNA screening images 
Bioinformatics  2010;26(18):i653-i658.
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 readout.
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 luciferase-based 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.
Supplementary information: Supplementary data are available at Bioinformatics online.
PMCID: PMC2935410  PMID: 20823335

Results 1-5 (5)