Identification of HNRNPK as regulator of hepatitis C virus particle production.

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.

The lipid kinase phosphatidylinositol-4 kinase III alpha regulates the phosphorylation status of hepatitis C virus NS5A.

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.

Interleukin-32: a new proinflammatory cytokine involved in hepatitis C virus-related liver inflammation and fibrosis.

UNLABELLED: Interleukin 32 (IL-32) is a recently described proinflammatory cytokine that activates p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB), thereby inducing proinflammatory cytokines such as IL-1ß and tumor necrosis factor alpha (TNF-α). We investigated the role of IL-32 in patients with chronic hepatitis C virus (HCV) infection. Steady-state hepatic messenger RNA (mRNA) levels of IL-32 were determined in a cohort of 90 subjects; anti-IL-32 staining was used in a second cohort of 132 consecutive untreated chronic HCV patients. Correlations with histological features of steatosis, inflammation, and fibrosis were made. In vitro, endogenous IL-32 in monocytes and in the human hepatoma cell line Huh-7.5 were examined. The effects of IL-32-overexpression and IL-32-silencing on HCV replication were studied using HCV luciferase reporter viruses. There were highly significant positive associations between hepatic IL-32 mRNA expression and liver steatosis, inflammation, fibrosis, smooth muscle actin (SMA) area, and serum alanine aminotransferase (ALT) levels. IL-32 protein expression was positively associated with portal inflammation, SMA area, and ALT. In vitro, IL-1ß and TNF-α significantly induced IL-32 expression in human Huh-7.5 cells. Alone, stimulation with interferon alpha (IFN-α) did not induce IL-32 expression in Huh-7.5. However, IFN-α exerted a significant additive effect on TNF-α-induced but not IL-1ß-induced IL-32 expression, particularly in CD14+ monocytes. This effect was dependent both on NF-κB and Jak/STAT signaling. Viral infection of Huh-7.5 cells resulted in a significant (11-fold) induction of IL-32 mRNA expression. However, modulation of IL-32 in Huh-7.5 cells by overexpression or silencing did not influence HCV virus replication as determined by luciferase assays. CONCLUSION: IL-32 is a novel proinflammatory cytokine involved in HCV-associated liver inflammation/fibrosis. IL-32 is expressed by human hepatocytes and hepatoma cells and its expression is regulated by proinflammatory stimuli.

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

BACKGROUND: 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. 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 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. 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.

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

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.

Recruitment and activation of a lipid kinase by hepatitis C virus NS5A is essential for integrity of the membranous replication compartment.

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 being 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.