Next-Generation Surrogate Wnts Support Organoid Growth and Deconvolute Frizzled Pleiotropy In Vivo.

Modulation of Wnt signaling has untapped potential in regenerative medicine due to its essential functions in stem cell homeostasis. However, Wnt lipidation and Wnt-Frizzled (Fzd) cross-reactivity have hindered translational Wnt applications. Here, we designed and engineered water-soluble, Fzd subtype-specific "next-generation surrogate" (NGS) Wnts that hetero-dimerize Fzd and Lrp6. NGS Wnt supports long-term expansion of multiple different types of organoids, including kidney, colon, hepatocyte, ovarian, and breast. NGS Wnts are superior to Wnt3a conditioned media in organoid expansion and single-cell organoid outgrowth. Administration of Fzd subtype-specific NGS Wnt in vivo reveals that adult intestinal crypt proliferation can be promoted by agonism of Fzd5 and/or Fzd8 receptors, while a broad spectrum of Fzd receptors can induce liver zonation. Thus, NGS Wnts offer a unified organoid expansion protocol and a laboratory "tool kit" for dissecting the functions of Fzd subtypes in stem cell biology.

Functional innate immunity restricts Hepatitis C Virus infection in induced pluripotent stem cell-derived hepatocytes.

Knowledge of activation and interplay between the hepatitis C virus (HCV) and the hosts' innate immunity is essential to understanding the establishment of chronic HCV infection. Human hepatoma cell lines, widely used as HCV cell culture system, display numerous metabolic alterations and a defective innate immunity, hindering the detailed study of virus-host interactions. Here, we analysed the suitability of induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iHLCs) as a physiologically relevant model to study HCV replication in vitro. Density gradients and triglyceride analysis revealed that iHLCs secreted very-low density lipoprotein (VLDL)-like lipoproteins, providing a putative platform for bona fide lipoviroparticles. iHLCs supported the full HCV life cycle, but in contrast to Huh7 and Huh7.5 cells, replication and viral RNA levels decreased continuously. Following HCV infection, interferon-stimulated gene (ISG)-expression significantly increased in iHLCs, whereas induction was almost absent in Huh7/7.5 cells. However, IFNα-stimulation equally induced ISGs in iHLCs and hepatoma cells. JAK-STAT pathway inhibition increased HCV replication in mature iHLCs, but not in Huh7 cells. Additionally, HCV replication levels where higher in STAT2-, but not STAT1-knockdown iHLCs. Our findings support iHLCs as a suitable model for HCV-host interaction regarding a functional innate immunity and lipoprotein synthesis.

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis.

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with "heavy" amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e. HCV-infected/"light" amino acids and uninfected control/"heavy" amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Quantitative Lipid Droplet Proteome Analysis Identifies Annexin A3 as a Cofactor for HCV Particle Production.

Lipid droplets are vital to hepatitis C virus (HCV) infection as the putative sites of virion assembly, but morphogenesis and egress of virions remain ill defined. We performed quantitative lipid droplet proteome analysis of HCV-infected cells to identify co-factors of that process. Our results demonstrate that HCV disconnects lipid droplets from their metabolic function. Annexin A3 (ANXA3), a protein enriched in lipid droplet fractions, strongly impacted HCV replication and was characterized further: ANXA3 is recruited to lipid-rich fractions in HCV-infected cells by the viral core and NS5A proteins. ANXA3 knockdown does not affect HCV RNA replication but severely impairs virion production with lower specific infectivity and higher density of secreted virions. ANXA3 is essential for the interaction of viral envelope E2 with apolipoprotein E (ApoE) and for trafficking, but not lipidation, of ApoE in HCV-infected cells. Thus, we identified ANXA3 as a regulator of HCV maturation and egress.

Visualization and analysis of hepatitis C virus structural proteins at lipid droplets by super-resolution microscopy.

Cytosolic lipid droplets are central organelles in the Hepatitis C Virus (HCV) life cycle. The viral capsid protein core localizes to lipid droplets and initiates the production of viral particles at lipid droplet-associated ER membranes. Core is thought to encapsidate newly synthesized viral RNA and, through interaction with the two envelope proteins E1 and E2, bud into the ER lumen. Here, we visualized the spatial distribution of HCV structural proteins core and E2 in vicinity of small lipid droplets by three-color 3D super-resolution microscopy. We observed and analyzed small areas of colocalization between the two structural proteins in HCV-infected cells with a diameter of approximately 100 nm that might represent putative viral assembly sites.

The intraviral protein interaction network of hepatitis C virus.

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-cytometry-based 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 Schindler, M. (2010) A flow cytometry-based FRET assay to identify and analyse protein-protein interactions in living cells. PLoS One 5, e9344). HCV proteins were constructed as fusions with the chromophores CFP and YFP. All HCV fusions were expressed and localized to specific subcellular compartments, indicating that they were functional. FACS-FRET measurements identified a total of 20 interactions; 13 of these were previously described and have now been confirmed in living cells via our method. Among the seven novel protein binding pairs, HCV p7 plays a pivotal role. It binds to the HCV capsid protein Core and the two glycoproteins E1 and E2. These interplays were further demonstrated in the relevant context of Huh7.5 liver cells expressing infectious HCV. Our work demonstrates the feasibility of rapidly generating small interaction networks via FACS-FRET and defines the network of intra-HCV protein interactions. Furthermore, our data support an important role of p7 in HCV assembly.