NF-κB inhibitor alpha controls SARS-CoV-2 infection in ACE2-overexpressing human airway organoids.

As SARS-CoV-2 continues to spread worldwide, tractable primary airway cell models that recapitulate the cell-intrinsic response to arising viral variants are needed. Here we describe an adult stem cell-derived human airway organoid model overexpressing the ACE2 receptor (ACE2-OE) that supports robust viral replication while maintaining 3D architecture and cellular diversity of the airway epithelium. ACE2-OE organoids were infected with SARS-CoV-2 variants and subjected to single-cell RNA-sequencing. Interferon-lambda was upregulated in cells with low-level infection while the NF-kB inhibitor alpha gene (encoding IkBa) was consistently upregulated in infected cells, and its expression positively correlated with infection levels. Confocal microscopy showed more IkBa expression in infected than bystander cells, but found concurrent nuclear translocation of NF-kB that IkBa usually prevents. Overexpressing a nondegradable IkBa mutant reduced NF-kB translocation and increased viral infection. These data demonstrate the functionality of ACE2-OE organoids in SARS-CoV-2 research and underscore that the strength of the NF-kB feedback loop in infected cells controls viral replication.

Hepatitis C virus infects and perturbs liver stem cells.

Hepatitis C virus (HCV) is the leading cause of death from liver disease. How HCV infection causes lasting liver damage and increases cancer risk remains unclear. Here, we identify bipotent liver stem cells as novel targets for HCV infection, and their erroneous differentiation as the potential cause of impaired liver regeneration and cancer development. We show 3D organoids generated from liver stem cells from actively HCV-infected individuals carry replicating virus and maintain low-grade infection over months. Organoids can be infected with a primary HCV isolate. Virus-inclusive single-cell RNA sequencing uncovered transcriptional reprogramming in HCV+ cells supporting hepatocytic differentiation, cancer stem cell development, and viral replication while stem cell proliferation and interferon signaling are disrupted. Our data add a new pathogenesis mechanism-infection of liver stem cells-to the biology of HCV infection that may explain progressive liver damage and enhanced cancer risk through an altered stem cell state.ImportanceThe hepatitis C virus (HCV) causes liver disease, affecting millions. Even though we have effective antivirals that cure HCV, they cannot stop terminal liver disease. We used an adult stem cell-derived liver organoid system to understand how HCV infection leads to the progression of terminal liver disease. Here, we show that HCV maintains low-grade infections in liver organoids for the first time. HCV infection in liver organoids leads to transcriptional reprogramming causing cancer cell development and altered immune response. Our finding shows how HCV infection in liver organoids mimics HCV infection and patient pathogenesis. These results reveal that HCV infection in liver organoids contributes to liver disease progression.

Human apoA-I[Lys107del] mutation affects lipid surface behavior of apoA-I and its ability to form large nascent HDL.

Population studies have found that a natural human apoA-I variant, apoA-I[K107del], is strongly associated with low HDL-C but normal plasma apoA-I levels. We aimed to reveal properties of this variant that contribute to its unusual phenotype associated with atherosclerosis. Our oil-drop tensiometry studies revealed that compared to WT, recombinant apoA-I[K107del] adsorbed to surfaces of POPC-coated triolein drops at faster rates, remodeled the surfaces to a greater extent, and was ejected from the surfaces at higher surface pressures on compression of the lipid drops. These properties may drive increased binding of apoA-I[K107del] to and its better retention on large triglyceride-rich lipoproteins, thereby increasing the variant's content on these lipoproteins. While K107del did not affect apoA-I capacity to promote ABCA1-mediated cholesterol efflux from J774 cells, it impaired the biogenesis of large nascent HDL particles resulting in the formation of predominantly smaller nascent HDL. Size-exclusion chromatography of spontaneously reconstituted 1,2-dimyristoylphosphatidylcholine-apoA-I complexes showed that apoA-I[K107del] had a hampered ability to form larger complexes but formed efficiently smaller-sized complexes. CD analysis revealed a reduced ability of apoA-I[K107del] to increase α-helical structure on binding to 1,2-dimyristoylphosphatidylcholine or in the presence of trifluoroethanol. This property may hinder the formation of large apoA-I[K107del]-containing discoidal and spherical HDL but not smaller HDL. Both factors, the increased content of apoA-I[K107del] on triglyceride-rich lipoproteins and the impaired ability of the variant to stabilize large HDL particles resulting in reduced lipid:protein ratios in HDL, may contribute to normal plasma apoA-I levels along with low HDL-C and increased risk for CVD.

NF-κB inhibitor alpha has a cross-variant role during SARS-CoV-2 infection in ACE2-overexpressing human airway organoids.

As SARS-CoV-2 continues to spread worldwide, tractable primary airway cell models that accurately recapitulate the cell-intrinsic response to arising viral variants are needed. Here we describe an adult stem cell-derived human airway organoid model overexpressing the ACE2 receptor that supports robust viral replication while maintaining 3D architecture and cellular diversity of the airway epithelium. ACE2-OE organoids were infected with SARS-CoV-2 variants and subjected to single-cell RNA-sequencing. NF-κB inhibitor alpha was consistently upregulated in infected epithelial cells, and its mRNA expression positively correlated with infection levels. Confocal microscopy showed more IκBα expression in infected than bystander cells, but found concurrent nuclear translocation of NF-κB that IκBα usually prevents. Overexpressing a nondegradable IκBα mutant reduced NF-κB translocation and increased viral infection. These data demonstrate the functionality of ACE2-OE organoids in SARS-CoV-2 research and identify an incomplete NF-κB feedback loop as a rheostat of viral infection that may promote inflammation and severe disease.

Modelling T-cell immunity against hepatitis C virus with liver organoids in a microfluidic coculture system.

Hepatitis C virus (HCV) remains a global public health challenge with an estimated 71 million people chronically infected, with surges in new cases and no effective vaccine. New methods are needed to study the human immune response to HCV since in vivo animal models are limited and in vitro cancer cell models often show dysregulated immune and proliferative responses. Here, we developed a CD8+ T cell and adult stem cell liver organoid system using a microfluidic chip to coculture 3D human liver organoids embedded in extracellular matrix with HLA-matched primary human T cells in suspension. We then employed automated phase contrast and immunofluorescence imaging to monitor T cell invasion and morphological changes in the liver organoids. This microfluidic coculture system supports targeted killing of liver organoids when pulsed with a peptide specific for HCV non-structural protein 3 (NS3) (KLVALGINAV) in the presence of patient-derived CD8+ T cells specific for KLVALGINAV. This demonstrates the novel potential of the coculture system to molecularly study adaptive immune responses to HCV in an in vitro setting using primary human cells.

A genome-wide CRISPR screen identifies UFMylation and TRAMP-like complexes as host factors required for hepatitis A virus infection.

Hepatitis A virus (HAV) is a positive-sense RNA virus causing acute inflammation of the liver. Here, using a genome-scale CRISPR screen, we provide a comprehensive picture of the cellular factors that are exploited by HAV. We identify genes involved in sialic acid/ganglioside biosynthesis and members of the eukaryotic translation initiation factor complex, corroborating their putative roles for HAV. Additionally, we uncover all components of the cellular machinery for UFMylation, a ubiquitin-like protein modification. We show that HAV translation specifically depends on UFM1 conjugation of the ribosomal protein RPL26. Furthermore, we find that components related to the yeast Trf4/5-Air1/2-Mtr4 polyadenylation (TRAMP) complex are required for viral translation independent of controlling viral poly(A) tails or RNA stability. Finally, we demonstrate that pharmacological inhibition of the TRAMP-like complex decreases HAV replication in hepatocyte cells and human liver organoids, thus providing a strategy for host-directed therapy of HAV infection.

Aromatic residues in the C terminus of apolipoprotein C-III mediate lipid binding and LPL inhibition.

Plasma apoC-III levels correlate with triglyceride (TG) levels and are a strong predictor of CVD outcomes. ApoC-III elevates TG in part by inhibiting LPL. ApoC-III likely inhibits LPL by competing for lipid binding. To probe this, we used oil-drop tensiometry to characterize binding of six apoC-III variants to lipid/water interfaces. This technique monitors the dependence of lipid binding on surface pressure, which increases during TG hydrolysis by LPL. ApoC-III adsorption increased surface pressure by upward of 18 mN/m at phospholipid/TG/water interfaces. ApoC-III was retained to high pressures at these interfaces, desorbing at 21-25 mN/m. Point mutants, which substituted alanine for aromatic residues, impaired the lipid binding of apoC-III. Adsorption and retention pressures decreased by 1-6 mN/m in point mutants, with the magnitude determined by the location of alanine substitutions. Trp42 was most critical to mediating lipid binding. These results strongly correlate with our previous results, linking apoC-III point mutants to increased LPL binding and activity at lipid surfaces. We propose that aromatic residues in the C-terminal half of apoC-III mediate binding to TG-rich lipoproteins. Increased apoC-III expression in the hypertriglyceridemic state allows apoC-III to accumulate on lipoproteins and inhibit LPL by preventing binding and/or access to substrate.

Entangled in a membranous web: ER and lipid droplet reorganization during hepatitis C virus infection.

Hepatitis C virus (HCV) is a major cause of liver disease worldwide. To establish and maintain chronic infection, HCV extensively rearranges cellular organelles to generate distinct compartments for viral RNA replication and virion assembly. Here, we review our current knowledge of how HCV proliferates and remodels ER-derived membranes while preserving and expanding associated lipid droplets during viral infection. Unraveling the molecular mechanisms responsible for HCV-induced membrane reorganization will enhance our understanding of the HCV life-cycle, the associated liver pathology, and the biology of the ER:lipid droplet interface in general.

A Pressure-dependent Model for the Regulation of Lipoprotein Lipase by Apolipoprotein C-II.

Apolipoprotein C-II (apoC-II) is the co-factor for lipoprotein lipase (LPL) at the surface of triacylglycerol-rich lipoproteins. LPL hydrolyzes triacylglycerol, which increases local surface pressure as surface area decreases and amphipathic products transiently accumulate at the lipoprotein surface. To understand how apoC-II adapts to these pressure changes, we characterized the behavior of apoC-II at multiple lipid/water interfaces. ApoC-II adsorption to a triacylglycerol/water interface resulted in large increases in surface pressure. ApoC-II was exchangeable at this interface and desorbed on interfacial compressions. These compressions increase surface pressure and mimic the action of LPL. Analysis of gradual compressions showed that apoC-II undergoes a two-step desorption, which indicates that lipid-bound apoC-II can exhibit at least two conformations. We characterized apoC-II at phospholipid/triacylglycerol/water interfaces, which more closely mimic lipoprotein surfaces. ApoC-II had a large exclusion pressure, similar to that of apoC-I and apoC-III. However, apoC-II desorbed at retention pressures higher than those seen with the other apoCs. This suggests that it is unlikely that apoC-I and apoC-III inhibit LPL via displacement of apoC-II from the lipoprotein surface. Upon rapid compressions and re-expansions, re-adsorption of apoC-II increased pressure by lower amounts than its initial adsorption. This indicates that apoC-II removed phospholipid from the interface upon desorption. These results suggest that apoC-II regulates the activity of LPL in a pressure-dependent manner. ApoC-II is provided as a component of triacylglycerol-rich lipoproteins and is the co-factor for LPL as pressure increases. Above its retention pressure, apoC-II desorbs and removes phospholipid. This triggers release of LPL from lipoproteins.

Changes in helical content or net charge of apolipoprotein C-I alter its affinity for lipid/water interfaces.

Amphipathic α-helices mediate binding of exchangeable apolipoproteins to lipoproteins. To probe the role of α-helical structure in protein-lipid interactions, we used oil-drop tensiometry to characterize the interfacial behavior of apolipoprotein C-I (apoC-I) variants at triolein/water (TO/W) and 1-palmitoyl-2-oleoylphosphatidylcholine/triolein/water (POPC/TO/W) interfaces. ApoC-I, the smallest apolipoprotein, has two amphipathic α-helices. Mutants had single Pro or Ala substitutions that resulted in large differences in helical content in solution and on phospholipids. The ability of apoC-I to bind TO/W and POPC/TO/W interfaces correlated strongly with α-helical propensity. On binding these interfaces, peptides with higher helical propensity increased surface pressure to a greater extent. Likewise, peptide exclusion pressure at POPC/TO/W interfaces increased with greater helical propensity. ApoC-I retention on TO/W and POPC/TO/W interfaces correlated strongly with phospholipid-bound helical content. On compression of these interfaces, peptides with higher helical content were ejected at higher pressures. Substitution of Arg for Pro in the N-terminal α-helix altered net charge and reduced apoC-I affinity for POPC/TO/W interfaces. Our results suggest that peptide-lipid interactions drive α-helix binding to and retention on lipoproteins. Point mutations in small apolipoproteins could significantly change α-helical propensity or charge, thereby disrupting protein-lipid interactions and preventing the proteins from regulating lipoprotein catabolism at high surface pressures.

Apolipoprotein C-I binds more strongly to phospholipid/triolein/water than triolein/water interfaces: a possible model for inhibiting cholesterol ester transfer protein activity and triacylglycerol-rich lipoprotein uptake.

Apolipoprotein C-I (apoC-I) is an important constituent of high-density lipoprotein (HDL) and is involved in the accumulation of cholesterol ester in nascent HDL via inhibition of cholesterol ester transfer protein and potential activation of lecithin:cholesterol acyltransferase (LCAT). As the smallest exchangeable apolipoprotein (57 residues), apoC-I transfers between lipoproteins via a lipid-binding motif of two amphipathic α-helices (AαHs), spanning residues 7-29 and 38-52. To understand apoC-I's behavior at hydrophobic lipoprotein surfaces, oil drop tensiometry was used to compare the binding to triolein/water (TO/W) and palmitoyloleoylphosphatidylcholine/triolein/water (POPC/TO/W) interfaces. When apoC-I binds to either interface, the surface tension (γ) decreases by ~16-18 mN/m. ApoC-I can be exchanged at both interfaces, desorbing upon compression and readsorbing on expansion. The maximal surface pressures at which apoC-I begins to desorb (Π(max)) were 16.8 and 20.7 mN/m at TO/W and POPC/TO/W interfaces, respectively. This suggests that apoC-I interacts with POPC to increase its affinity for the interface. ApoC-I is more elastic on POPC/TO/W than TO/W interfaces, marked by higher values of the elasticity modulus (ε) on oscillations. At POPC/TO/W interfaces containing an increasing POPC:TO ratio, the pressure at which apoC-I begins to be ejected increases as the phospholipid surface concentration increases. The observed increase in apoC-I interface affinity due to higher degrees of apoC-I-POPC interactions may explain how apoC-I can displace larger apolipoproteins, such as apoE, from lipoproteins. These interactions allow apoC-I to remain bound to the interface at higher Π values, offering insight into apoC-I's rearrangement on triacylglycerol-rich lipoproteins as they undergo Π changes during lipoprotein maturation by plasma factors such as lipoprotein lipase.