Cell death-inducing DFFA-like effector b is required for hepatitis C virus entry into hepatocytes.

UNLABELLED: The molecular mechanism of the hepatic tropism of hepatitis C virus (HCV) remains incompletely defined. In vitro hepatic differentiation of pluripotent stem cells produces hepatocyte-like cells (HLCs) permissive for HCV infection, providing an opportunity for studying liver development and host determinants of HCV susceptibility. We previously identified the transition stage of HCV permissiveness and now investigate whether a host protein whose expression is induced during this transition stage is important for HCV infection. We suppressed the expression of a liver-specific protein, cell death-inducing DFFA-like effector b (CIDEB), and performed hepatocyte function and HCV infection assays. We also used a variety of cell-based assays to dissect the specific step of the HCV life cycle that potentially requires CIDEB function. We found CIDEB to be an essential cofactor for HCV entry into hepatocytes. Genetic interference with CIDEB in stem cells followed by hepatic differentiation leads to HLCs that are refractory to HCV infection, and infection time course experiments revealed that CIDEB functions in a late step of HCV entry, possibly to facilitate membrane fusion. The role of CIDEB in mediating HCV entry is distinct from those of the well-established receptors, as it is not required for HCV pseudoparticle entry. Finally, HCV infection effectively downregulates CIDEB protein through a posttranscriptional mechanism. IMPORTANCE: This study identifies a hepatitis C virus (HCV) entry cofactor that is required for HCV infection of hepatocytes and potentially facilitates membrane fusion between viral and host membranes. CIDEB and its interaction with HCV may open up new avenues of investigation of lipid droplets and viral entry.

Phosphatidylinositol 4-kinase IIIß is required for severe acute respiratory syndrome coronavirus spike-mediated cell entry.

Phosphatidylinositol kinases (PI kinases) play an important role in the life cycle of several viruses after infection. Using gene knockdown technology, we demonstrate that phosphatidylinositol 4-kinase IIIß (PI4KB) is required for cellular entry by pseudoviruses bearing the severe acute respiratory syndrome-coronavirus (SARS-CoV) spike protein and that the cell entry mediated by SARS-CoV spike protein is strongly inhibited by knockdown of PI4KB. Consistent with this observation, pharmacological inhibitors of PI4KB blocked entry of SARS pseudovirions. Further research suggested that PI4P plays an essential role in SARS-CoV spike-mediated entry, which is regulated by the PI4P lipid microenvironment. We further demonstrate that PI4KB does not affect virus entry at the SARS-CoV S-ACE2 binding interface or at the stage of virus internalization but rather at or before virus fusion. Taken together, these results indicate a new function for PI4KB and suggest a new drug target for preventing SARS-CoV infection.

Intrinsic antiviral immunity of barrier cells revealed by an iPSC-derived blood-brain barrier cellular model.

Physiological blood-tissue barriers play a critical role in separating the circulation from immune-privileged sites and denying access to blood-borne viruses. The mechanism of virus restriction by these barriers is poorly understood. We utilize induced pluripotent stem cell (iPSC)-derived human brain microvascular endothelial cells (iBMECs) to study virus-blood-brain barrier (BBB) interactions. These iPSC-derived cells faithfully recapitulate a striking difference in in vivo neuroinvasion by two alphavirus isolates and are selectively permissive to neurotropic flaviviruses. A model of cocultured iBMECs and astrocytes exhibits high transendothelial electrical resistance and blocks non-neurotropic flaviviruses from getting across the barrier. We find that iBMECs constitutively express an interferon-induced gene, IFITM1, which preferentially restricts the replication of non-neurotropic flaviviruses. Barrier cells from blood-testis and blood-retinal barriers also constitutively express IFITMs that contribute to the viral resistance. Our application of a renewable human iPSC-based model for studying virus-BBB interactions reveals that intrinsic immunity at the barriers contributes to virus exclusion.

An hPSC-Derived Tissue-Resident Macrophage Model Reveals Differential Responses of Macrophages to ZIKV and DENV Infection.

Zika virus (ZIKV) and dengue virus (DENV) are two closely related flaviviruses that lead to different clinical outcomes. The mechanism for the distinct pathogenesis of ZIKV and DENV is poorly understood. Here, we investigate ZIKV and DENV infection of macrophages using a human pluripotent stem cell (hPSC)-derived macrophage model and discover key virus-specific responses. ZIKV and DENV productively infect hPSC-derived macrophages. DENV, but not ZIKV, infection of macrophages strongly activates macrophage migration inhibitory factor (MIF) secretion and decreases macrophage migration. Neutralization of MIF leads to improved migratory ability of DENV-infected macrophages. In contrast, ZIKV-infected macrophages exhibit prolonged migration and express low levels of pro-inflammatory cytokines and chemokines. Mechanistically, ZIKV disrupts the nuclear factor κB (NF-κB)-MIF positive feedback loop by inhibiting the NF-κB signaling pathway. Our results demonstrate the utility of hPSC-derived macrophages in infectious disease modeling and suggest that the distinct impact of ZIKV and DENV on macrophage immune response may underlie different pathogenesis of Zika and dengue diseases.

Modeling Dengue Virus-Hepatic Cell Interactions Using Human Pluripotent Stem Cell-Derived Hepatocyte-like Cells.

The development of dengue antivirals and vaccine has been hampered by the incomplete understanding of molecular mechanisms of dengue virus (DENV) infection and pathology, partly due to the limited suitable cell culture or animal models that can capture the comprehensive cellular changes induced by DENV. In this study, we differentiated human pluripotent stem cells (hPSCs) into hepatocytes, one of the target cells of DENV, to investigate various aspects of DENV-hepatocyte interaction. hPSC-derived hepatocyte-like cells (HLCs) supported persistent and productive DENV infection. The activation of interferon pathways by DENV protected bystander cells from infection and protected the infected cells from massive apoptosis. Furthermore, DENV infection activated the NF-κB pathway, which led to production of proinflammatory cytokines and downregulated many liver-specific genes such as albumin and coagulation factor V. Our study demonstrates the utility of hPSC-derived hepatocytes as an in vitro model for DENV infection and reveals important aspects of DENV-host interactions.

Inhibition of SARS pseudovirus cell entry by lactoferrin binding to heparan sulfate proteoglycans.

It has been reported that lactoferrin (LF) participates in the host immune response against Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) invasion by enhancing NK cell activity and stimulating neutrophil aggregation and adhesion. We further investigated the role of LF in the entry of SARS pseudovirus into HEK293E/ACE2-Myc cells. Our results reveal that LF inhibits SARS pseudovirus infection in a dose-dependent manner. Further analysis suggested that LF was able to block the binding of spike protein to host cells at 4°C, indicating that LF exerted its inhibitory function at the viral attachment stage. However, LF did not disrupt the interaction of spike protein with angiotensin-converting enzyme 2 (ACE2), the functional receptor of SARS-CoV. Previous studies have shown that LF colocalizes with the widely distributed cell-surface heparan sulfate proteoglycans (HSPGs). Our experiments have also confirmed this conclusion. Treatment of the cells with heparinase or exogenous heparin prevented binding of spike protein to host cells and inhibited SARS pseudovirus infection, demonstrating that HSPGs provide the binding sites for SARS-CoV invasion at the early attachment phase. Taken together, our results suggest that, in addition to ACE2, HSPGs are essential cell-surface molecules involved in SARS-CoV cell entry. LF may play a protective role in host defense against SARS-CoV infection through binding to HSPGs and blocking the preliminary interaction between SARS-CoV and host cells. Our findings may provide further understanding of SARS-CoV pathogenesis and aid in treatment of this deadly disease.

Identification of peptide mimetics of xenoreactive alpha-Gal antigenic epitope by phage display.

The prevention of hyperacute rejection (HAR) triggered by interaction between the human natural antibody and xenoreactive antigenic epitope (Gal-alpha1, 3Gal) present on pig cells is the key to success in pig-to-human xenotransplantation. The phage display technology offers an effective strategy for screening peptides which can interact with the anti-Gal antibody to block alpha-Gal antigen binding site. Two peptide libraries, linear 7 peptide library and C7C library, were panned on the anti-B monoclonal antibody which has the characteristic of binding to the alpha-Gal antigenic epitope. After four rounds of panning, 22 positive phage clones were selected. Highly homologous sequence PT and STL existed among these selected peptides. Stachyose competitive ELISAs revealed that these peptides specifically bound to alpha-Gal antigen binding site. Eight peptide mimics of alpha-Gal antigenic epitope could inhibit the agglutination of pig red blood cells mediated by human sera in a dose-dependent manner. These results demonstrated that the selected peptides can mimic the conformational structure of alpha-Gal antigenic epitope and have the therapeutic potential in xenotransplantation.