A robust cell culture system supporting the complete life cycle of hepatitis B virus

Michailidis, Eleftherios; Pabon, Jonathan; Xiang, Kuanhui; Park, Paul; Ramanan, Vyas; Hoffmann, Hans-Heinrich; Schneider, William M.; Bhatia, Sangeeta N.; Jong, Ype P. de; Shlomai, Amir; Rice, Charles M.

doi:10.1038/s41598-017-16882-5

Cited by 64 publications

(89 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Huh7 cells were seeded, transduced with individual lentiviruses, and 48 hours post-transduction infected with HCoV-229E at 33°C. Infection was stopped 24 hours (MOI=0.1) or 48 hours (MOI=0.01) postinfection and plates were immuno-stained as described previously 61 with an anti-HCoV-229E N protein antibody and a AlexaFluor488-conjugated donkey anti-mouse secondary antibody (Extended methods;…”

Section: Isg Screenmentioning

confidence: 99%

LY6E impairs coronavirus fusion and confers immune control of viral disease

Pfaender

Mar

Michailidis

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

Zoonotic coronaviruses (CoVs) are significant threats to global health, as exemplified by the recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 1 . Host immune responses to CoV are complex and regulated in part through antiviral interferons.However, the interferon-stimulated gene products that inhibit CoV are not well characterized 2 . Here, we show that interferon-inducible lymphocyte antigen 6 complex, locus E (LY6E) potently restricts cellular infection by multiple CoVs, including SARS-CoV, SARS-CoV-2, and Middle East respiratory syndrome coronavirus (MERS-CoV). Mechanistic studies revealed that LY6Einhibits CoV entry into cells by interfering with spike protein-mediated membrane fusion. Importantly, mice lacking Ly6e in hematopoietic cells were highly susceptible to murine CoV infection. Exacerbated viral pathogenesis in Ly6e knockout mice was accompanied by loss of hepatic and splenic immune cells and reduction in global antiviral gene pathways. Accordingly, we found that Ly6e directly protects primary B cells and dendritic cells from murine CoV infection. Our results demonstrate that LY6E is a critical antiviral immune effector that controlsCoV infection and pathogenesis. These findings advance our understanding of immune-mediated control of CoV in vitro and in vivo, knowledge that could help inform strategies to combat infection by emerging CoV. author/funder. All rights reserved. No reuse allowed without permission.

show abstract

Section: Isg Screenmentioning

confidence: 99%

LY6E impairs coronavirus fusion and confers immune control of viral disease

Pfaender

Mar

Michailidis

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…These individuals displayed a broad spectrum of anti-HBs titers (x-axis in Figure 1A and Figure S1B; Table S1). To determine their neutralizing activity, we tested their ability to block HBV infection in sodium taurocholate co-transporting polypeptide (NTCP)-overexpressing HepG2 cells (Michailidis et al, 2017; Yan et al, 2012) (y-axis in Figure 1A and Figure S1B and S1C; Table S1). Sera or antibodies purified from individuals with high neutralizing titers (elite neutralizers) were then compared across a wide range of dilutions (Figure 1B and 1C).…”

Section: Resultsmentioning

confidence: 99%

“…To determine whether the new monoclonals neutralize HBV in vitro , we performed neutralization assays using HepG2-NTCP cells (Figure 5A and 5B). The 50% inhibitory concentration (IC 50 ) values were calculated based on HBsAg/HBeAg ELISA or immunofluorescence staining for HBcAg expression (Michailidis et al, 2017) (Figure 5C). Neutralizing activity was further verified by in vitro neutralization assays using primary human hepatocytes (Michailidis et al, 2020) (Figure 5C and 5D).…”

Section: Resultsmentioning

confidence: 99%

Public broadly neutralizing antibodies against hepatitis B virus in individuals with elite serologic activity

Wang

Michailidis

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

SUMMARYAlthough there is no effective cure for chronic hepatitis B virus (HBV) infection, antibodies are protective and constitute clinical correlates of recovery from infection. To examine the human neutralizing antibody response to HBV in elite neutralizers we screened 144 individuals. The top individuals produced shared clones of broadly neutralizing antibodies (bNAbs) that targeted 3 non-overlapping epitopes on the HBV S antigen (HBsAg). Single bNAbs protected humanized mice against infection, but selected for resistance mutations in mice with established infection. In contrast, infection was controlled by a combination of bNAbs targeting non-overlapping epitopes with complementary sensitivity to mutations that commonly emerge during human infection. The co-crystal structure of one of the bNAbs with a peptide epitope containing residues frequently mutated in human immune escape variants revealed a loop anchored by oppositely charged residues. The structure provides a molecular explanation for why immunotherapy for HBV infection may require combinations of complementary bNAbs.

show abstract

“…Cell culture: Human HCC cell lines HepAD38 (25), HepG2 (7), HepG2-NTCP clone 7 (26) and HepaRG cells (27) were grown as described. Cell lines were routinely tested for mycoplasma.…”

Section: Methodsmentioning

confidence: 99%

“…Plasmids used are listed in Supporting Table S1. Infection assays of HepaRG and HepG2-NTCP cell lines were performed as described (26,27), employing 100 HBV genome equivalents per cell. Wnt Reporter Assay: HBV replicating HepAD38 cells (5x10 4 cells per well of 24 well-plate, day 3 of HBV replication) were co-transfected with TOPflash vector (25ng) containing TCF-binding sites upstream of firefly luciferase, and Renilla luciferase vector (25ng).…”

Section: Transfection and Infection Assays: Hepg2 And Hepad38 Cells (mentioning

confidence: 99%

RNA Helicase DDX5 Negatively Regulates Wnt Signaling and Hepatocyte Reprogramming in Hepatitis B Virus-related Hepatocellular Carcinoma

Mani

Cui

Yan

et al. 2019

Preprint

View full text Add to dashboard Cite

Keywords: Hepatitis B Virus, Hepatocellular Carcinoma, RNA helicase DDX5/p68, miR17~92, miR106b~25, RNA-seq, Wnt/β-catenin/ hepatocyte reprogramming/ hepatic cancer stem cell/ antagomirs Word count: 6,200 (without references) ABSTRACT Background and Aims: RNA helicase DEAD box protein 5 (DDX5) is downregulated during hepatitis B virus (HBV) replication, and associates with poor prognosis HBV-related hepatocellular carcinoma (HCC). The aim of this study is to determine the mechanism and significance of DDX5 downregulation for HBV-driven HCC. Approach & Results: We used established cellular models of HBV replication, HBV infection, as well as HBV-related liver tumors. HBV replicating DDX5 knockdown hepatocytes were analyzed by RNAseq; differentially expressed genes were validated by qRT-PCR, and bioinformatic analyses of HCCs from The Cancer Genome Atlas. Our results show reduced expression of DDX5 in HCCs of all etiologies is associated with poor survival. In HBV replicating hepatocytes, downregulation of DDX5 is mediated by miR17~92 and miR106b~25, induced by HBV infection. Increased expression of these miRNAs was quantified in HBV-associated HCCs expressing a hepatic cancer stem cell (hCSC)-like gene signature and reduced DDX5 mRNA, suggesting a role for DDX5 in hCSC formation. Interestingly, DDX5 knockdown in HBV replicating hepatocyte cell lines resulted in hepatosphere formation, sorafenib and cisplatin resistance, Wnt signaling activation and pluripotency gene expression, all characteristics of hCSCs. Moreover, DDX5 knockdown increased viral replication. RNA-seq analyses of HBV-replicating DDX5 knockdown cells, identified enhanced expression of key genes of the Wnt/β-catenin pathway, including Frizzled7 (FZD7) and Matrix Metallopeptidase7 (MMP7), indicative of Wnt signaling activation. Clinically, elevated FZD7 expression correlates with poor patient survival. Importantly, inhibitors to miR17~92 and miR106b~25 restored DDX5 levels and suppressed both Wnt/β-catenin activation and viral replication. Conclusion: DDX5 is a negative regulator of Wnt signaling and hepatocyte reprogramming in HCCs. Restoration of DDX5 levels in HBV-infected patients can exert both antitumor and antiviral effects.

show abstract

A robust cell culture system supporting the complete life cycle of hepatitis B virus

Cited by 64 publications

References 27 publications

LY6E impairs coronavirus fusion and confers immune control of viral disease

LY6E impairs coronavirus fusion and confers immune control of viral disease

Public broadly neutralizing antibodies against hepatitis B virus in individuals with elite serologic activity

RNA Helicase DDX5 Negatively Regulates Wnt Signaling and Hepatocyte Reprogramming in Hepatitis B Virus-related Hepatocellular Carcinoma

Contact Info

Product

Resources

About