scholarly journals Establishment of a novel hepatitis B virus culture system using immortalized human hepatocytes

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yuichi Akahori ◽  
Hiroki Kato ◽  
Takashi Fujita ◽  
Kohji Moriishi ◽  
Yasuhito Tanaka ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Cell Lines ◽  
Culture System ◽  
Sodium Taurocholate ◽  
Three Dimensional ◽  
Human Hepatocytes ◽  
Host Cells ◽  
B Virus ◽  
Almost All

AbstractRecent development of hepatitis B virus (HBV) culture systems has made it possible to analyze the almost all steps of the viral life cycle. However, the reproducibility of interaction between HBV and host cells seemed inaccurate in those systems because of utilization of cancer cell lines with a difference from hepatocytes in the majority of cases. In this study, in order to resolve this point, a novel HBV culture system using non-cancer-derived immortalized human hepatocytes derived cell lines, producing exogenous human sodium taurocholate cotransporting polypeptide, was developed. One of the cell clones, E/NtG8 cells, was permissive to both blood-borne HBV (HBVbb) and culture-derived recombinant HBV when cultured in the three-dimensional condition. Furthermore, the production of infectious HBV particles, which showed the similar physicochemical properties to HBVbb, was observed for about a month after HBVbb infection in this system, suggesting that it may reproduce whole steps of the HBV lifecycle under the condition analogous to human liver cells infected with HBV. This system seemed to contribute not only to find novel interactions between HBV and host cells but also to understand mechanism of HBV pathogenesis.

NTCP oligomerization occurs downstream of the NTCP-EGFR interaction during hepatitis B virus internalization

2021 ◽  
Author(s):  
Kento Fukano ◽  
Mizuki Oshima ◽  
Senko Tsukuda ◽  
Hideki Aizaki ◽  
Mio Ohki ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Bile Acid ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Viral Entry ◽  
Sodium Taurocholate ◽  
Growth Factor Receptor ◽  
Host Cells ◽  
B Virus ◽  
Epidermal Growth

Sodium taurocholate cotransporting polypeptide (NTCP) is a receptor that is essential for hepatitis B virus (HBV) entry into the host cell. A number of HBV entry inhibitors targeting NTCP have been reported to date; these inhibitors have facilitated a mechanistic analysis of the viral entry process. However, the mechanism of HBV internalization into host cells after interaction of virus with NTCP remains largely unknown. Recently, we reported that troglitazone, a thiazolidinedione derivative, specifically inhibits both HBV internalization and NTCP oligomerization, resulting in inhibition of HBV infection. Here, using troglitazone as a chemical probe to investigate entry process, the contribution of NTCP oligomerization to HBV internalization was evaluated. Using surface plasmon resonance and transporter kinetics, we found that troglitazone directly interacts with NTCP and non-competitively interferes with NTCP-mediated bile acid uptake, suggesting that troglitazone allosterically binds to NTCP, rather than to the bile acid-binding pocket. Additionally, alanine scanning mutagenesis showed that a mutation at phenylalanine 274 of NTCP (F274A) caused a loss of HBV susceptibility and disrupted both the oligomerization of NTCP and HBV internalization without affecting viral attachment to the cell surface. An inhibitor of the interaction between NTCP and epidermal growth factor receptor (EGFR), another host cofactor essential for HBV internalization, impeded NTCP oligomerization. Meanwhile, co-immunoprecipitation analysis revealed that neither troglitazone nor the F274A mutation in NTCP affect the NTCP-EGFR interaction. These findings suggest that NTCP oligomerization is initiated downstream of the NTCP-EGFR interaction, and then triggers HBV internalization. This study provides significant insight into the HBV entry mechanisms. Importance Hepatitis B virus (HBV) infection is mediated by a specific interaction with sodium taurocholate cotransporting polypeptide (NTCP), a viral entry receptor. Although the virus-receptor interactions are believed to trigger viral internalization into host cells, the exact molecular mechanisms of HBV internalization are not understood. In this study, we revealed the mode of action whereby troglitazone, a specific inhibitor of HBV internalization, impedes NTCP oligomerization, and identified NTCP phenylalanine 274 as a residue essential for this oligomerization. We further analyzed the association between NTCP oligomerization and HBV internalization, a process that is mediated by epidermal growth factor receptor (EGFR), another essential host cofactor for HBV internalization. Our study provides critical information on the mechanism of HBV entry, and suggests that oligomerization of the viral receptor serves as an attractive target for drug discovery.

scholarly journals Murine Retroviral Pseudotype Virus Containing Hepatitis B Virus Large and Small Surface Antigens Confers Specific Tropism for Primary Human Hepatocytes: a Potential Liver-Specific Targeting System

2002 ◽  
Vol 76 (2) ◽  
pp. 912-917 ◽  
Author(s):  
Vicky M.-H. Sung ◽  
Michael M. C. Lai
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Cell Lines ◽  
Surface Antigens ◽  
Human Hepatocytes ◽  
Virus Infectivity ◽  
Specific Targeting ◽  
Primary Human Hepatocytes ◽  
Pseudotype Virus ◽  
B Virus

ABSTRACT We have developed a system for producing murine leukemia virus (MLV) pseudotyped with human hepatitis B virus (HBV) large (L) and small (S) surface antigens (HBsAg) for targeting primary human hepatocytes. Using the MLV(HBV) pseudotype virus containing a β-galactosidase reporter gene, we demonstrated that this pseudotype virus exhibits strict tropism for primary human hepatocytes, similar to the natural target cell specificity of HBV. It does not infect any of the established tissue culture cell lines, including human hepatoma cell lines (HepG2 and Huh-7), or rat primary hepatocytes. The infectivity of MLV(HBV) for human hepatocytes was inhibited by anti-HBs antibody. The L form of HBsAg was both necessary and sufficient for virus infectivity, but the presence of both L and S forms enhanced the surface expression of HBsAg and thus increased virus production. The middle form of HBsAg was not necessary. This pseudotype virus bypasses the requirement for the liver-specific transcription factors for HBV replication, enabling direct study of HBV tissue tropism conferred by the viral envelope proteins. This virus also offers a potential liver-specific targeting system for gene therapy.

scholarly journals A short HBV RNA region induces RNR-R2 expression in non-cycling cells and in primary human hepatocytes

2018 ◽  
Author(s):  
Inna Ricardo-Lax ◽  
Karin Broennimann ◽  
Julia Adler ◽  
Eleftherios Michailidis ◽  
Ype P de Jong ◽  
...  
Keyword(s):  
Dna Damage ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Dna Damage Response ◽  
Human Hepatocytes ◽  
Host Cells ◽  
Primary Human Hepatocytes ◽  
B Virus ◽  
Damage Response ◽  
Cellular Dna

AbstractHepatitis B virus infects non-dividing cells in which dNTPs are scarce. HBV replication requires dNTPs. To cope with this constraint the virus induces the DNA damage response (DDR) pathway culminating in RNR-R2 expression and the generation of an active RNR holoenzyme, the key regulator of dNTP levels. Previously we reported that the HBx open reading frame (ORF) triggers this pathway. Unexpectedly however, we report here that the production of HBx protein is not essential. We found that a small region of 125 bases within the HBx transcript is sufficient to induce RNR-R2 expression in growth arrested HepG2 cells and in primary human hepatocytes (PHH). The observed HBx embedded regulatory element is named ERE. We demonstrate that ERE is functional as a positive strand RNA polymerase-II transcript. Remarkably, ERE is sufficient to induce the Chk1-E2F1-RNR-R2 DDR pathway, previously reported to be activated by HBV. Furthermore, we found that ERE activates ATR but not ATM in eliciting this DDR pathway in upregulating RNR-R2. These findings demonstrate the multitasking role of HBV transcripts in mediating virus-host cell interaction, a mechanism that explains how such a small genome effectively serves such a pervasive virus.Author summaryThe hepatitis B virus (HBV) infects the human liver and over 250 million people worldwide are chronically infected with HBV and at risk for cirrhosis and liver cancer. HBV has a very small DNA genome with only four genes, much fewer than other viruses. For propagation the virus consumes dNTPs, the building blocks of DNA, in much higher amounts than the infected cells provide. To cope with this constraint, the virus manipulates the cells to increase the production of dNTPs. We found that the virus activates the cellular response to DNA damage upon which the cells increase the production of dNTPs, but instead of repairing cellular DNA, the virus uses them for production of its own DNA. Usually viruses manipulate host cells with one or more of their unique proteins, however the small HBV genome cannot afford having such a unique gene and protein. Instead, we found that here the virus relies on RNA to manipulate the host cells. Our findings highlight the unprecedented principle of a multitasking viral RNA that is not only designed to be translated into proteins but also harbors an independent role in activating the cellular DNA damage response.

scholarly journals E-cadherin binds glycosylated sodium-taurocholate cotransporting polypeptide to facilitate hepatitis B virus entry

2019 ◽  
Author(s):  
Qin Hu ◽  
Fei-Fei Zhang ◽  
Liang Duan ◽  
Bo Wang ◽  
Pu Li ◽  
...  
Keyword(s):  
Public Health ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Sodium Taurocholate ◽  
Hbv Infection ◽  
Host Cells ◽  
Heparg Cells ◽  
B Virus ◽  
Hepatocyte Polarity ◽  
E Cadherin

AbstractHepatitis B virus (HBV) continues to pose a serious public health risk and is one of the major causes of chronic liver disease and hepatocellular carcinoma. Current antiviral therapy does not effectively eradicate HBV and, thus, further investigation into the mechanisms employed by HBV to allow for invasion of host cells, is critical for the development of novel therapeutic agents. Sodium-taurocholate cotransporting polypeptide (NTCP) has been identified as a functional receptor for HBV. However, the specific mechanism by which HBV and NTCP interact remains unclear. Herein we show that the expression of E-cadherin was upregulated in cells expressing HBV, while knockdown of E-cadherin in HepG2-NTCP cells, HepaRG cells and primary human hepatocytes served to significantly inhibit infection by HBV and HBV pseudotyped particles. Alternatively, exogenous E-cadherin expression was found to significantly enhance HBV uptake by HepaRG cells. Further, mechanistic studies identified glycosylated NTCP localized to the cell membrane via E-cadherin binding, which subsequently allowed for more efficient binding between NTCP and the preS1 of the large HBV surface proteins. E-cadherin was also found to play a key role in establishing and maintaining hepatocyte polarity, which is essential for efficient HBV infection. These observations suggest that E-cadherin facilitates HBV entry through regulation of NTCP distribution and hepatocyte polarity.Author SummaryHepatitis B Virus (HBV) still seriously endangers public health. It is very important to understand the mechanism of HBV invading host cells for developing new therapy target. Sodium-taurocholate cotransporting polypeptide (NTCP) is the key receptor mediating HBV invasion, while other molecules also exhibit important roles in ensuring efficient and productive HBV infection. This study reports that E-cadherin facilitates HBV entry by directly interacting with glycosylated NTCP to mediate its distribution on the hepatocyte membrane and also affects the efficacy of HBV invasion by influncing hepatocyte polarity.

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