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Defined host factors support HBV infection in non-hepatic 293T cells.

ABSTRACT: Hepatitis B virus (HBV) is a human hepatotropic virus. However, HBV infection also occurs at extrahepatic sites, but the relevant host factors required for HBV infection in non-hepatic cells are only partially understood. In this article, a non-hepatic cell culture model is constructed by exogenous expression of four host genes (NTCP, HNF4?, RXR? and PPAR?) in human non-hepatic 293T cells. This cell culture model supports HBV entry, transcription and replication, as evidenced by the detection of HBV pgRNA, HBV cccDNA, HBsAg, HBeAg, HBcAg and HBVDNA. Our results suggest that the above cellular factors may play a key role in HBV infection of non-hepatic cells. This model will facilitate the identification of host genes that support extrahepatic HBV infection.

SUBMITTER: Yang X

PROVIDER: S-EPMC7028854 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Defined host factors support HBV infection in non-hepatic 293T cells.

Yang Xiaoqiang X Cai Weiwen W Sun Xiaoyue X Bi Yanwei Y Zeng Chui C Zhao XiaoYu X Zhou Qi Q Xu Tian T Xie Qingdong Q Sun Pingnan P Zhou Xiaoling X

Journal of cellular and molecular medicine 20200112 4

Hepatitis B virus (HBV) is a human hepatotropic virus. However, HBV infection also occurs at extrahepatic sites, but the relevant host factors required for HBV infection in non-hepatic cells are only partially understood. In this article, a non-hepatic cell culture model is constructed by exogenous expression of four host genes (NTCP, HNF4α, RXRα and PPARα) in human non-hepatic 293T cells. This cell culture model supports HBV entry, transcription and replication, as evidenced by the detection of ...[more]

PMID: 31930674

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Project description:BACKGROUND: The common marmoset (Callithrix jacchus) is a New World primate sharing many similarities with humans. Recently developed technology for generating transgenic marmosets has opened new avenues for faithful recapitulation of human diseases, which could not be achieved in rodent models. However, the longer lifespan of common marmosets compared with rodents may result in an extended period for in vivo analysis of common marmoset disease models. Therefore, establishing rapid and efficient techniques for obtaining neuronal cells from transgenic individuals that enable in vitro analysis of molecular mechanisms underlying diseases are required. Recently, several groups have reported on methods, termed direct reprogramming, to generate neuronal cells by defined factors from somatic cells of various kinds of species, including mouse and human. The aim of the present study was to determine whether direct reprogramming technology was applicable to common marmosets. RESULTS: Common marmoset induced neuronal (cjiN) cells with neuronal morphology were generated from common marmoset embryonic skin fibroblasts (cjF) by overexpressing the neuronal transcription factors: ASCL1, BRN2, MYT1L and NEUROD1. Reverse transcription-polymerase chain reaction of cjiN cells showed upregulation of neuronal genes highly related to neuronal differentiation and function. The presence of neuronal marker proteins was also confirmed by immunocytochemistry. Electrical field stimulation to cjiN cells increased the intracellular calcium level, which was reversibly blocked by the voltage-gated sodium channel blocker, tetrodotoxin, indicating that these cells were functional. The neuronal function of these cells was further confirmed by electrophysiological analyses showing that action potentials could be elicited by membrane depolarization in current-clamp mode while both fast-activating and inactivating sodium currents and outward currents were observed in voltage-clamp mode. The 5-bromodeoxyuridine (BrdU) incorporation assay showed that cjiN cells were directly converted from cjFs without passing a proliferative state. CONCLUSIONS: Functional common marmoset neuronal cells can be obtained directly from embryonic fibroblasts by overexpressing four neuronal transcription factors under in vitro conditions. Overall, direct conversion technology on marmoset somatic cells provides the opportunity to analyze and screen phenotypes of genetically-modified common marmosets.

Project description:Background and aims: Chronic hepatitis B virus (HBV) infection is a major health burden potentially evolving toward cirrhosis and hepatocellular carcinoma. HBV physiopathology is strongly related to the host immunity, yet the mechanisms of viral evasion from immune-surveillance are still misunderstood. The immune response elicited at early stages of viral infection is believed to be important for subsequent disease outcome. Dendritic cells (DCs) are crucial immune sentinels which orchestrate antiviral immunity, which offer opportunity to pathogens to subvert them to escape immunity. Despite the pivotal role of DCs in orientating antiviral responses and determining the outcome of infection, their precise involvement in HBV pathogenesis is not fully explored. Methods: One hundred thirty chronically HBV infected patients and 85 healthy donors were enrolled in the study for blood collection, together with 29 chronically HBV infected patients and 33 non-viral infected patients that were included for liver biopsy collection. In a pioneer way, we investigated the phenotypic and functional features of both circulating and intrahepatic BDCA1+ cDC2, BDCA2+ pDCs, and BDCA3+ cDC1 simultaneously in patients with chronic HBV infection by designing a unique multi-parametric flow cytometry approach. Results: We showed modulations of the frequencies and basal activation status of blood and liver DCs associated with impaired expressions of specific immune checkpoints and TLR molecules on circulating DC subsets. Furthermore, we highlighted an impaired maturation of circulating and hepatic pDCs and cDCs following stimulation with specific TLR agonists in chronic HBV patients, associated with drastic dysfunctions in the capacity of circulating DC subsets to produce IL-12p70, TNFα, IFNα, IFNλ1, and IFNλ2 while intrahepatic DCs remained fully functional. Most of these modulations correlated with HBsAg and HBV DNA levels. Conclusion: We highlight potent alterations in the distribution, phenotype and function of all DC subsets in blood together with modulations of intrahepatic DCs, revealing that HBV may hijack the immune system by subverting DCs. Our findings provide innovative insights into the immuno-pathogenesis of HBV and the mechanisms of virus escape from immune control. Such understanding is promising for developing new therapeutic strategies restoring an efficient immune control of the virus.

Dataset Information

Defined host factors support HBV infection in non-hepatic 293T cells.

Publications

Defined host factors support HBV infection in non-hepatic 293T cells.

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