Editor’s Note: Hepatitis B Virus (HBV) infection is one of the main causes of liver diseases, affecting approximately 292 million people worldwide who are chronic carriers of HBV, leading to severe conditions like liver cirrhosis and primary liver cancer. The infection process of HBV relies on the binding of the viral envelope proteins to receptors on the cell surface. Therefore, interfering with the binding of the viral envelope proteins and receptors can be an effective antiviral strategy. During the 15th Chronic Viral Hepatitis Antiviral Treatment Challenges and Hot Topics Academic Conference, held in Chongqing from November 17-19, 2023, and organized by the Chinese Medical Journal Company Ltd., Editorial Office of the Chinese Journal of Hepatology, and co-organized by the Hepatitis Group of the Chinese Society of Hepatology, the Second Affiliated Hospital of Chongqing Medical University, and the Chongqing Medical University Institute of Viral Hepatitis, Dr. Ailong Huang, President of Chongqing Medical University, shared his team’s latest research findings on how HBV depends on various receptors and auxiliary receptors to infect cells. This research provides new insights into the mechanism of HBV invasion and the development of novel drugs to block HBV infection.
HBV infection is one of the primary causes of liver diseases, with about 292 million chronic HBV carriers globally, leading to serious conditions like liver cirrhosis and primary liver cancer. Since the discovery of HBV by Dr. Baruch S. Blumberg in 1964, scientists have studied it for decades, clarifying processes like transcription and replication, providing a crucial research foundation for controlling chronic hepatitis B. However, how HBV infects liver cells, a significant scientific question, has puzzled the academic community for nearly half a century. It was not until 2012 that Chinese scientist Dr. Wenhui Li discovered and reported the Sodium Taurocholate Cotransporting Polypeptide (NTCP) as a functional receptor for HBV infection, effectively elucidating the process of HBV invasion into liver cells. This discovery significantly advanced the research on HBV infection mechanisms, providing new ideas and methods for constructing natural infection cell models and developing novel antiviral drugs.
However, in the HBV infection system based on NTCP, researchers gradually identified new issues. For example, in vivo, HBV infection is highly efficient, with only a small amount of virus needed to infect a large number of liver cells. Yet, in cell models of HBV infection, infecting NTCP-expressing cells with high titers of HBV particles still results in limited infection efficiency, requiring the addition of substances like polyethylene glycol (PEG) and dimethyl sulfoxide (DMSO) to enhance cell susceptibility. From understanding other viruses, it’s clear that to establish an efficient infection process and adapt to different cell types, viruses often utilize multiple receptors or auxiliary receptors. These receptors and auxiliary receptors provide more opportunities and pathways for interaction between the virus and the cell surface, thus increasing the success rate of infection. For example, in the invasion process of HIV, the virus needs to bind not only to CD4 but also to auxiliary receptors such as CCR5 or CXCR4 to successfully enter the host cell. Therefore, this strategy of relying on multiple receptors and auxiliary receptors ensures the efficiency and adaptability of viral infection. However, in the HBV system, besides NTCP, whether other receptors or auxiliary receptors are involved in the HBV infection process is still unclear. Therefore, further research is necessary to deepen our understanding of the HBV infection mechanism.
By analyzing the viral antigen (HBcAg, HBsAg) and viral nucleic acid (HBV DNA, cccDNA) detection data published by various laboratories, it was found that HBV infection exhibits heterogeneity both in HepG2-NTCP stable cell lines and liver biopsy tissues. This means there are differences in the presence or intensity of HBV infection within the same cell line or tissue. Single-cell sequencing technology, which can isolate the gene expression profile of individual cells, reveals the unique gene expression patterns and states of each cell.
Therefore, our team, considering this heterogeneity in HBV infection among cells, conducted single-cell sequencing and susceptibility analysis on liver biopsy samples from children who had not undergone antiviral treatment. We found that besides NTCP, the gene expression of multiple membrane proteins was also related to HBV infection, indicating their potential role in the infection process. Subsequently, in various cell models like PHH and HepG2-NTCP, we verified these membrane proteins using digital PCR, fluorescence confocal, and hybridization experiments. It was discovered that overexpressing the single-pass transmembrane glycoprotein Neuropilin 1 (NRP1) before virus infection significantly promoted HBV attachment and internalization on cells, enhancing the virus’s infection in an NTCP-dependent manner. Conversely, reducing or knocking out endogenous NRP1 in cells using shRNA or CRISPR/Cas9 technology significantly lowered HBV infection efficiency.
Since the interaction between viral envelope proteins and host molecules on the cell membrane is a key step in viral infection, we further investigated the relationship between NRP1 and HBV surface proteins. The results showed that NRP1 could bind to HBV’s large surface protein (LHBs), facilitating viral attachment to the cell surface. This NRP1-viral protein interaction further strengthened the binding between HBV and NTCP, ultimately promoting viral infection.
Lastly, our team also explored whether NRP1 could serve as a new antiviral target. Immunoprecipitation experiments showed that NRP1 antagonists could disrupt the NRP1-HBV interaction, and their pre-incubation with HepG2-NTCP cells reduced HBV infection in a concentration-dependent manner. Additionally, given the limited host range of HBV, our team also constructed a liver humanized HBV infection mouse model based on uPA/SCID. In this model, NRP1 antagonists also significantly reduced virological indicators in the serum and liver of mice.
In conclusion, the results of this study indicate that NRP1 is an important auxiliary factor in the HBV infection process. This discovery will be beneficial for further understanding the mechanism of HBV invasion and for providing new strategies in constructing efficient viral infection models and developing drugs to block viral infection.
