Chronic hepatitis B remains one of the most prevalent infectious diseases worldwide, with an estimated 257 million people worldwide being carriers of chronic hepatitis B. Sodium taurocholate cotransport polypeptide (NTCP) is a differentiation-related, hepatocyte- and species-specific HBV cell membrane receptor. NTCP expression predisposes human hepatocellular carcinoma cells and undifferentiated HepaRG cells to HBV. Recent studies have shown that despite persistent levels of NTCP expression, in vitro HBV infection remains relatively inefficient and requires a large excess of virus to establish an effective infection.
Neurocilidin-1 (NRP1) is a single-shot transmembrane glycoprotein with extracellular, transmembrane, and intracellular domains, with NRP1 acting as a coreceptor that binds to various extracellular ligands, such as signaling protein (SEMA), hepatocyte growth factor, platelet-derived growth factor, and transforming growth factor β (TGF-β). However, the role of NRP1 in HBV infection remains unclear. Recent studies have reported single-cell transcriptional profiles of liver biopsies in HBV-infected children to investigate whether other host factors regulate the course of HBV infection. Yimaitong is hereby excerpted for clinicians' reference.
1. Research Methodology
Single-cell sequencing and susceptibility analysis were performed on liver biopsy samples from children with hepatitis B, and the effects of candidate host factors on HBV infection were analyzed using primary human hepatocytes, HepG2-NTCP cells, and human liver chimeric mice.
2. Findings
1. Single-cell transcriptome map of the liver of a child infected with hepatitis B virus
Single-cell RNA sequencing analysis was performed on liver biopsy samples from 5 HBV-infected children who did not receive antiviral therapy (Figure 1A). After quality control and filtration, a dataset containing 14,028 single-cell transcriptomes was obtained. Using dimensionality reduction and cluster analysis, UMAP identified 16 cell populations (Figure 1B). These populations cover eight different cell lineages, including B cells, endothelial cells, hepatocytes, macrophages, monocytes, neutrophils, NK cells, and T cells (Figure 1C). Each pedigree expresses its classical hallmark (Fig. 1D, E). Three hepatocyte subclusters targeted by HBV were identified, and HBV transcription and overall gene expression of these three cell clusters (hepatocytes C1-C3) were analyzed comparatively (Figure 1F).
HBV transcript levels and NTCP levels in cluster 1 (C1) were consistently higher than those in clusters 2 (C2) and cluster 3 (C3), suggesting that the levels of NTCP in cells were positively correlated with the number of viral transcripts. Cells in cluster 2 had an overall increase in HBV transcript levels compared to cluster 3, although NTCP levels were similarly low in both clusters (Figure 1G, H).
Fig.1 Single-cell transcriptome profile of the liver of HBV-infected children
2. HBV induces host membrane protein expression for effective infection
Differentially expressed transcripts were further analyzed between the two identified hepatocyte subsets (clusters 2 and 3). A total of 597 genes were dysregulated in cluster 2 compared to cluster 3, of which 368 genes were upregulated and 229 genes downregulated (Figure 2A). When examining the function of upregulated genes in cluster 2, receptor-mediated endocytosis, viral entry into host cells, and cell adhesion were particularly abundant compared to cluster 3 (Figure 2B). These data suggest that host cofactors may be involved in the early steps of viral entry during HBV infection.
Fig.2 NRP1 was identified as an important host factor for HBV infection
3. NRP1 is involved in the process of HBV infection
Figure 3 shows the role of NRP1 in HBV entry in two cell lines (HepG2 and Huh-7) transfected with plasmids encoding two identified host factors (human NTCP and NRP1). The results showed that NRP1 enhanced HBV infection and promoted HBV attachment and internalization in the early stage of viral entry in the presence of NTCP.
Fig.3 The important role of NRP1 in promoting HBV infection
4. NRP1 directly interacts with HBV LHBs
Figure 4 shows the interaction between NRP1 and LHBs, suggesting that there is a direct interaction between NRP1 and LHBs, and that the b-domain of NRP1 is essential for mediating its interaction with LHBs during HBV infection.
Fig.4 Interaction between NRP1 and HBV LHBs
5. Amino acid residues 88 and 92 in preS1 are essential for NRP1 binding
Figure 5 shows the preS1 region-specific interaction of NRP1 with LHB labeled with GFP or Flag. The results showed that R88 and R92 in the preS1 domain were essential for the interaction between LHBs and NRP1.
Figure 5 Arginine residues 88 and 92 in preS1 are essential for binding to NRP1
6. The interaction between NRP1 and preS1 regulates NTCP-mediated HBV infectivity
Figure 6 shows the importance of the NRP1-preS1 interaction for NTCP-mediated viral entry. In the presence of NRP1, NTCP exhibits increased binding affinity for preS1 (Figure 6A). The results suggest that NRP1-preS1 interaction regulates the ability of NTCP to bind to preS1 and promotes viral infection.
Fig.6 The interaction between NRP1 and preS1 regulates NTCP-mediated HBV infection
3. Conclusions of the study
Hepatitis B virus infection is a global public health problem, but the understanding of the early process of hepatitis B virus infection is still limited. By single-cell sequencing, a novel host factor, NRP1, was identified, which regulates hepatitis B virus entry by interacting with hepatitis B virus preS1 and NTCP. In addition, antagonists against NRP1 can inhibit hepatitis B virus infection in vivo and in vitro. This study could further deepen our understanding of the early infection process of the hepatitis B virus.
Bibliography:
Yu H, Ren J, Deng H, Li L, Zhang Z, Cheng S, Guo Z, Huang A, Dang Y, Song K, Wu D, Yao X, Qin Y, Yang Z, Xu K, He X, Chen J. Neuropilin-1 is a novel host factor modulating the entry of hepatitis B virus. J Hepatol. 2024 Jul 1:S0168-8278(24)02339-0. doi: 10.1016/j.jhep.2024.06.032. Epub ahead of print. PMID: 38960374.
Source: Yimaitong Hepatology