This study detected the breakpoints of cancer tissues and adjacent tissues in 27 HCC patients. It was found that the intersection of the breakpoints between cancer and adjacent tissues was 3 (Supplementary Tables 5 and 8). At the same time, the intersection of the breakpoints-related genes between them was 38 (Supplementary Fig. 1). Therefore, in terms of breakpoints and gene levels, there are very few intersections between cancer and adjacent tissues, and given that the project mainly focused on the virus-integrated sites in the cancer tissues, we carried out a comprehensive analysis of downstream functional characterization using virus integrated sites in the cancer tissues. The virus integration hotspots were identified as TERT and KMT2B3. This study not only discovered two new integration genes, N4BP1 and WASHP (both integrated three times), but also found that the sample integration frequencies of the TERT gene were 23.7% (101/426), 20.5% (18/88), 27.1% (48/177), and 14.8% (4/27), respectively, after comparing with previous studies. In addition, we found that the integration frequencies of KMT2B were 7.3% (31/426), 10.2% (9/88), 1.7% (3/177), and 22.2% (6/27), respectively1,3,4. This indicated significant differences among different studies, and the integration frequency of the KMT2B gene in Asia was much higher than that in Europe. Moreover, a comparison between the Asian and European populations revealed that there were a series of common hotspot genes (TERT, KMT2B, EMBP1, LOC441666, MTRNR2L1, ANKRD26P1, RSPO2, and GABRB3), indicating that there were some common characteristics of virus integration among the different ethnic groups. This suggests that the common virus integration events have certain regularity in enhancing tumor evolution, but the hypothesis requires further study. In addition, we found that N4BP1 enables mRNA binding activity, ribonuclease activity, and ubiquitin-binding activity. It is also involved in the cellular response to UV and negative regulation of viral genome replication7. It acts as a restriction factor against some viruses, such as HIV-1: restricts HIV-1 replication by binding to HIV-1 mRNAs and mediating their degradation via its ribonuclease activity8. WASH8P (WAS Protein Family Homolog 8) is a pseudogene. Although little research has been done on this gene, it was recently been linked to rectal cancer9. Considering the limited knowledge of N4BP1 and WASH8P genes and virus integration information, this study could not elaborate on how virus integration affects the two genes.

Herein, it was found that the regions of the HBV genome where breakpoints easily appear are the 0–1 K region and 1.6–2.0 K region. The regions before and after 1.8 K are the linear ends of the HBV genome. Moreover, the virus integration of linear double-stranded HBV genome is frequent10. The integration in the 0–1 K region could be due to abnormal rupture or secondary recombination during integration11. After conducting a comprehensive pathway analysis of all the integration genes in the four existing studies, we found that the virus integration sites were significantly enriched within the Rap1 signaling pathway, Axon guidance, Calcium signaling pathway, Cholinergic synapse, GABAergic synapse, Glutamatergic synapse, and other pathways. In addition, GO analysis demonstrated that the virus integration sites have specific effects on synapse organization, cell junction assembly, regulation of neuron projection development, and other pathways. Interestingly, the virus integration enriches neural transmission and key synaptic pathways. Thus, we speculated that in addition to the above three reasons which can lead to the tumor, the virus integration event may cause cancer by inducing the abnormality of neural transmission and the transmitter system. Current studies also indicate that neurotransmitters and growth factors scattered in the peripheral nervous system can trigger many cancers under experimental conditions, including pancreatic, gastric, colon, prostate, breast, oral, and skin12,13. Meanwhile, researchers have observed that the signal transduction among sympathetic nerve, parasympathetic nerve, and malignant cells in the tumor microenvironment usually regulates the onset or metastasis of cancer via the neurotransmitter-dependent signal transduction cascades. Studies have also revealed that in tumors, axonogenesis is promoted by the feedforward mechanism due to enhanced adrenergic or cholinergic signal transduction12,14. Therefore, virus integration events likely cause tumor onset and progression through interaction with neural signal pathways. Since virus integration events occur earlier than tumors6, they may affect relevant neural pathways during the early stage of tumors. Therefore, there is a need to explore the correlation between the onset and progression of early tumors with abnormal mental symptoms, including anxiety and depression, and psychosomatic symptoms.

This study found that the same direction connection often accompanies the connection site between the HBV genome and the human genome (++, −−) or reverse connection (+−, −+) during HBV integration. We identified 775 points and found that the same direction connection accounted for 388/775. In contrast, the reverse connection accounted for 387/775, indicating that insertion of virus integration happens in various directions. The proportion of reverse connection cases shows that virus integration leads to the frequent reverse connection of genomes. The reverse connection at both endpoints after virus integration indicates similar inversion events. However, although this phenomenon has been observed in many studies on HBV integration models4,15, the mechanism and impact behind this inversion event have not yet been elucidated. Thus, we speculate that the internal mechanism (Fig. 5a, b and Supplementary Tables 6 and 7) of the virus causing inversion is by developing a hairpin structure to replicate virus integration through an in-depth analysis of HBV integration on two typical sites. Furthermore, this inversion event is highly prone to form a cross structure which is easy to break and create a bare end16,17. When this end contacts the distal chromosome or other viruses to integrate and develop a bare end, it can cause translocation through non-homologous recombination (Fig. 5c). The above structural changes will lead to a high-degree structural variation of the human genome structure, resulting in a higher level of instability of the chromosome structure17. Therefore, HBV virus integration can lead to abnormal expression of integrated genes and the abnormal production of viral proteins. Moreover, it can induce the generation of tumors by directly causing a higher degree of genomic instability through the inversion and translocation events.

a The mechanism behind HBV integration in TERT, b HBV integration in DNAH9. c The mechanism of induced inversion translocation.

Conclusion: This study identified a series of new virus integration genes and expanded the latest knowledge of virus integration hotspots in the field of HBV integration. There is an extensive background of random integration and evident hotspot genes, such as TERT and KMT2B. We found that these hotspot genes are universal across different ethnic groups, providing an effective target for better research on the pathogenic mechanism and treatment of liver cancer through virus integration. Virus integration is widely believed to cause changes in genome structure, genomic instability, and abnormal expression of tumor suppressor genes, oncogenes, and viral genes. This study established that virus integration could directly cause inversion and translocation at the genomic structural level and found that virus integration can be closely associated with nerve axons and crucial synapses. Although the mechanism of virus integration affecting nerve axons and synaptic pathways requires further study, the close relationship with the nervous system has been gradually uncovered. Li et al. also observed that genes shared by HBV and HPV integration are enriched in nerve axons and essential synapses. These phenomena indicate that virus integration is closely linked with nervous system abnormalities during the carcinogenic process11. This study confirmed the commonness of virus integration hotspots among different ethnic groups and discovered new hotspot genes, describing the close relationship between virus integration and the nervous system. Moreover, the study explained the mechanism of inversion and translocation due to HBV integration. Overall, the research achievements will be significant for the clinical application of HBV integration and for studying carcinogenic mechanisms and drug targets.