Pathological Implications of Hepatitis B Viral DNA Integration Into Host Cells

Qian Yang1 2†, Yunzhi Pan2†,AliceS.S.Li3, Jia Zhang4,LiXiao2, and Pierre Sirois5 ∗ 1Department of Pathology, Kunming First People’s Hospital, Kunming, Yunnan, China 2Laboratory of Molecular Medicine, College of Pharmaceutical Science, Soochow University, Suzhou 215123, China 3University of Waterloo, 200 University Ave W, Waterloo, ON, Canada, N2L 3G1 4Novartis Institute of Genomic Functional Study, San Diego, CA 92131, USA 5CHUL Research Center, Laval University, Quebec, Canada G1V 4G2

Infections with Hepatitis B virus (HBV) and hepatitis C virus (HCV) are the main causes of human hepatocellular carcinoma HCC in many Asian countries. Viral DNA integration into the chromosomes of hepatic cells has been exclusively observed in HCC following chronic HBV infection. As the carcinogenesis is a slow and complicated process, it may be attributed to the activation of oncogenes, inactivation of tumor suppressor genes, and overexpression of viral genes by HBV DNA integration. Both HBV and HCV infections show individual and ethnic differences, it thus seems that where and how frequent the viral DNA integrated into hepatic chromosomes is of great relevance on its carcinogenicity. Better understanding of the pathologies following viral DNA integration has high clinical implications such as in the prevention, diagnosis, therapy, and prognosisIP: of 192.168.39.211 HCC in HBV infected On: Fri, individuals. 01 Oct 2021 Newly 06:32:53 developed technologies applied in HCC research have promising values. OneCopyright: of these technologies American Scientific is circulating Publishers DNA assay that may become a useful tool in studying both the viral DNA integration and the generalDelivered mutation by Ingenta load in the liver. Another technology is the engineered nuclease such as zinc ﬁnger nuclease and TAL nuclease. These nucleases may be used in slowing down or block the viral DNA integration process, thereby preventing and even treating HBV related HCC.

KEYWORDS: HBV Integration, HCC, Hepatitis B, Carcinogenesis.

INTRODUCTION closed circular supercoiled DNA (cccDNA) molecule. The Hepatitis virus and human hepatocellular carcinoma circularity of HBV DNA is maintained by its 5 cohesive (HCC) have a close relationship. Infections with Hepati- termini. The cccDNA is the original template that HBV tis B virus and hepatitis C virus are thought to be the replicates in hepatic cells. Transcripts are polyadenylated main causes of HCC in many Asian countries as docu- and transported to the cytoplasm where they are trans- mented by an increasing number of epidemiological data.1 lated into the viral nucleocapsid and precore antigen. HBV An epidemiological survey suggested that the main cause DNA has a very compact coding organization with four of HCC in China is hepatitis virus infection (mainly HBV partially overlapping open reading frames (ORFs) that are and HCV) with HBV contributing to 75–85% cases of the translated into seven known proteins: pre-S1, pre-S2, S, total amount of HCCs.2 pre-C, C, P, X protein and probably some other unknown HBV virion DNA is a relaxed circular, partially duplex products. The discovery of HBV DNA integration into the molecule of 3.2 kb. In the nucleus of infected cells, host cell genome is a milestone in HCC genetics research. when the second-strand DNA synthesis is completed, the During the course of chronic hepatitis B infection, HBV gaps in both strands are repaired to yield a covalently DNA randomly integrates into the hepatic cell genome at low frequency. The viral DNA integration usually leads the host gene to replacement, copy reverse, or deletion, etc.; ∗ Author to whom correspondence should be addressed. sequentially, it may inﬂuence genes involved in genetic E-mail: [email protected] †These two authors contributed equally to this work. reparation, cell cycling, cell differentiation and apopto- Received: 5 January 2014 sis of the host. These genetic changes may consequently Accepted: 25 January 2014 lead to carcinogenesis. This paper summarizes the present

Gene Gene Edit. 2015, Vol. 1, No. 1 2376-3949/2015/1/059/008 doi:10.1166/gge.2015.1008 59 Pathological Implications of Hepatitis B Viral DNA Integration Into Host Cells Yang et al. progress of HBV integration into host cells and discusses the carcinogenesis effect of the HBV DNA integration.

HBV INTEGRATION IN HCC An increasing number of data on HBV DNA integration into host cells suggest its possible relationship with liver carcinogenesis. HCC carcinogenesis is a complicated and relatively slow process in which HBV DNA continuously integrates into host chromosomes in the period of acute and chronic hepatitis or in HBV carriers. In a study of 68 cases of HCC, the integration of HBV DNA in host hepatic cells was detected in 61 cases.3 The viral insertion site was spread over all chromosomes except chromosomes 13, X, and Y. The genes involved include oncogenes, anti-oncogenes, apoptosis- related genes, and genes coding unknown proteins. Some other genes such as human telomerase reverse transcriptase (hTERT) gene, calcium signaling related gene, 60 s ribosomal protein encoding gene, platelet derived growth factor gene, and mixed lineage leukemia (MLL) encoding gene sites were interrupted by HBV DNA integration in selected cases. Another study with 40 HCC cases also showed a high percentage of HBV DNA integration into hepatic cells, in which 68 HBV integration loci were identified in 34 cases.4 These involved loci mostly related to the cell cycle and gene controlling, suchIP:as: 192.168.39.211 cell cycle control- On: Fri, 01 Oct 2021 06:32:53 ling gene CDKN2C; apoptosis-relatedCopyright: gene PAWR, American NTNl, Scientific Publishers MAPK8IP2, TNFsFlA, TNFRsFlA, and N7ITNl;Delivered signal by Ingenta conduction gene GNATl, MAPKl, and MAPK8 IP2; transcription related gene MTFl, MAML3, ROCK2, RAB5A, NSPCI, TBXl, and wTl; protein composition gene EIF5 and TINPl; DNA repairment gene (GTSEl); and protein degeneration and transfer gene RAB5A, etc. Among them, MLL4, GNATl, and FNl are most frequently identified in different specimens of selected cases. The chromosomes and genes involved in HBV integration are widespread and may not be selective. Tamori et al. studied 15 HCC cases and detected the gene integration site in ras-responsive element binding protein 1, Figure 1. Carcinogenesis of hepatocellular carcinoma and calmodulin 1, mixed lineage leukemia 2 (MLL2).5 Tokino HBV infection. Hepatocelluar carcinomas are associated with et al. reported that HBV DNA could integrate in chro- chronic HBV infection. Following HBV infection, most of the patients can completely recover from acute hepatitis by mosomes including chromosome X; at chromosome 11 removing all HBV with host immunity. However, 10–20% of the 6 and 17, HBV integration was detected at a higher rate. infected individuals become HBV carrier or chronic patients. The HBV DNA integration at human telomerase reverse The process of HBV DNA integration into host hepatic cells transcriptase (hTERT) gene site was identified in 2 HCC thus occurs over years during viral replication and hepatic cases.7 Interestingly, in cervix cancer, human papillary cell proliferation. Some of the viral DNA integration may have little or no carcinogenic effects, whereas mutagenic and car- virus (HPV) integration into hTERT gene loci was also cinogenic effects are significant when pro-oncogenes are acti- detected. It appears that although HBV DNA may integrate vated, cancer suppressor genes are inhibited, and certain viral into the hepatic cell genome randomly in HCCs, some par- genes are overexpressed. ticular genes may play an important role in carcinogenesis following HBV integration (Fig. 1). change the expression of oncogene, anti-oncogene and It was suggested that many integration events occur in micro RNA. HBV DNA integration can induce wide-range unsteady human genome or genes surrounding it.8 Integra- genetic changes in the host genome including chromosome tion also takes place near the cancer-related genes and may deletion, translocation, fusion gene transcripts, genomic

60 Gene Gene Edit. 1, 59–66, 2015 Yang et al. Pathological Implications of Hepatitis B Viral DNA Integration Into Host Cells

DNA amplification, and genomic instability. Some of the cell carcinoma are more closely related to each other in integration loci are tabulated in Table I. chronic as compared to acute viral hepatitis. In an 18 year Recently, a whole-genome sequencing of 4 HCC follow up study, Giacchino investigated 325 children car- patients was performed; a total of 255 HBV integra- rying HBV; three of them converted to chronic hepatitis tion sites were identified in 4 HCC which revealed and then to hepatic cell carcinoma after 6–11 years, and the widespread viral integration events. They observed HBV DNA integration was detected in one of these three a diverse collection of genomic perturbations near viral patients. integration sites, including direct gene disruption, viral promoter-driven human transcription, viral-human tran- Chronic Hepatitis script fusion, and DNA copy number alteration. Such Viral DNA integration is more frequently identified in widespread random viral integration will likely increase chronic hepatitis patients studied. In a study of 16 chronic carcinogenic opportunities in HBV-infected individuals. hepatitis, Takada et al. detected random integration of The initial HBV integration reports have likely been under- HBV DNA in 15 cases by Southern blot in which all estimated by previous PCR-based approaches.9 integrants had some flanking sequence DNA deletion and some had gene rearrangements.14 In another report, Acute Hepatitis Murakami et al. reported the HBV DNA integration in all In a study with 19 cases of acute hepatitis, HBV DNA of the 12 chronic hepatitis cases.10 integrations were detected in 3 cases in which one integra- The clinical consequences of viral DNA integration are tion site was in the tumor necrosis factor (TNF)-induced of great importance. Cao et al. studied HCC tissues and protein gene intronic sequence.10 Viral DNA integration surrounding “normal” hepatic tissues around HCC mass, is not necessarily associated with carcinogenesis in acute a total of 10 specimens of these “normal” hepatic tis- hepatitis. From a small sample observation, Kimbi et al. sues were found with integrated HBV DNA. These inte- reported the complete recovery from illness in 5 patients grated DNA sequences included 6 known viral genes suffering from acute hepatitis.11 In this report, only one encoding for gamma interferon, sigma-1 receptor, V-fos patient had HBV S and X gene sequences integrated into transfer effect protein, Glycoprotein G, RABAPTIN-5 pro- 7q11.23. It seems that the type of genes integrated and the tein, Dishevelled-3 (DUI.3) protein and 4 unknown DNA location of the chromosome being integrated are two fac- sequences. It was noted that the integration loci identified tors that determine whether viralIP: integration 192.168.39.211 is related On: to Fri, in01 surrounding Oct 2021 06:32:53 nontumor liver tissues were different from carcinogenesis. Copyright: American Scientificloci detected Publishers in tumor tissues of same patients. It is pos- Delivered by Ingenta In the period of acute HBV infection, viral DNA inte- sible that different integration loci may lead to different gration may occur as a random event. Viral DNA integra- outcome.9–15 tion could be detected even in very early stages of HBV The integration loci of HBV DNA into the host cell infection.12 13 It is not yet known whether or not HBV genome provide clues for HCC carcinogenesis mechanism. viral integration is involved in the transition from acute Although HBsAg turned negative in serum, the integra- to chronic hepatitis. However, viral integration and hepatic tion of HBV DNA may be detectable in hepatic cells in

Table I. Reports on HBV DNA integration into host cells in HCC.

Cases HBV integration First author of HCC events Chromosome(s) involved Gene(s) involved

Murakami 68 61 All except for 13,X,Y Oncogene, cell gene, Calcium signaling genes, PDGF, MILE, Apoptosis-related genes Hong Tu 40 34 MLL4, GNATl, FNl, CDKN2C, PAWR, NTNl, MAPK8IP2, TNFsFlA, TNFRsFlA, N7ITNl, GNATl, MAPKl, MAPK8IP2MTFl, MAML3, ROCK2, RAB5A, NSPCI, TBXl, wTlEIF5, TINPl, GTSEl, RAB5A Akihiro Tamori 15 15 ras-binding protein 1 response unit, calmodulin 1, MLL2, FLJ333655, LOC220272 M J Ferber 8 2 hTERT* S Zhong 54 43 TUrashima 28 16 Xie Junxia 24 15 1–3, 5–7, 10, 12–15, 17, 22 Paterlini- Brechot 22 15 1–3, 5, 9, 12, 14, 16, 18, 20, 22 NTRK2, IRAK2, MAPK1, IP3Rtype2, IP3R type 1, ST3GAL VI, TRUP, EMX2-like, hTERT, NMP84p, TRAP150 , MCM8, FR7, SERCA1 Takashi Tokino 1–7, 9, 11, 12, 15–18, X Onco-genes int-2 and hst-1

Note: Only hTERT gene detection was performed.

Gene Gene Edit. 1, 59–66, 2015 61 Pathological Implications of Hepatitis B Viral DNA Integration Into Host Cells Yang et al. some HBsAb positive individuals. The causal relationship level was observed in the patients with HCC (47.1 ± between HBV viral integration and HCC was indirectly 43.7 ng/mL), compared with healthy volunteers (17.6 ± supported by an observation related to alcoholic cirrhosis. 9.5 ng/mL).26 Recently, Huang et al. also quantified the As controls, Horiike et al. investigated 13 HBsAg negative circulating DNA from 72 patients with HCC, 37 with liver HCC transferred from alcoholic cirrhosis where none of cirrhosis or chronic hepatitis, and 41 healthy volunteers by them had HBV DNA integration; these results suggested a real-time quantitative PCR method.27 DNA levels in the that HCC transformed from HBV negative diseases were HCC plasma (173 ng/mL) were significantly higher than independent of HBV integration.16 those in patients without HCC (46 ng/mL) or the healthy controls (9 ng/mL). It is noteworthy that the level of circulating DNA is closely associated with tumor size and TNM HBV INTEGRATION OUTSIDE THE LIVER stage and may be a valuable tool for the detecting and HBV was found to exist mainly in a dissociated form in predicting HCC. To explore the features of microsatellite tissues outside the liver. Davison et al. detected HBV DNA alterations of circulating DNA of HCC patients, a study in 14 out of 18 sperm samples, 12 out of 18 saliva sam- analyzed blood samples collected from 62 HCC patients ples and 10 out of 18 urine samples; all of these detected and the corresponding tumor tissues.28 Microsatellite alter- viral genes were in dissociated forms, of which 10 were 17 ations showed a highly concordant pattern between plasma macromolecules. The dissociated HBV DNA was also DNA and the DNA of cancer tissues, which indicated identified in peripheral leucocytes from hepatitis patients. that the microsatellite alterations of tumor tissues were In addition to the dissociated form, HBV DNA can also reflected in plasma DNA. In addition to being a tumor integrate into cells outside the liver. Laskus et al. detected marker, circulating DNA could be a useful specimen in 2 HBV DNA integration in peripheral monocytes in a 18 dissecting the HBV DNA integration into hepatic tissues cohort of 10 HBV hepatitis patients. The integration loci before the carcinogenesis of HCC. were located in the adhesion end of the repeated sequence These viral DNA integration data from tissues outside of DR1 and DR2. In another study with peripheral mono- the liver may merely suggest that HBV DNA has the abil- cytes from active chronic hepatitis patients, 4 out of 7 ity to integrate into human chromosomes. The viral DNA HBV integrations were identified. HBV integrations were integration outside the liver might partly be attributed to also reported in other tissues such as in kidneys, pancreas, 19 20 host genome properties and theoretically referred to the placenta and skin. IP: 192.168.39.211 On: Fri, similar01 Oct integration2021 06:32:53 which occurred in the liver. However, Due to the ease of sample collection,Copyright: viral integration American Scientificthis hypothesis Publishers is difficult to confirm. With the limited in sperm had been studied in a relatively large numberDelivered of by dataIngenta available, the relationship between HBV viral DNA 21 cases. Huang investigated sperm chromosomes of 9 HBV integration into sperm or other non-hepatic tissues and positive patients with fluorescence in situ hybridization. increased susceptibility of HCC is not yet established. The cases included 1 acute hepatitis B, 2 active chronic hepatitis B, 4 persistent chronic hepatitis B and 2 HBsAg carriers with no clinical symptoms. Among them, specific THE SEGMENTS AND THE TYPE OF HBV fluorescent signal spots of HBV DNA were seen in sperm DNA INTEGRATION chromosomes in one patient with persistent chronic hep- HBx Gene atitis B. Similar results were obtained by Xu et al. in a HBx sub-gene was shown to integrate in HCC cells at a study with more patients.22 In the 27 sperm specimens they higher frequency than other HBV segments.29 HBx works analyzed, 6 were HBV positive, 3 were HBV dissociative as HBV transcription factor and can change the expression type and 3 were HBV integrative type. levels of both viral and host genes. HBx gene plays its Circulating free DNA is another convenient specimen role by coding HBx protein. HBx is a 154 amino acid pro- used to study HBV viral DNA integration as well as muta- tein which transactivates the cell promoter and enhancer of genesis following HBV infection. Simon et al.23 reported HBV gene expression. It also restrains the function of cel- that an isolated DNA chromosome from a HCC patient lular P53. HBx protein transactivates many known genes peripheral blood had multiple signs of chromosome insta- including oncogenes (c-Myc, c-Jun/fos, K-ras, IGF-II,IGF- bility including DNA breaks, minutes, chromosome pulver- IR, EGFR), transcript factor gene NF-kappa B, AP-1, ization and accentric fragments, some of which localized at AP-2, Subunit of RNA polymerase II, RPB5, TATA bind- the same chromosomal sites in the tumor cell line derived ing protein, ATF/CREB, and virus oncogenes (e.g., HBV from the biopsy specimen of the identical patient’s HCC enhancer). HBx could also inactivate cancer suppressor mass. It is highly possible that this circulating chromosome gene (such as p53, Rb, bcl-2, and DNA mismatch repair is released from hepatic cells, rather than from cells outside genes). By activating oncogenes and inactivating cancer the liver. Actually, circulating free DNA samples are now suppressor genes, the overexpression of HBx is thus asso- used for the early diagnosis of cancer.24 25 ciated with the development of HCC. In a study aimed to quantify circulating DNA from However, not all integrations of HBx genes can induce patients with HCC, a significantly higher circulating DNA the hepatic cell malignant conversion. Integration of HBx

62 Gene Gene Edit. 1, 59–66, 2015 Yang et al. Pathological Implications of Hepatitis B Viral DNA Integration Into Host Cells gene and expression of HBx protein can be found in HCC integration products. The second type, called “complex cells as well as HBV carriers without HCC. Reifenberg type,” is essentially the same as the first type, except that et al. introduced HBx gene into the embryo of C57B/6J the viral genome structure is more complicated. The sec- mice and found that while HBx gene was expressed, the ond type of integration may have been produced via the histological study did not indicate the presence of HCC.30 same process, using preformed complex viral genomes as This discrepancy could be explained by the biological substrates. The third type called integron, has a simple effects of wild type HBx gene versus the truncated type. viral genome, but with a larger cell DNA deletion. Differ- The complete length of the HBx gene may not induce ent forms of viral DNA may be used as substrates in the the host cell to acquire a growth advantage. On the con- integration process which is characterized by eliciting of trary, it may promote cell apoptosis. When 3 end of HBx deletions of different size in the target cell DNA. was deleted, the short HBx protein could transform hepatic cells to HCC cells by incorporating with other cell genes. CARCINOGENIC MECHANISMS OF HBV Therefore, the truncated x protein has larger potential to VIRAL INTEGRATION induce HCC than the wild type.31–33 HCC occurrence is the result of a complicated process. HBV DNA integration into the human hepatic cell chro- HBS Gene mosome is an important step, possibly the first genetic Truncated pre-S2/S gene products behave as transacti- change in multi-staged hepatocarcinogenesis. Most stud- vating factors and are often found in HBV DNA inte- ies on viral DNA integration have suggested that it is a gration loci in HCC patients. Pre-S/S gene encodes the random process. During the infection acute phase, there hepatitis B virus small, middle and large surface protein are no specific integration loci and the viral integration (SHBs, MHBs, LHBs). These gene products have both seems to be unlikely selected. However, during the infec- glycosylative and none-glycosylative types. Pre-S2/S pro- tion chronic phase, selected cell genes nearby, within, tein can trans-activate cell genes such as c-myc, c-fos, and or flanking integrated HBV DNA, may abnormally be c-Haras. Accumulation of overexpressed HBV surface pro- expressed by the influence of inserted virus sequences. teins, especially LHBs, in hepatic cells, is associated with When these abnormalities accumulate, hepatocarcinogene- cell degeneration, and carcinogenesis. It has been shown sis may finally occur. Although HBV DNA integration was that pre-S2 up-regulated human telomerase reverse tran- detected in almost all chromosomes in human HCCs, inte- scriptase and induced telomeraseIP: activation 192.168.39.211 which trans- On: Fri, 01 Oct 2021 06:32:53 gration hotspots, such as MLL4, tolemerase gene, GNATl formed hepatic cell line to the malignantCopyright: form. 34AmericanPre-S2 Scientific Publishers and FNl were reported. As an example of the hotspots protein could activate protein kinase C, which inDelivered turn after by Ingenta integration, WU et al. found in a transgenic carp study that phosphorylation activates c-Raf-1/MEK/ ERK signal trans- transgenes mostly located in repeated sequences, A/T rich fer cascades. Truncated pre-S2/S protein could activate sequences, TOPO isomerase I, II function sites and some MAPK signal pathway and transcription factor (e.g.,: AP-1 nearby structure sequences (SAR,MAR).36 The biologi- and NF-kappaB), which then favors hepatic cell division. cal effect of these integration hotspots on hepatocarcino- Urashima et al. studied HBV integration using Southern genesis is not known yet. In addition to interfering with blot with the probe of whole length HBV DNA, X and host cellular genes, the integration status of HBV DNA pre-S2 gene segments.29 The whole length of HBV DNA may also play a role in carcinogenesis. However we still integrated in 16 out of 28 (57.1%) cases; X gene integrated have questions on how much integrons are required, what in 14 out of 16 (87.5%) cases; preS2/S gene integrated in are the integration loci and what is the role of inserted 6 out of 16 (37.5%) cases; and C gene was rarely detected. virus gene (e.g., HBx gene) integrality in carcinogenesis Generally the HBV gene segments that integrate in HCC development. are trans-activated genes, including HBX and HBs gene, Experimental evidence from molecular biological studies especially C-terminal truncated HBS gene. suggests that the integration of HBV DNA modulates cell proliferation through at least four mechanisms.23 37–40 First, THE TYPES OF VIRAL DNA INTEGRATION the HBV-encoded trans-activator (e.g., hbx) could play a HBV DNA integrations are divided into 3 types based role in activating oncogenes and cellular genes to make cell on their structures.35 The first type in which the structure growth out of control. Oncogene such as c-myc, n-myc, of the viral sequence is simple is called “simple type,” c-fos, c-jun, and jun-B expression is usually detected.39–41 and part of it is with deletion. The viral cohesive end Secondly, HBV combines with transcription genes to mod- sequence appears at one of the virus-cell DNA junctions, ulate cellular signal pathway in activating gene expression. and the integration has elicited a microdeletion of the tar- In HepG2 cell line, Zhang et al. reported that HBV up- get cell DNA sequence. This structure suggests the pres- regulated HLJ1 expression mediated by YY1 transcription ence of viral DNA replication intermediates as substrates factor in HepG2 cells.42 Thirdly, HBV may influence cel- for integration. Judging from its frequency in HCC, this lular DNA repairing machinery by eliciting DNA repair may represent the most preferred type among the primary factors. Groisman et al. reported that HBx-induced DNA

Gene Gene Edit. 1, 59–66, 2015 63 Pathological Implications of Hepatitis B Viral DNA Integration Into Host Cells Yang et al. repair inhibition occurred through a p53-independent reg- (HDR) or non-homologous end joining (NHEJ) pathways. ulatory pathway and suggested that the inhibition of DNA In addition to the application on gene modification in repair mechanisms by HBV products may contribute to cells and intact organisms, a number of recent papers the observed synergistic interaction between chronic infec- have reported that this gene editing technology can be tion with HBV and exposure to liver carcinogens.43 Finally, applied effectively to human diseases including viral HIV HBV integrations usually cause chromosomal transloca- infection.48 49 These two studies used the same antiviral tion, deletion, and inversion/duplication which may play a strategy of knockout host CCR5 gene with genetically key role during a multi-step liver carcionogenesis.44 45 engineered nuclease. Although it is not practically avail- DNA repair system of the host may clear away some able for HBV infection at this moment, we have reason of the hepatic cells infected with HBV DNA. However, to expect the application of engineered nuclease such as when hepatic cells with integrated HBV DNA have sur- zinc finger nuclease or transcription activator-like effector vived immunity surveillance, their proliferation and further nuclease in the treatment of HBV infection or in the pre- accumulation of genetic abnormalities may finally lead to vention or hindrance of the viral DNA integration. With the onset of HCC (Fig. 1). Albeit the carcinogenesis of a better understanding the interaction between HBV and HCC is a slow and a complicated process, it seems that host cells, both viral genes and host genes can be the target the HBV viral DNA integration into hepatic cells is crucial of future gene therapy by gene editing technology. in the development of most, if not all, HCC in individuals In conclusion, HBV viral integration into host cells suffered from HBV infection based on the clinical obser- is a well confirmed pathological phenomenon following vation as far. HBV infection. Although its pathological roles in acute and chronic hepatitis are still to be elucidated, HBV viral CLINICAL IMPLICATION OF DNA integration into host cells may play a crucial role HBV VIRAL INTEGRATION in the carcinogenesis of HCC in individuals with chronic The mutation rate for HBV DNA is higher than that of HBV infection based on the increasing evidence from clin- other DNA viruses. On a clinical point of view, there ical observation. The HBV viral integration may activate is increasing evidence that the clinical profile including pro-oncogenes or inhibit cancer suppressor genes. Addi- the response to treatment and the long-term prognosis tionally, the overexpression of some of the duplicated viral genes may have carcinogenetic effects themselves. Better may differ depending on whichIP: genotype 192.168.39.211 has infected On: the Fri, 01 Oct 2021 06:32:53 patient.46 47 The various sequences of integrated HBV may understanding of the interactions between HBV and host Copyright: American Scientificgenome Publishers by analyzing the HBV viral integrated segments have different potential influence on hepatocarcinogenesis.Delivered by Ingenta Whether the variable consequences following the infection may pave a new avenue to gene therapy, either targeting of different subtypes is related or partially related to the the viral genome or the home genome by modern engi- altered viral DNA integration rates is still to be elucidated. neered nuclease of ZFNs and TALENs with their gene Further investigation leading to the better correlation editing functionalities. between HBV viral integration and HCC would be greatly Acknowledgments: This study is partially supported valuable for the prevention and treatment of HBV related HCC. HBV insertional tags provide a new tool for identi- by National Natural Science Foundation of China fying more human cancer related genes. The integration- (No. 81301267), Chinese National 863 Major Grant related viral genes and host genes offer clues to study (No. 2012AA020905), the Priority Academic Program the molecular biological mechanisms and development of Development of Jiangsu Higher Education Institutions and HCC. Analysis of HBV viral integration will help to eluci- Jiangsu Province’s clinical medical science and technol- date the HBV relevant carcinogenesis and possibly reveal ogy program (No. BL2013016). Qian Yang and Yunzhi the key gene or genes that trigger the occurrence of HCC, Pan organized the literature and drafted the first version. either viral gene or host gene. It is possible that some Alice SS Li drafted the second version. Jia Zhang and Li of the loci associated with HBV viral integrations may Xiao proofread the manuscript and prepared the third ver- become the new marker for early diagnosis by circulating sion. Pierre Sirois finalized the manuscript overall both in DNA assay, and even become the targets of chemotherapy scientifically and in language. or gene therapy. Current technological progress allows the knockout of REFERENCES both viral genes and host genes in vitro and in vivo 1. Pollicino T., Saitta C., and Raimondo G. (2011). Hepatocellular car- using engineered nucleases such as ZFNs (zinc-finger cinoma: The point of view of the hepatitis B virus. Carcinogenesis nucleases) and TALENs (transcription activator-like effec- 32, 1122–32. 2. tor nucleases). These engineered nucleases enable effi- Kudo M., Han K. H., Kokudo N., Cheng A. L., Choi B. I., Furuse J., Izumi N., Park J. W., Poon R. T., and Sakamoto M. (2010). 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