1 3529 210 ILLUMINATING THE LIVE-CELL DYNAMICS OF HEPATITIS B VIRUS COVALENTLY CLOSED CIRCULAR DNA USING THE CRISPR-TAG SYSTEM. THE COVALENTLY CLOSED CIRCULAR DNA (CCCDNA) OF HEPATITIS B VIRUS (HBV) IS THE MAJOR OBSTACLE TO CURING CHRONIC HEPATITIS B (CHB). CURRENT CCCDNA DETECTION METHODS ARE MOSTLY BASED ON BIOCHEMICAL EXTRACTION AND BULK MEASUREMENTS. THEY NEVERTHELESS GENERATED A GENERAL SKETCH OF ITS BIOLOGICAL FEATURES. HOWEVER, AN UNDERSTANDING OF THE SPATIOTEMPORAL FEATURES OF CCCDNA IS STILL LACKING. TO ACHIEVE THIS, WE ESTABLISHED A SYSTEM COMBINING CRISPR-TAG AND RECOMBINANT HBV MINICIRCLE TECHNOLOGY TO VISUALIZE CCCDNA AT SINGLE-CELL LEVEL IN REAL TIME. USING THIS SYSTEM, WE FOUND THAT THE OBSERVED RECOMBINANT CCCDNA (RCCCDNA) CORRELATED QUANTITATIVELY WITH ITS ACTIVE TRANSCRIPTS WHEN A LOW TO MEDIUM NUMBER OF FOCI (<20) ARE PRESENT, BUT THIS CORRELATION WAS LOST IN CELLS HARBORING HIGH COPY NUMBERS (>/=20) OF RCCCDNA. THE DISRUPTION OF HBX EXPRESSION SEEMS TO DISPLACE CCCDNA FROM THE DCAS9-ACCESSIBLE REGION, WHILE HBX COMPLEMENTATION RESTORED THE NUMBER OF OBSERVABLE CCCDNA FOCI. THIS INDICATED REGULATION OF CCCDNA ACCESSIBILITY BY HBX. SECOND, OBSERVABLE HBV AND DUCK HBV (DHBV) CCCDNA MOLECULES ARE SUBSTANTIALLY LOST DURING CELL DIVISION, AND THE REMAINING ONES WERE DISTRIBUTED RANDOMLY TO DAUGHTER CELLS. IN CONTRAST, KAPOSI'S SARCOMA-ASSOCIATED HERPESVIRUS (KSHV)-DERIVED EPISOMES CAN BE RETAINED IN A LANA (LATENCY-ASSOCIATED NUCLEAR ANTIGEN)-DEPENDENT MANNER. LAST, THE DYNAMICS OF RCCCDNA EPISOMES IN NUCLEI DISPLAYED CONFINED DIFFUSION AT SHORT TIME SCALES, WITH DIRECTIONAL TRANSPORT OVER LONGER TIME SCALES. IN CONCLUSION, THIS SYSTEM ENABLES THE STUDY OF PHYSIOLOGICAL KINETICS OF CCCDNA AT THE SINGLE-CELL LEVEL. THE DIFFERENTIAL ACCESSIBILITY OF RCCCDNA TO DCAS9 UNDER VARIOUS PHYSIOLOGICAL CONDITIONS MAY BE EXPLOITED TO ELUCIDATE THE COMPLEX TRANSCRIPTIONAL AND EPIGENETIC REGULATION OF THE HBV MINICHROMOSOME. IMPORTANCE UNDERSTANDING THE FORMATION AND MAINTENANCE OF HBV CCCDNA HAS ALWAYS BEEN A CENTRAL ISSUE IN THE STUDY OF HBV PATHOBIOLOGY. HOWEVER, LITTLE PROGRESS HAS BEEN MADE DUE TO THE LACK OF ROBUST ASSAY SYSTEMS AND ITS RESISTANCE TO GENETIC MODIFICATION. HERE, A LIVE-CELL IMAGING SYSTEM BY GRAFTING CRISPR-TAG INTO THE RECOMBINANT CCCDNA WAS ESTABLISHED TO VISUALIZE ITS MOLECULAR BEHAVIOR IN REAL TIME. WE FOUND THAT THE ACCESSIBILITY OF RCCCDNA TO DCAS9-BASED IMAGING IS RELATED TO HBX-REGULATED MECHANISMS. WE ALSO CONFIRMED THE SUBSTANTIAL LOSS OF OBSERVABLE RCCCDNA IN ONE-ROUND CELL DIVISION AND RANDOM DISTRIBUTION OF THE REMAINING MOLECULES. MOLECULAR DYNAMICS ANALYSIS REVEALED THE CONFINED MOVEMENT OF THE RCCCDNA EPISOME, SUGGESTING ITS JUXTAPOSITION TO CHROMATIN DOMAINS. OVERALL, THIS NOVEL SYSTEM OFFERS A UNIQUE PLATFORM TO INVESTIGATE THE INTRANUCLEAR DYNAMICS OF CCCDNA WITHIN LIVE CELLS. 2023 2 3186 51 HBV COVALENTLY CLOSED CIRCULAR DNA MINICHROMOSOMES IN DISTINCT EPIGENETIC TRANSCRIPTIONAL STATES DIFFER IN THEIR VULNERABILITY TO DAMAGE. BACKGROUND AND AIMS: HBV COVALENTLY CLOSED CIRCULAR DNA (CCCDNA) IS A MAJOR OBSTACLE FOR A CURE OF CHRONIC HEPATITIS B. ACCUMULATING EVIDENCE SUGGESTS THAT EPIGENETIC MODIFICATIONS REGULATE THE TRANSCRIPTIONAL ACTIVITY OF CCCDNA MINICHROMOSOMES. HOWEVER, IT REMAINS UNCLEAR HOW THE EPIGENETIC STATE OF CCCDNA AFFECTS ITS STABILITY. APPROACHES AND RESULTS: BY USING HBV INFECTION CELL MODELS AND IN VITRO AND IN VIVO RECOMBINANT CCCDNA (RCCCDNA) AND HBVCIRCLE MODELS, THE REDUCTION RATE OF HBV CCCDNA AND THE EFFICACY OF APOLIPOPROTEIN B MRNA EDITING ENZYME CATALYTIC SUBUNIT 3A (APOBEC3A)-MEDIATED AND CRISPR/CRISPR-ASSOCIATED 9 (CAS9)-MEDIATED CCCDNA TARGETING WERE COMPARED BETWEEN CCCDNAS WITH DISTINCT TRANSCRIPTIONAL ACTIVITIES. INTERFERON-ALPHA TREATMENT AND HEPATITIS B X PROTEIN (HBX) DELETION WERE APPLIED AS TWO STRATEGIES FOR CCCDNA REPRESSION. CHROMATIN IMMUNOPRECIPITATION AND MICROCOCCAL NUCLEASE ASSAYS WERE PERFORMED TO DETERMINE THE EPIGENETIC PATTERN OF CCCDNA. HBV CCCDNA LEVELS REMAINED STABLE IN NONDIVIDING HEPATOCYTES; HOWEVER, THEY WERE SIGNIFICANTLY REDUCED DURING CELL DIVISION, AND THE REDUCTION RATE WAS SIMILAR BETWEEN CCCDNAS IN TRANSCRIPTIONALLY ACTIVE AND TRANSCRIPTIONALLY REPRESSED STATES. STRIKINGLY, HBV RCCCDNA WITHOUT HBX EXPRESSION EXHIBITED A SIGNIFICANTLY LONGER PERSISTENCE IN MICE. THE CCCDNA WITH LOW TRANSCRIPTIONAL ACTIVITY EXHIBITED AN EPIGENETICALLY INACTIVE PATTERN AND WAS MORE DIFFICULT TO ACCESS BY APOBEC3A AND ENGINEERED CRISPR-CAS9. THE EPIGENETIC REGULATOR ACTIVATING CCCDNA INCREASED ITS VULNERABILITY TO APOBEC3A. CONCLUSIONS: HBV CCCDNA MINICHROMOSOMES IN DISTINCT EPIGENETIC TRANSCRIPTIONAL STATES SHOWED A SIMILAR REDUCTION RATE DURING CELL DIVISION BUT SIGNIFICANTLY DIFFERED IN THEIR ACCESSIBILITY AND VULNERABILITY TO TARGETED NUCLEASES AND ANTIVIRAL AGENTS. EPIGENETIC SENSITIZATION OF CCCDNA MAKES IT MORE SUSCEPTIBLE TO DAMAGE AND MAY POTENTIALLY CONTRIBUTE TO AN HBV CURE. 2022 3 6337 32 THE ROLE OF DNA-METHYLTRANSFERASES IN THE LIFE CYCLE OF HEPATITIS B VIRUS AND PATHOGENESIS OF CHRONIC HEPATITIS B. CHRONIC HEPATITIS B IS CAUSED BY A PERSISTENT FORM OF HEPATITIS B VIRUS, COVALENTLY CLOSED CIRCULAR DNA (CCCDNA). STABILITY OF CCCDNA IS ASSOCIATED WITH INTRACELLULAR LOCALIZATION OF CCCDNA AND FORMATION OF MINICHROMOSOME, REGULATED BY EPIGENETIC MECHANISMS. ONE OF THE KEY MECHANISMS IN EPIGENETICS IS METHYLATION OF DNA ON CPG ISLANDS. EXPRESSION LEVELS OF DNA-METHYLTRANSFERASES (DNMTS) IN CHRONIC HEPATITIS B PATIENTS WERE SHOWN TO BE UPREGULATED. NEVERTHELESS, THE ROLE OF DNMTS IN THE LIFE CYCLE OF HBV AND THEIR EFFECTS ON THE CELL REMAIN ELUSIVE. IN THIS REVIEW, WE DISCUSS LATEST ACHIEVEMENTS ON THE ROLE OF DNMTS IN CHRONIC HEPATITIS B AND HBV IN VITRO MODELS. 2018 4 4056 53 MAPPING THE HETEROGENEITY OF HISTONE MODIFICATIONS ON HEPATITIS B VIRUS DNA USING LIVER NEEDLE BIOPSIES OBTAINED FROM CHRONICALLY INFECTED PATIENTS. COVALENTLY CLOSED CIRCULAR DNA (CCCDNA) FORMS THE BASIS FOR REPLICATION AND PERSISTENCE OF HEPATITIS B VIRUS (HBV) IN THE CHRONICALLY INFECTED LIVER. WE HAVE PREVIOUSLY SHOWN THAT VIRAL TRANSCRIPTION IS SUBJECT TO REGULATION BY POSTTRANSLATIONAL MODIFICATIONS (PTMS) OF HISTONE PROTEINS BOUND TO CCCDNA THROUGH ANALYSIS OF DE NOVO HBV-INFECTED CELL LINES. WE NOW REPORT THE SUCCESSFUL ADAPTATION OF THIS CHROMATIN IMMUNOPRECIPITATION SEQUENCING (CHIPSEQ) APPROACH FOR ANALYSIS OF FINE-NEEDLE PATIENT LIVER BIOPSY SPECIMENS TO INVESTIGATE THE ROLE OF HISTONE PTMS IN CHRONICALLY HBV-INFECTED PATIENTS. USING 18 SPECIMENS FROM PATIENTS IN DIFFERENT STAGES OF CHRONIC HBV INFECTION, OUR WORK SHOWS THAT THE PROFILE OF HISTONE PTMS IN CHRONIC INFECTION IS MORE NUANCED THAN PREVIOUSLY OBSERVED IN IN VITRO MODELS OF ACUTE INFECTION. IN LINE WITH OUR PREVIOUS FINDINGS, WE FIND THAT THE MAJORITY OF HBV-DERIVED SEQUENCES ARE ASSOCIATED WITH THE ACTIVATING HISTONE PTM H3K4ME3. HOWEVER, WE SHOW A STRIKING INTERPATIENT VARIABILITY OF ITS DEPOSITION IN THIS PATIENT COHORT CORRELATED WITH VIRAL TRANSCRIPTION AND PATIENT HBV EARLY ANTIGEN (HBEAG) STATUS. UNEXPECTEDLY, WE DETECTED DEPOSITION OF THE CLASSICAL INHIBITORY HISTONE PTM H3K9ME3 ON HBV-DNA IN AROUND HALF OF THE PATIENT BIOPSY SPECIMENS, WHICH COULD NOT BE LINKED TO REDUCED LEVELS OF VIRAL TRANSCRIPTS. OUR RESULTS SHOW THAT CURRENT IN VITRO MODELS ARE UNABLE TO FULLY RECAPITULATE THE COMPLEX EPIGENETIC LANDSCAPE OF CHRONIC HBV INFECTION OBSERVED IN VIVO AND DEMONSTRATE THAT FINE-NEEDLE LIVER BIOPSY SPECIMENS CAN PROVIDE SUFFICIENT MATERIAL TO FURTHER INVESTIGATE THE INTERACTION OF VIRAL AND HOST PROTEINS ON HBV-DNA.IMPORTANCE HEPATITIS B VIRUS (HBV) IS A MAJOR GLOBAL HEALTH CONCERN, CHRONICALLY INFECTING MILLIONS OF PATIENTS AND CONTRIBUTING TO A RISING BURDEN OF LIVER DISEASE. THE VIRAL GENOME FORMS THE BASIS FOR CHRONIC INFECTION AND HAS BEEN SHOWN TO BE SUBJECT TO REGULATION BY EPIGENETIC MECHANISMS, SUCH AS POSTTRANSLATIONAL MODIFICATION OF HISTONE PROTEINS. HERE, WE CONFIRM AND EXPAND ON PREVIOUS RESULTS BY ADAPTING A HIGH-RESOLUTION TECHNIQUE FOR ANALYSIS OF HISTONE MODIFICATIONS FOR USE WITH PATIENT-DERIVED FINE-NEEDLE LIVER BIOPSY SPECIMENS. OUR WORK HIGHLIGHTS THAT THE SITUATION IN VIVO IS MORE COMPLEX THAN PREDICTED BY CURRENT IN VITRO MODELS, FOR EXAMPLE, BY SUGGESTING A NOVEL, NONCANONICAL ROLE OF THE HISTONE MODIFICATION H3K9ME3 IN THE HBV LIFE CYCLE. IMPORTANTLY, ENABLING THE USE OF FINE-NEEDLE LIVER BIOPSY SPECIMENS FOR SUCH HIGH-RESOLUTION ANALYSES MAY FACILITATE FURTHER RESEARCH INTO THE EPIGENETIC REGULATION OF THE HBV GENOME. 2019 5 1178 31 CONTROL OF CCCDNA FUNCTION IN HEPATITIS B VIRUS INFECTION. THE TEMPLATE OF HEPATITIS B VIRUS (HBV) TRANSCRIPTION, THE COVALENTLY CLOSED CIRCULAR DNA (CCCDNA), PLAYS A KEY ROLE IN THE LIFE CYCLE OF THE VIRUS AND PERMITS THE PERSISTENCE OF INFECTION. NOVEL MOLECULAR TECHNIQUES HAVE OPENED NEW POSSIBILITIES TO INVESTIGATE THE ORGANIZATION AND THE ACTIVITY OF THE CCCDNA MINICHROMOSOME IN VIVO, AND RECENT ADVANCES HAVE STARTED TO SHED LIGHT ON THE COMPLEXITY OF THE MECHANISMS CONTROLLING CCCDNA FUNCTION. NUCLEAR CCCDNA ACCUMULATES IN HEPATOCYTE NUCLEI AS A STABLE MINICHROMOSOME ORGANIZED BY HISTONE AND NON-HISTONE VIRAL AND CELLULAR PROTEINS. IDENTIFICATION OF THE MOLECULAR MECHANISMS REGULATING CCCDNA STABILITY AND ITS TRANSCRIPTIONAL ACTIVITY AT THE RNA, DNA AND EPIGENETIC LEVELS IN THE COURSE OF CHRONIC HEPATITIS B (CH-B) INFECTION MAY REVEAL NEW POTENTIAL THERAPEUTIC TARGETS FOR ANTI-HBV DRUGS AND HENCE ASSIST IN THE DESIGN OF STRATEGIES AIMED AT SILENCING AND EVENTUALLY DEPLETING THE CCCDNA RESERVOIR. 2009 6 6115 36 THE EPIGENETIC CONTROL OF HEPATITIS B VIRUS MODULATES THE OUTCOME OF INFECTION. EPIGENETIC MODIFICATIONS ARE STABLE ALTERATIONS IN GENE EXPRESSION THAT DO NOT INVOLVE MUTATIONS OF THE GENETIC SEQUENCE ITSELF. IT HAS BECOME INCREASINGLY CLEAR THAT EPIGENETIC FACTORS CONTRIBUTE TO THE OUTCOME OF CHRONIC HEPATITIS B VIRUS (HBV) INFECTION BY AFFECTING CELLULAR AND VIRION GENE EXPRESSION, VIRAL REPLICATION AND THE DEVELOPMENT OF HEPATOCELLULAR CARCINOMA. HBV PERSISTS IN THE NUCLEUS OF INFECTED HEPATOCYTES AS A STABLE NON-INTEGRATED COVALENTLY CLOSED CIRCULAR DNA (CCCDNA) WHICH FUNCTIONS AS A MINICHROMOSOME. THERE ARE TWO MAJOR FORMS OF HBV EPIGENETIC REGULATION: POSTTRANSLATIONAL MODIFICATION OF HISTONE PROTEINS ASSOCIATED WITH THE CCCDNA MINICHROMOSOME AND DNA METHYLATION OF VIRAL AND HOST GENOMES. THIS REVIEW EXPLORES HOW HBV CAN INTERPHASE WITH HOST EPIGENETIC REGULATION IN ORDER TO EVADE HOST DEFENCES AND TO PROMOTE ITS OWN SURVIVAL AND PERSISTENCE. WE FOCUS ON THE EFFECT OF CCCDNA BOUND-HISTONE MODIFICATIONS AND THE METHYLATION STATUS OF HBV DNA IN REGULATING VIRAL REPLICATION. INVESTIGATION OF HBV EPIGENETIC CONTROL HAS IMPORTANT CLINICAL CORRELATES WITH REGARDS TO THE DEVELOPMENT OF POTENTIAL THERAPEUTIC REGIMENS THAT WILL SUCCESSFULLY ERADICATE HBV INFECTION AND DEAL WITH HBV REACTIVATION IN THOSE UNDERGOING TREATMENT WITH DEMETHYLATING AGENTS. 2015 7 5368 31 RECENT ADVANCES IN THE STUDY OF HEPATITIS B VIRUS COVALENTLY CLOSED CIRCULAR DNA. CHRONIC HEPATITIS B INFECTION IS CAUSED BY HEPATITIS B VIRUS (HBV) AND A TOTAL CURE IS YET TO BE ACHIEVED. THE VIRAL COVALENTLY CLOSED CIRCULAR DNA (CCCDNA) IS THE KEY TO ESTABLISH A PERSISTENT INFECTION WITHIN HEPATOCYTES. CURRENT ANTIVIRAL STRATEGIES HAVE NO EFFECT ON THE PRE-EXISTING CCCDNA RESERVOIR. THEREFORE, THE STUDY OF THE MOLECULAR MECHANISM OF CCCDNA FORMATION IS BECOMING A MAJOR FOCUS OF HBV RESEARCH. THIS REVIEW SUMMARIZES THE CURRENT ADVANCES IN CCCDNA MOLECULAR BIOLOGY AND THE LATEST STUDIES ON THE ELIMINATION OR INACTIVATION OF CCCDNA, INCLUDING THREE MAJOR AREAS: (1) EPIGENETIC REGULATION OF CCCDNA BY HBV X PROTEIN, (2) IMMUNE-MEDIATED DEGRADATION, AND (3) GENOME-EDITING NUCLEASES. ALL THESE ASPECTS PROVIDE CLUES ON HOW TO FINALLY ATTAIN A CURE FOR CHRONIC HEPATITIS B INFECTION. 2017 8 4055 47 MAPPING OF HISTONE MODIFICATIONS IN EPISOMAL HBV CCCDNA UNCOVERS AN UNUSUAL CHROMATIN ORGANIZATION AMENABLE TO EPIGENETIC MANIPULATION. CHRONIC HEPATITIS B VIRUS (HBV) INFECTION AFFECTS 240 MILLION PEOPLE WORLDWIDE AND IS A MAJOR RISK FACTOR FOR LIVER FAILURE AND HEPATOCELLULAR CARCINOMA. CURRENT ANTIVIRAL THERAPY INHIBITS CYTOPLASMIC HBV GENOMIC REPLICATION, BUT IS NOT CURATIVE BECAUSE IT DOES NOT DIRECTLY AFFECT NUCLEAR HBV CLOSED CIRCULAR DNA (CCCDNA), THE GENOMIC FORM THAT TEMPLATES VIRAL TRANSCRIPTION AND SUSTAINS VIRAL PERSISTENCE. NOVEL APPROACHES THAT DIRECTLY TARGET CCCDNA REGULATION WOULD THEREFORE BE HIGHLY DESIRABLE. CCCDNA IS ASSEMBLED WITH CELLULAR HISTONE PROTEINS INTO CHROMATIN, BUT LITTLE IS KNOWN ABOUT THE REGULATION OF HBV CHROMATIN BY HISTONE POSTTRANSLATIONAL MODIFICATIONS (PTMS). HERE, USING A NEW CCCDNA CHIP-SEQ APPROACH, WE REPORT, TO OUR KNOWLEDGE, THE FIRST GENOME-WIDE MAPS OF PTMS IN CCCDNA-CONTAINING CHROMATIN FROM DE NOVO INFECTED HEPG2 CELLS, PRIMARY HUMAN HEPATOCYTES, AND FROM HBV-INFECTED LIVER TISSUE. WE FIND HIGH LEVELS OF PTMS ASSOCIATED WITH ACTIVE TRANSCRIPTION ENRICHED AT SPECIFIC SITES WITHIN THE HBV GENOME AND, SURPRISINGLY, VERY LOW LEVELS OF PTMS LINKED TO TRANSCRIPTIONAL REPRESSION EVEN AT SILENT HBV PROMOTERS. WE SHOW THAT TRANSCRIPTION AND ACTIVE PTMS IN HBV CHROMATIN ARE REDUCED BY THE ACTIVATION OF AN INNATE IMMUNITY PATHWAY, AND THAT THIS EFFECT CAN BE RECAPITULATED WITH A SMALL MOLECULE EPIGENETIC MODIFYING AGENT, OPENING THE POSSIBILITY THAT CHROMATIN-BASED REGULATION OF CCCDNA TRANSCRIPTION COULD BE A NEW THERAPEUTIC APPROACH TO CHRONIC HBV INFECTION. 2015 9 2240 42 EPIGENETIC MODULATION IN CHRONIC HEPATITIS B VIRUS INFECTION. THE HUMAN HEPATITIS B VIRUS (HBV) IS A SMALL-ENVELOPED DNA VIRUS CAUSING ACUTE AND CHRONIC HEPATITIS. DESPITE THE EXISTENCE OF AN EFFECTIVE PROPHYLACTIC VACCINE AND THE STRONG CAPACITY OF APPROVED ANTIVIRAL DRUGS TO SUPPRESS VIRAL REPLICATION, CHRONIC HBV INFECTION (CHB) CONTINUES TO BE A MAJOR HEALTH BURDEN WORLDWIDE. BOTH THE INABILITY OF THE IMMUNE SYSTEM TO RESOLVE CHB AND THE UNIQUE REPLICATION STRATEGY EMPLOYED BY HBV, WHICH FORMS A STABLE VIRAL COVALENTLY CLOSED CIRCULAR DNA (CCCDNA) MINICHROMOSOME IN THE HEPATOCYTE NUCLEUS, ENABLE INFECTION PERSISTENCE. KNOWLEDGE OF THE COMPLEX NETWORK OF INTERACTIONS THAT HBV ENGAGES WITH ITS HOST IS STILL LIMITED BUT ACCUMULATING EVIDENCE INDICATES THAT EPIGENETIC MODIFICATIONS OCCURRING BOTH ON THE CCCDNA AND ON THE HOST GENOME IN THE COURSE OF INFECTION ARE ESSENTIAL TO MODULATE VIRAL ACTIVITY AND LIKELY CONTRIBUTE TO PATHOGENESIS AND CANCER DEVELOPMENT. THUS, A DEEPER UNDERSTANDING OF EPIGENETIC REGULATORY PROCESSES MAY OPEN NEW VENUES TO CONTROL AND EVENTUALLY CURE CHB. THIS REVIEW SUMMARIZES MAJOR FINDINGS IN HBV EPIGENETIC RESEARCH, FOCUSING ON THE EPIGENETIC MECHANISMS REGULATING CCCDNA ACTIVITY AND THE MODIFICATIONS DETERMINED IN INFECTED HOST CELLS AND TUMOR LIVER TISSUES. 2020 10 5689 43 SILENCING HEPATITIS B VIRUS COVALENTLY CLOSED CIRCULAR DNA: THE POTENTIAL OF AN EPIGENETIC THERAPY APPROACH. GLOBAL PROPHYLACTIC VACCINATION PROGRAMMES HAVE HELPED TO CURB NEW HEPATITIS B VIRUS (HBV) INFECTIONS. HOWEVER, IT IS ESTIMATED THAT NEARLY 300 MILLION PEOPLE ARE CHRONICALLY INFECTED AND HAVE A HIGH RISK OF DEVELOPING HEPATOCELLULAR CARCINOMA. AS SUCH, HBV REMAINS A SERIOUS HEALTH PRIORITY AND THE DEVELOPMENT OF NOVEL CURATIVE THERAPEUTICS IS URGENTLY NEEDED. CHRONIC HBV INFECTION HAS BEEN ATTRIBUTED TO THE PERSISTENCE OF THE COVALENTLY CLOSED CIRCULAR DNA (CCCDNA) WHICH ESTABLISHES ITSELF AS A MINICHROMOSOME IN THE NUCLEUS OF HEPATOCYTES. AS THE VIRAL TRANSCRIPTION INTERMEDIATE, THE CCCDNA IS RESPONSIBLE FOR PRODUCING NEW VIRIONS AND PERPETUATING INFECTION. HBV IS DEPENDENT ON VARIOUS HOST FACTORS FOR CCCDNA FORMATION AND THE MINICHROMOSOME IS AMENABLE TO EPIGENETIC MODIFICATIONS. TWO HBV PROTEINS, X (HBX) AND CORE (HBC) PROMOTE VIRAL REPLICATION BY MODULATING THE CCCDNA EPIGENOME AND REGULATING HOST CELL RESPONSES. THIS INCLUDES VIRAL AND HOST GENE EXPRESSION, CHROMATIN REMODELING, DNA METHYLATION, THE ANTIVIRAL IMMUNE RESPONSE, APOPTOSIS, AND UBIQUITINATION. ELIMINATION OF THE CCCDNA MINICHROMOSOME WOULD RESULT IN A STERILIZING CURE; HOWEVER, THIS MAY BE DIFFICULT TO ACHIEVE. EPIGENETIC THERAPIES COULD PERMANENTLY SILENCE THE CCCDNA MINICHROMOSOME AND PROMOTE A FUNCTIONAL CURE. THIS REVIEW EXPLORES THE CCCDNA EPIGENOME, HOW HOST AND VIRAL FACTORS INFLUENCE TRANSCRIPTION, AND THE RECENT EPIGENETIC THERAPIES AND EPIGENOME ENGINEERING APPROACHES THAT HAVE BEEN DESCRIBED. 2021 11 1052 38 CLINICAL IMPLICATIONS OF HEPATITIS B VIRUS RNA AND COVALENTLY CLOSED CIRCULAR DNA IN MONITORING PATIENTS WITH CHRONIC HEPATITIS B TODAY WITH A GAZE INTO THE FUTURE: THE FIELD IS UNPREPARED FOR A STERILIZING CURE. . CHRONIC HEPATITIS B VIRUS (HBV) INFECTION HAS LONG REMAINED A CRITICAL GLOBAL HEALTH ISSUE. COVALENTLY CLOSED CIRCULAR DNA (CCCDNA) IS A PERSISTENT FORM OF THE HBV GENOME THAT MAINTAINS HBV CHRONICITY. DECADES OF EXTENSIVE RESEARCH RESULTED IN THE TWO THERAPEUTIC OPTIONS CURRENTLY AVAILABLE: NUCLEOT(S)IDE ANALOGS AND INTERFERON (IFN) THERAPY. A PLETHORA OF RELIABLE MARKERS TO MONITOR HBV PATIENTS HAS BEEN ESTABLISHED, INCLUDING THE RECENTLY DISCOVERED ENCAPSIDATED PREGENOMIC RNA IN SERUM, WHICH CAN BE USED TO DETERMINE TREATMENT END-POINTS AND TO PREDICT THE SUSCEPTIBILITY OF PATIENTS TO IFN. ADDITIONALLY, HBV RNA SPLICE VARIANTS AND CCCDNA AND ITS EPIGENETIC MODIFICATIONS ARE ASSOCIATED WITH THE CLINICAL COURSE AND RISKS OF HEPATOCELLULAR CARCINOMA (HCC) AND LIVER FIBROSIS. HOWEVER, NEW ANTIVIRALS, INCLUDING CRISPR/CAS9, APOBEC-MEDIATED DEGRADATION OF CCCDNA, AND T-CELL THERAPIES AIM AT COMPLETELY ELIMINATING HBV, AND IT IS CLEAR THAT THE DIAGNOSTIC ARSENAL FOR DEFINING THE LONG-AWAITED STERILIZING CURE IS MISSING. IN THIS REVIEW, WE DISCUSS THE CURRENTLY AVAILABLE TOOLS FOR DETECTING AND MEASURING HBV RNAS AND CCCDNA, AS WELL AS THE STATE-OF-THE-ART IN CLINICAL IMPLICATIONS OF THESE MARKERS, AND DEBATE NEEDS AND GOALS WITHIN THE CONTEXT OF THE STERILIZING CURE THAT IS SOON TO COME. 2018 12 2324 35 EPIGENETIC REGULATION OF HEPATITIS B VIRUS COVALENTLY CLOSED CIRCULAR DNA: IMPLICATIONS FOR EPIGENETIC THERAPY AGAINST CHRONIC HEPATITIS B. HEPATITIS B VIRUS (HBV) INFECTION REPRESENTS A SIGNIFICANT PUBLIC HEALTH BURDEN WORLDWIDE. ALTHOUGH CURRENT THERAPEUTICS MANAGE TO CONTROL THE DISEASE PROGRESSION, LIFELONG TREATMENT AND SURVEILLANCE ARE REQUIRED BECAUSE DRUG RESISTANCE DEVELOPS DURING TREATMENT AND REACTIVATIONS FREQUENTLY OCCUR FOLLOWING MEDICATION CESSATION. THUS, THE OCCURRENCE OF HEPATOCELLULAR CARCINOMA IS DECREASED, BUT NOT ELIMINATED. ONE MAJOR REASON FOR FAILURE OF HBV TREATMENT IS THE INABILITY TO ERADICATE OR INACTIVATE THE VIRAL COVALENTLY CLOSED CIRCULAR DNA (CCCDNA), WHICH IS A STABLE EPISOMAL FORM OF THE VIRAL GENOME DECORATED WITH HOST HISTONES AND NONHISTONE PROTEINS. ACCUMULATING EVIDENCE SUGGESTS THAT EPIGENETIC MODIFICATIONS OF CCCDNA CONTRIBUTE TO VIRAL REPLICATION AND THE OUTCOME OF CHRONIC HBV INFECTION. HERE, WE SUMMARIZE CURRENT PROGRESS ON HBV EPIGENETICS RESEARCH AND THE THERAPEUTIC IMPLICATIONS FOR CHRONIC HBV INFECTION BY LEARNING FROM THE EPIGENETIC THERAPIES FOR CANCER AND OTHER VIRAL DISEASES, WHICH MAY OPEN A NEW VENUE TO CURE CHRONIC HEPATITIS B. (HEPATOLOGY 2017;66:2066-2077). 2017 13 3806 45 INTRACELLULAR INTERFERON SIGNALLING PATHWAYS AS POTENTIAL REGULATORS OF COVALENTLY CLOSED CIRCULAR DNA IN THE TREATMENT OF CHRONIC HEPATITIS B. INFECTION WITH THE HEPATITIS B VIRUS (HBV) IS STILL A MAJOR GLOBAL HEALTH THREAT AS 250 MILLION PEOPLE WORLDWIDE CONTINUE TO BE CHRONICALLY INFECTED WITH THE VIRUS. WHILE PATIENTS MAY BE TREATED WITH NUCLEOSIDE/NUCLEOTIDE ANALOGUES, THIS ONLY SUPPRESSES HBV TITRE TO SUB-DETECTION LEVELS WITHOUT ELIMINATING THE PERSISTENT HBV COVALENTLY CLOSED CIRCULAR DNA (CCCDNA) GENOME. AS A RESULT, HBV INFECTION CANNOT BE CURED, AND THE VIRUS REACTIVATES WHEN CONDITIONS ARE FAVORABLE. INTERFERONS (IFNS) ARE CYTOKINES KNOWN TO INDUCE POWERFUL ANTIVIRAL MECHANISMS THAT CLEAR VIRUSES FROM INFECTED CELLS. THEY HAVE BEEN SHOWN TO INDUCE CCCDNA CLEARANCE, BUT THEIR USE IN THE TREATMENT OF HBV INFECTION IS LIMITED AS HBV-TARGETING IMMUNE CELLS ARE EXHAUSTED AND HBV HAS EVOLVED MULTIPLE MECHANISMS TO EVADE AND SUPPRESS IFN SIGNALLING. THUS, TO FULLY UTILIZE IFN-MEDIATED INTRACELLULAR MECHANISMS TO EFFECTIVELY ELIMINATE HBV, INSTEAD OF DIRECT IFN ADMINISTRATION, NOVEL STRATEGIES TO SUSTAIN IFN-MEDIATED ANTI-CCCDNA AND ANTIVIRAL MECHANISMS NEED TO BE DEVELOPED. THIS REVIEW WILL CONSOLIDATE WHAT IS KNOWN ABOUT HOW IFNS ACT TO ACHIEVE ITS INTRACELLULAR ANTIVIRAL EFFECTS AND HIGHLIGHT THE CRITICAL INTERFERON-STIMULATED GENE TARGETS AND EFFECTOR MECHANISMS WITH POTENT ANTI-CCCDNA FUNCTIONS. THESE INCLUDE CCCDNA DEGRADATION BY APOBECS AND CCCDNA SILENCING AND TRANSCRIPTION REPRESSION BY EPIGENETIC MODIFICATIONS. IN ADDITION, THE MECHANISMS THAT HBV EMPLOYS TO DISRUPT IFN SIGNALLING WILL BE DISCUSSED. DRUGS THAT HAVE BEEN DEVELOPED OR ARE IN THE PIPELINE FOR COMPONENTS OF THE IFN SIGNALLING PATHWAY AND HBV TARGETS THAT DETRACT IFN SIGNALLING MECHANISMS WILL ALSO BE IDENTIFIED AND DISCUSSED FOR UTILITY IN THE TREATMENT OF HBV INFECTIONS. TOGETHER, THESE WILL PROVIDE USEFUL INSIGHTS INTO DESIGN STRATEGIES THAT SPECIFICALLY TARGET CCCDNA FOR THE ERADICATION OF HBV. 2021 14 5547 39 ROLE OF EPIGENETIC MODIFICATION IN INTERFERON TREATMENT OF HEPATITIS B VIRUS INFECTION. HUMAN HEPATITIS B VIRUS (HBV) IS A SMALL, ENVELOPED DNA VIRUS THAT CAUSES ACUTE AND CHRONIC HEPATITIS. CHRONIC HEPATITIS B (CHB) IS ASSOCIATED WITH HEPATOCELLULAR CARCINOMA PATHOGENESIS. INTERFERONS (IFNS) HAVE BEEN USED FOR THE TREATMENT OF CHB FOR A LONG TIME, WITH ADVANTAGES INCLUDING LESS TREATMENT DURATION AND SUSTAINED VIROLOGICAL RESPONSE. PRESENTLY, VARIOUS EVIDENCE SUGGESTS THAT EPIGENETIC MODIFICATION OF THE VIRAL COVALENTLY CLOSED CIRCULAR DNA (CCCDNA) AND THE HOST GENOME IS CRUCIAL FOR THE REGULATION OF VIRAL ACTIVITY. THIS MODIFICATION INCLUDES HISTONE ACETYLATION, DNA METHYLATION, N6-METHYLADENOSINE, AND NON-CODING RNA MODIFICATION. IFN TREATMENT FOR CHB CAN STIMULATE MULTIPLE IFN-STIMULATED GENES FOR INHIBITING VIRUS REPLICATION. IFNS CAN ALSO AFFECT THE HBV LIFE CYCLE THROUGH EPIGENETIC MODULATION. IN THIS REVIEW, WE SUMMARIZED THE DIFFERENT MECHANISMS THROUGH WHICH IFN-ALPHA INHIBITS HBV REPLICATION, INCLUDING EPIGENETIC REGULATION. MOREOVER, THE MECHANISMS UNDERLYING IFN ACTIVITY ARE DISCUSSED, WHICH INDICATED ITS POTENTIAL AS A NOVEL TREATMENT FOR CHB. IT IS PROPOSED THAT EPIGENETIC CHANGES SUCH AS HISTONE ACETYLATION, DNA METHYLATION, M6A METHYLATION COULD BE THE TARGETS OF IFN, WHICH MAY OFFER A NOVEL APPROACH TO HBV TREATMENT. 2022 15 5936 34 TARGETING HEPATITIS B VIRUS COVALENTLY CLOSED CIRCULAR DNA AND HEPATITIS B VIRUS X PROTEIN: RECENT ADVANCES AND NEW APPROACHES. CHRONIC HEPATITIS B VIRUS (HBV) INFECTION REMAINS A WORLDWIDE CONCERN AND PUBLIC HEALTH PROBLEM. TWO KEY ASPECTS OF THE HBV LIFE CYCLE ARE ESSENTIAL FOR VIRAL REPLICATION AND THUS THE DEVELOPMENT OF CHRONIC INFECTIONS: THE ESTABLISHMENT OF THE VIRAL MINICHROMOSOME, COVALENTLY CLOSED CIRCULAR (CCC) DNA, WITHIN THE NUCLEUS OF INFECTED HEPATOCYTES AND THE EXPRESSION OF THE REGULATORY HEPATITIS B VIRUS X PROTEIN (HBX). INTERESTINGLY, NUCLEAR HBX REDIRECTS HOST EPIGENETIC MACHINERY TO ACTIVATE CCCDNA TRANSCRIPTION. IN THIS PERSPECTIVE, WE PROVIDE AN OVERVIEW OF RECENT ADVANCES IN UNDERSTANDING THE REGULATION OF CCCDNA AND THE MECHANISTIC AND FUNCTIONAL ROLES OF HBX. WE ALSO DESCRIBE THE PROGRESS TOWARD TARGETING BOTH CCCDNA AND HBX FOR THERAPEUTIC PURPOSES. FINALLY, WE OUTLINE STANDING QUESTIONS IN THE FIELD AND PROPOSE COMPLEMENTARY CHEMICAL BIOLOGY APPROACHES TO ADDRESS THEM. 2019 16 6862 36 [OCCULT HEPATITIS B VIRUS INFECTION]. OCCULT HEPATITIS B VIRUS (HBV) INFECTION IS A PECULIAR FORM OF CHRONIC VIRAL INFECTION IDENTIFIED SINCE THE EARLY 80'S AND CAN BE DEFINED AS THE PRESENCE OF HBV DNA IN THE SERUM AND/OR IN THE LIVER TISSUE OF PATIENTS NEGATIVE FOR THE HBV SURFACE ANTIGEN (HBSAG) USING USUAL SEROLOGICAL TESTS. THE DATA ABOUT THE PREVALENCE OF OCCULT HBV INFECTION ARE CONTRASTING AND THE REPORTED PREVALENCES IN VARIOUS CATEGORIES OF INDIVIDUALS ARE HIGHLY DIVERSE. THE MOLECULAR BASIS OF THE OCCULT HBV INFECTION IS THE COVALENTLY CLOSED-CIRCULAR DNA (CCCDNA) THAT PERSISTS IN THE CELL NUCLEI AND THAT SERVES AS A TEMPLATE FOR GENE TRANSCRIPTION. THE PHYSIOPATHOLOGY OF OCCULT HBV INFECTION IS STILL UNCLEAR. HOWEVER, THE AVAILABLE DATA SUGGEST THAT THE HOST'S IMMUNE RESPONSE, THE CO-INFECTIONS WITH OTHER INFECTIOUS AGENTS AND EPIGENETIC FACTORS MAY PLAY IMPORTANT ROLES IN INDICING THE OCCULT STATUS. THE CLINICAL RELEVANCE OF OCCULTHBVINFECTION REMAINS DEBATED BUT IT MAY IMPACT IN FOUR CLINICAL CONTEXTS: 1) THE TRANSMISSION OF THE INFECTION BY BLOOD TRANSFUSION OR ORGAN TRANSPLANTATION; 2) THE ACUTE REACTIVATION WHEN AN IMMUNOSUPPRESSIVE STATUS OCCURS MAINLY IN PATIENTS WITH ISOLATED ANTI-HBC (CHEMOTHERAPY, TRANSPLANTATIONS, IMMUNODEPRESSION, NEW IMMUNOSUPPRESSIVE THERAPY AS ANTI-CD20 OR ANTI TNF); 3) THE POTENT BUT NON PROVED PROGRESSION OF LIVER FIBROSIS IN HCV INFECTED PATIENTS OR IN PATIENTS WITH CRYPTOGENETIC LIVER DISEASE; AND, 4. THE RISK FACTOR FOR HEPATOCELLULAR CARCINOMA DEVELOPMENT. 2008 17 3782 48 INTERFERON THERAPY OF CHRONIC HEPATITIS B. CHRONIC HEPATITIS B (CHB) RESULTS FROM THE INABILITY OF THE HOST'S IMMUNE SYSTEM TO CONTROL VIRAL REPLICATION. INTERFERON-ALPHA (IFN-ALPHA) THERAPY CAN CONVERT CHB INTO INACTIVE HEPATITIS B VIRUS (HBV) INFECTION IN 20-30% OF THE TREATED PATIENTS. IN SPITE OF THE LOW RESPONSE RATE, IFN-ALPHA THERAPY HAS THE ADVANTAGE OF HAVING A LIMITED DURATION AND BEING EFFECTIVE EVEN AFTER THERAPY, AS DEMONSTRATED BY A MUCH HIGHER INCIDENCE OF HBSAG CLEARANCE IN RESPONDERS TO IFN-ALPHA THAN IN NATURALLY OCCURRING INACTIVE HBSAG CARRIERS. IFN-ALPHA HAS MULTIPLE ANTIVIRAL, ANTIPROLIFERATIVE, AND IMMUNOMODULATORY ACTIVITIES AND TARGETS: CELLULAR GENES (IFN-STIMULATED GENES) ACTIVATING DIFFERENT PATHWAYS OF ANTIVIRAL DEFENSE IN INFECTED AND NONINFECTED CELLS, HBV REPLICATION BLOCKING THE RNA-CONTAINING CORE PARTICLE FORMATION AND ACCELERATING THEIR DECAY, DEGRADING PREGENOMIC RNA, AND MODULATING THE NUCLEAR VIRAL MINICHROMOSOME (COVALENTLY CLOSED CIRCULAR DNA) ACTIVITY BY TARGETING ITS EPIGENETIC REGULATION AND BOTH INNATE AND ADAPTIVE IMMUNE RESPONSE. THE INTERFERENCE OF VIRAL HETEROGENEITY AND GENETIC POLYMORPHISMS OF THE HOST ON IFN-ALPHA SUSCEPTIBILITY IS UNDER INVESTIGATION. ONLY A BETTER UNDERSTANDING OF THE COMPLEX INTERPLAY BETWEEN THE DIFFERENT ACTIVITIES OF IFN-ALPHA WOULD WARRANT THE AMELIORATION OF CURRENT THERAPEUTIC STRATEGIES AND THE DESIGN OF NEW THERAPEUTIC APPROACHES. THE STUDY OF ON-TREATMENT DYNAMICS OF HBV INFECTION BY MEANS OF COMBINED QUANTITATIVE MONITORING OF SERUM HBV DNA AND HBSAG WARRANT TAILORING TREATMENT AT THE SINGLE-PATIENT LEVEL AND CAN HELP TO MAKE TREATMENT MORE COST-EFFECTIVE BY USING THE DIFFERENT COMBINATIONS OF CURRENTLY AVAILABLE ANTIVIRALS, INCLUDING IFN, MORE APPROPRIATELY. INTEGRATED MOLECULAR AND CLINICAL KNOWLEDGE IN A SYSTEMS MEDICINE FASHION IS MANDATORY TO FURTHER IMPROVE ANTIVIRAL THERAPY IN CHB. 2014 18 5952 42 TARGETING VIRAL CCCDNA FOR CURE OF CHRONIC HEPATITIS B. PURPOSE OF REVIEW: CHRONIC HEPATITIS B (CHB), CAUSED BY HEPATITIS B VIRUS (HBV), IS A MAJOR CAUSE OF ADVANCED LIVER DISEASE AND HEPATOCELLULAR CARCINOMA (HCC) WORLDWIDE. HBV REPLICATION IS CHARACTERIZED BY THE SYNTHESIS OF COVALENTLY CLOSED CIRCULAR (CCC) DNA WHICH IS NOT TARGETED BY ANTIVIRAL NUCLEOS(T)IDE ANALOGUES (NUCS) THE KEY MODALITY OF STANDARD OF CARE. WHILE HBV REPLICATION IS SUCCESSFULLY SUPPRESSED IN TREATED PATIENTS, THEY REMAIN AT RISK FOR DEVELOPING HCC. WHILE FUNCTIONAL CURE, CHARACTERIZED BY LOSS OF HBSAG, IS THE FIRST GOAL OF NOVEL ANTIVIRAL THERAPIES, CURATIVE TREATMENTS ELIMINATING CCCDNA REMAIN THE ULTIMATE GOAL. THIS REVIEW SUMMARIZES RECENT ADVANCES IN THE DISCOVERY AND DEVELOPMENT OF NOVEL THERAPEUTIC STRATEGIES AND THEIR IMPACT ON CCCDNA BIOLOGY. RECENT FINDINGS: WITHIN THE LAST DECADE, SUBSTANTIAL PROGRESS HAS BEEN MADE IN THE UNDERSTANDING OF CCCDNA BIOLOGY INCLUDING THE DISCOVERY OF HOST DEPENDENCY FACTORS, EPIGENETIC REGULATION OF CCCDNA TRANSCRIPTION AND IMMUNE-MEDIATED DEGRADATION. SEVERAL APPROACHES TARGETING CCCDNA EITHER IN A DIRECT OR INDIRECT MANNER ARE CURRENTLY AT THE STAGE OF DISCOVERY, PRECLINICAL OR EARLY CLINICAL DEVELOPMENT. EXAMPLES INCLUDE GENOME-EDITING APPROACHES, STRATEGIES TARGETING HOST DEPENDENCY FACTORS OR EPIGENETIC GENE REGULATION, NUCLEOCAPSID MODULATORS AND IMMUNE-MEDIATED DEGRADATION. SUMMARY: WHILE DIRECT-TARGETING CCCDNA STRATEGIES ARE STILL LARGELY AT THE PRECLINICAL STAGE OF DEVELOPMENT, CAPSID ASSEMBLY MODULATORS AND IMMUNE-BASED APPROACHES HAVE REACHED THE CLINICAL PHASE. CLINICAL TRIALS ARE ONGOING TO ASSESS THEIR EFFICACY AND SAFETY IN PATIENTS INCLUDING THEIR IMPACT ON VIRAL CCCDNA. COMBINATION THERAPIES PROVIDE ADDITIONAL OPPORTUNITIES TO OVERCOME CURRENT LIMITATIONS OF INDIVIDUAL APPROACHES. 2020 19 3255 33 HEPATITIS B VIRUS X PROTEIN MEDIATED EPIGENETIC ALTERATIONS IN THE PATHOGENESIS OF HEPATOCELLULAR CARCINOMA. CHRONIC HEPATITIS B VIRUS (HBV) INFECTION IS A WORLDWIDE HEALTH PROBLEM. HEPATITIS B VIRUS X PROTEIN (HBX), A PLEIOTROPIC REGULATORY PROTEIN ENCODED BY HBV, IS NECESSARY FOR THE TRANSCRIPTION OF HBV COVALENTLY CLOSED CIRCULAR DNA (CCCDNA) MINICHROMOSOMES, AND AFFECTS THE EPIGENETIC REGULATION OF HOST CELLS. THE EPIGENETIC REPROGRAMMING OF HBX ON HOST CELL GENOME IS STRONGLY INVOLVED IN HBV-RELATED HCC CARCINOGENESIS. HERE, WE REVIEW THE LATEST FINDINGS OF THE EPIGENETIC REGULATION INDUCED BY HBX PROTEIN IN HEPATOCELLULAR CARCINOMA (HCC), INCLUDING DNA METHYLATION, HISTONE MODIFICATION AND NON-CODING RNA EXPRESSION. THE INFLUENCE OF HBX ON THE EPIGENETIC REGULATION OF CCCDNA IS ALSO SUMMARIZED. IN ADDITION, PRELIMINARY STUDIES OF TARGETED DRUGS FOR EPIGENETIC CHANGES INDUCED BY HBX ARE ALSO DISCUSSED. THE EXPLORATION OF EPIGENETIC MARKERS AS POTENTIAL TARGETS WILL HELP TO DEVELOP NEW PREVENTION AND/OR TREATMENT METHODS FOR HBX-RELATED HCC. 2022 20 6016 30 THE ASSOCIATION BETWEEN HEPATOCARCINOGENESIS AND INTRACELLULAR ALTERATIONS DUE TO HEPATITIS B VIRUS INFECTION. CHRONIC HEPATITIS B VIRUS (HBV) INFECTION IS A WORLDWIDE HEALTH PROBLEM LEADING TO SEVERE LIVER DYSFUNCTION, INCLUDING LIVER CIRRHOSIS AND HEPATOCELLULAR CARCINOMA. ALTHOUGH CURRENT ANTIVIRAL THERAPIES FOR CHRONIC HBV INFECTION HAVE BEEN IMPROVED AND CAN LEAD TO A STRONG SUPPRESSION OF VIRAL REPLICATION, IT IS DIFFICULT TO COMPLETELY ELIMINATE THE VIRUS WITH THESE THERAPIES ONCE CHRONIC HBV INFECTION IS ESTABLISHED IN THE HOST. FURTHERMORE, CHRONIC HBV INFECTION ALTERS INTRACELLULAR METABOLISM AND SIGNALLING PATHWAYS, RESULTING IN THE ACTIVATION OF CARCINOGENESIS IN THE LIVER. HBV PRODUCES FOUR VIRAL PROTEINS: HEPATITIS B SURFACE-, HEPATITIS B CORE-, HEPATITIS B X PROTEIN, AND POLYMERASE; EACH PLAYS AN IMPORTANT ROLE IN HBV REPLICATION AND THE INTRACELLULAR SIGNALLING PATHWAYS ASSOCIATED WITH HEPATOCARCINOGENESIS. IN VITRO AND IN VIVO EXPERIMENTAL MODELS FOR ANALYZING HBV INFECTION AND REPLICATION HAVE BEEN ESTABLISHED, AND GENE EXPRESSION ANALYSES USING MICROARRAYS OR NEXT-GENERATION SEQUENCING HAVE ALSO BEEN DEVELOPED. THUS, IT IS POSSIBLE TO CLARIFY THE MOLECULAR MECHANISMS FOR INTRACELLULAR ALTERATIONS, SUCH AS ENDOPLASMIC RETICULUM STRESS, OXIDATIVE STRESS, AND EPIGENETIC MODIFICATIONS. IN THIS REVIEW, THE IMPACT OF HBV VIRAL PROTEINS AND INTRACELLULAR ALTERATIONS IN HBV-ASSOCIATED HEPATOCARCINOGENESIS ARE DISCUSSED. 2021