1 4055 145 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 2 5547 44 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 3 6337 27 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 5368 34 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 5 2324 45 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 6 1178 37 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 7 5689 51 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 8 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 9 6115 43 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 10 5936 35 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 11 6016 36 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 12 4056 65 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 13 3186 42 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 14 3255 34 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 15 5952 43 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 16 3401 26 HOW DID HEPATITIS B VIRUS EFFECT THE HOST GENOME IN THE LAST DECADE? THE PRINCIPAL REASON OF CHRONIC LIVER DISEASE, CIRRHOSIS AND HEPATOCELLULAR CARCINOMA IS CHRONIC VIRAL HEPATITIS ALL OVER THE WORLD. HEPATITIS B VIRUS (HBV) HAS SOME MUTAGENIC EFFECTS ON THE HOST GENOME. HBV MAY BE EXHIBITING THESE MUTAGENIC EFFECTS THROUGH INTEGRATING INTO THE HOST GENOME, THROUGH ITS VIRAL PROTEINS OR THROUGH SOME EPIGENETIC MECHANISMS RELATED WITH HBV PROTEINS. THIS REVIEW AIMS TO SUMMARIZE THE MOLECULAR MECHANISMS USED BY HBV FOR EFFECTING HOST GENOME DETERMINED IN THE LAST DECADE. THE FOCUS WILL BE ON THE EFFECTS OF INTEGRATION, HBV PROTEINS, ESPECIALLY HBV X PROTEIN AND EPIGENETIC MECHANISMS ON THE HOST GENOME. THESE INTERACTIONS BETWEEN HBV AND THE HOST GENOME ALSO FORMS THE UNDERLYING MECHANISMS OF THE EVOLUTION OF HEPATOCELLULAR CARCINOMA. 2014 17 4127 44 MECHANISMS OF DNA METHYLATION IN VIRUS-HOST INTERACTION IN HEPATITIS B INFECTION: PATHOGENESIS AND ONCOGENETIC PROPERTIES. HEPATITIS B VIRUS (HBV), THE WELL-STUDIED ONCOVIRUS THAT CONTRIBUTES TO THE MAJORITY OF HEPATOCELLULAR CARCINOMAS (HCC) WORLDWIDE, CAN CAUSE A SEVERE INFLAMMATORY MICROENVIRONMENT LEADING TO GENETIC AND EPIGENETIC CHANGES IN HEPATOCYTE CLONES. HBV REPLICATION CONTRIBUTES TO THE REGULATION OF DNA METHYLTRANSFERASE GENE EXPRESSION, PARTICULARLY BY X PROTEIN (HBX), AND SUBSEQUENT METHYLATION CHANGES MAY LEAD TO ABNORMAL TRANSCRIPTION ACTIVATION OF ADJACENT GENES AND GENOMIC INSTABILITY. UNDOUBTEDLY, THE ALTERED EXPRESSION OF THESE GENES HAS BEEN KNOWN TO CAUSE DIVERSE ASPECTS OF INFECTED HEPATOCYTES, INCLUDING APOPTOSIS, PROLIFERATION, REACTIVE OXYGEN SPECIES (ROS) ACCUMULATION, AND IMMUNE RESPONSES. ADDITIONALLY, POLLUTANT-INDUCED DNA METHYLATION CHANGES AND ABERRANT METHYLATION OF IMPRINTED GENES IN HEPATOCYTES ALSO COMPLICATE THE PROCESS OF TUMORIGENESIS. MEANWHILE, HEPATOCYTES ALSO CONTRIBUTE TO EPIGENETIC MODIFICATION OF THE VIRAL GENOME TO AFFECT HBV REPLICATION OR VIRAL PROTEIN PRODUCTION. MEANWHILE, METHYLATION LEVELS OF HBV INTEGRANTS AND SURROUNDING HOST REGIONS ALSO PLAY CRUCIAL ROLES IN THEIR ABILITY TO PRODUCE VIRAL PROTEINS IN AFFECTED HEPATOCYTES. BOTH HOST AND VIRAL CHANGES CAN PROVIDE NOVEL INSIGHTS INTO TUMORIGENESIS, INDIVIDUALIZED RESPONSES TO THERAPEUTIC INTERVENTION, DISEASE PROGRESS, AND EARLY DIAGNOSIS. AS SUCH, DNA METHYLATION-MEDIATED EPIGENETIC SILENCING OF CANCER-RELATED GENES AND VIRAL REPLICATION IS A COMPELLING THERAPEUTIC GOAL TO REDUCE MORBIDITY AND MORTALITY FROM LIVER CANCER CAUSED BY CHRONIC HBV INFECTION. IN THIS REVIEW, WE SUMMARIZE THE MOST RECENT RESEARCH ON ABERRANT DNA METHYLATION ASSOCIATED WITH HBV INFECTION, WHICH IS INVOLVED IN HCC DEVELOPMENT, AND PROVIDE AN OUTLOOK ON THE FUTURE DIRECTION OF THE RESEARCH. 2021 18 2165 42 EPIGENETIC MECHANISMS IN HEPATITIS B VIRUS-ASSOCIATED HEPATOCELLULAR CARCINOMA. CHRONIC INFECTION OF THE LIVER BY THE HEPATITIS B VIRUS (HBV) IS ASSOCIATED WITH INCREASED RISK FOR DEVELOPING HEPATOCELLULAR CARCINOMA (HCC). A MULTITUDE OF STUDIES HAVE INVESTIGATED THE MECHANISM OF LIVER CANCER PATHOGENESIS DUE TO CHRONIC HBV INFECTION. CHRONIC INFLAMMATION, EXPRESSION OF SPECIFIC VIRAL PROTEINS SUCH AS HBX, THE INTEGRATION SITE OF THE VIRAL GENOME INTO THE HOST GENOME, AND THE VIRAL GENOTYPE, ARE KEY PLAYERS CONTRIBUTING TO HCC PATHOGENESIS. IN ADDITION, THE GENETIC BACKGROUND OF THE HOST AND EXPOSURE TO ENVIRONMENTAL CARCINOGENS ARE ALSO PREDISPOSING PARAMETERS IN HEPATOCARCINOGENESIS. DESPITE THE PLETHORA OF STUDIES, THE MOLECULAR MECHANISM OF HCC PATHOGENESIS REMAINS INCOMPLETELY UNDERSTOOD. IN THIS REVIEW, THE FOCUS IS ON EPIGENETIC MECHANISMS INVOLVED IN THE PATHOGENESIS OF HBV-ASSOCIATED HCC. EPIGENETIC MECHANISMS ARE DYNAMIC MOLECULAR PROCESSES THAT REGULATE GENE EXPRESSION WITHOUT ALTERING THE HOST DNA, ACTING BY MODIFYING THE HOST CHROMATIN STRUCTURE VIA COVALENT POST-TRANSLATIONAL HISTONE MODIFICATIONS, CHANGING THE DNA METHYLATION STATUS, EXPRESSION OF NON-CODING RNAS SUCH AS MICRORNAS AND LONG NONCODING RNAS, AND ALTERING THE SPATIAL, 3-D ORGANIZATION OF THE CHROMATIN OF THE VIRUS-INFECTED CELL. HEREIN, STUDIES ARE DESCRIBED THAT PROVIDE EVIDENCE IN SUPPORT OF DEREGULATION OF EPIGENETIC MECHANISMS IN THE HBV-INFECTED/-REPLICATING HEPATOCYTE AND THEIR CONTRIBUTION TO HEPATOCYTE TRANSFORMATION. IN CONTRAST TO GENETIC MUTATIONS WHICH ARE PERMANENT, EPIGENETIC ALTERATIONS ARE DYNAMIC AND REVERSIBLE. ACCORDINGLY, THE IDENTIFICATION OF ESSENTIAL MOLECULAR EPIGENETIC TARGETS INVOLVED IN HBV-MEDIATED HCC PATHOGENESIS OFFERS THE OPPORTUNITY FOR THE DESIGN AND DEVELOPMENT OF NOVEL EPIGENETIC THERAPEUTIC APPROACHES. 2021 19 3806 47 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 20 6271 40 THE ONCOGENIC ROLE OF HEPATITIS B VIRUS. THE HEPATITIS B VIRUS (HBV) IS A SMALL ENVELOPED DNA VIRUS THAT CAUSES ACUTE AND CHRONIC HEPATITIS. HBV INFECTION IS A WORLD HEALTH PROBLEM, WITH 350 MILLION CHRONICALLY INFECTED PEOPLE AT INCREASED RISK OF DEVELOPING LIVER DISEASE AND HEPATOCELLULAR CARCINOMA (HCC). HBV HAS BEEN CLASSIFIED AMONG HUMAN TUMOR VIRUSES BY VIRTUE OF A ROBUST EPIDEMIOLOGIC ASSOCIATION BETWEEN CHRONIC HBV CARRIAGE AND HCC OCCURRENCE. IN THE ABSENCE OF CYTOPATHIC EFFECT IN INFECTED HEPATOCYTES, THE ONCOGENIC ROLE OF HBV MIGHT INVOLVE A COMBINATION OF DIRECT AND INDIRECT EFFECTS OF THE VIRUS DURING THE MULTISTEP PROCESS OF LIVER CARCINOGENESIS. LIVER INFLAMMATION AND HEPATOCYTE PROLIFERATION DRIVEN BY HOST IMMUNE RESPONSES ARE RECOGNIZED DRIVING FORCES OF LIVER CELL TRANSFORMATION. GENETIC AND EPIGENETIC ALTERATIONS CAN ALSO RESULT FROM VIRAL DNA INTEGRATION INTO HOST CHROMOSOMES AND FROM PROLONGED EXPRESSION OF VIRAL GENE PRODUCTS. NOTABLY, THE TRANSCRIPTIONAL REGULATORY PROTEIN HBX ENCODED BY THE X GENE IS ENDOWED WITH TUMOR PROMOTER ACTIVITY. HBX HAS PLEIOTROPIC ACTIVITIES AND PLAYS A MAJOR ROLE IN HBV PATHOGENESIS AND IN LIVER CARCINOGENESIS. BECAUSE HEPATIC TUMORS CARRY A DISMAL PROGNOSIS, THERE IS URGENT NEED TO DEVELOP EARLY DIAGNOSTIC MARKERS OF HCC AND EFFECTIVE THERAPIES AGAINST CHRONIC HEPATITIS B. DECIPHERING THE ONCOGENIC MECHANISMS THAT UNDERLIE HBV-RELATED TUMORIGENESIS MIGHT HELP DEVELOPING ADAPTED THERAPEUTIC STRATEGIES. 2014