1  2401 165 EPIGENETIC REPROGRAMMING PROMOTES THE ANTIVIRAL ACTION OF IFNALPHA IN HBV-INFECTED CELLS. CHRONIC HEPATITIS B VIRUS (HBV) INFECTIONS REMAIN A HEALTH BURDEN AFFECTING ~250 MILLION PEOPLE WORLDWIDE. THUS FAR, AVAILABLE INTERFERON-ALPHA (IFNALPHA)-BASED THERAPIES HAVE SHOWN UNSATISFACTORY CURE RATES, AND ALTERNATIVE THERAPEUTIC MOLECULES ARE STILL REQUIRED. HOWEVER, THEIR DEVELOPMENT HAS BEEN HAMPERED BECAUSE ACCESSIBLE CELL MODELS SUPPORTING RELEVANT HBV REPLICATION AND APPROPRIATE ANTIVIRAL ACTIVITY ARE LACKING. STRATEGIES THAT REVERSE EPIGENETIC ALTERATIONS OFFER A UNIQUE OPPORTUNITY FOR CELL REPROGRAMMING, WHICH IS VALUABLE FOR RESTORING ALTERED CELLULAR FUNCTIONS IN HUMAN CELL LINES. THIS WORK AIMED TO INVESTIGATE THE FEASIBILITY OF CONVERTING HEPG2 CELLS THAT STABLY OVEREXPRESS THE HBV ENTRY RECEPTOR (SODIUM/TAUROCHOLATE COTRANSPORTING POLYPEPTIDE, NTCP) TOWARD IFNALPHA-RESPONSIVE CELLS USING EPIGENETIC REPROGRAMMING. HEREIN, WE SHOWED THAT AN EPIGENETIC REGIMEN WITH NON-CYTOTOXIC DOSES OF THE DEMETHYLATING COMPOUND 5-AZACYTIDINE RESTORED THE ANTI-HBV ACTION OF IFNALPHA IN EPIGENETICALLY REPROGRAMMED HEPG2-NTCP-C4 CELLS, NAMED REP-HEPG2-NTCP CELLS. THUS, A SIGNIFICANT INHIBITION IN HBV DNA LEVELS WAS MEASURED IN REP-HEPG2-NTCP CELLS AFTER IFNALPHA TREATMENT. THIS INHIBITORY EFFECT WAS ASSOCIATED WITH THE ENHANCEMENT OF IFNALPHA-MEDIATED INDUCTION OF CRITICAL INTERFERON-STIMULATED GENES (ISGS), WHICH WAS LIMITED IN NON-REPROGRAMMED CELLS. IN PARTICULAR, OUR DATA INDICATED THAT RE-EXPRESSION OF 2'-5'-OLIGOADENYLATE SYNTHETASE 1 (OAS1) AND INTERFERON REGULATORY FACTOR 9 (IRF9) WAS THE RESULT OF AN EPIGENETICALLY DRIVEN UNMASKING OF THESE GENES IN REPROGRAMMED CELLS. AT LAST, WE EVALUATED THE THERAPEUTIC POTENTIAL OF THE IFN ANALOG CDM-3008 IN REP-HEPG2-NTCP CELLS AND DEMONSTRATED THE EFFICIENCY OF THIS CHEMICAL COMPOUND IN TRIGGERING ISG INDUCTION AND HBV INHIBITION. IN SUMMARY, THIS STUDY SHOWS THAT EPIGENETIC REPROGRAMMING PROMOTES THE IFNALPHA RESPONSE IN HBV-INFECTED CELLS AND IS POTENTIALLY ATTRACTIVE FOR CELL-BASED EXPERIMENTAL SCREENING OF IFN-LIKE COMPOUNDS.	2021	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
2  5510  39 RIBAVIRIN RESTORES IFNALPHA RESPONSIVENESS IN HCV-INFECTED LIVERS BY EPIGENETIC REMODELLING AT INTERFERON STIMULATED GENES. OBJECTIVES: CAVEATS IN THE UNDERSTANDING OF RIBAVIRIN (RBV) MECHANISMS OF ACTION HAS SOMEHOW PREVENTED THE DEVELOPMENT OF BETTER ANALOGUES ABLE TO FURTHER IMPROVE ITS THERAPEUTIC CONTRIBUTION IN INTERFERON (IFN)-BASED AND DIRECT ANTIVIRAL AGENT-BASED REGIMENS FOR CHRONIC HCV OR OTHER INDICATIONS. HERE, WE DESCRIBE A NEW MECHANISM BY WHICH RBV MODULATES IFN-STIMULATED GENES (ISGS) AND CONTRIBUTES TO RESTORE HEPATIC IMMUNE RESPONSIVENESS. DESIGN: RBV EFFECT ON ISG EXPRESSION WAS MONITORED IN VITRO AND IN VIVO, THAT IS, IN NON-TRANSFORMED HEPATOCYTES AND IN THE LIVER OF RBV MONO-TREATED PATIENTS, RESPECTIVELY. MODULATION OF HISTONE MODIFICATIONS AND RECRUITMENT OF HISTONE-MODIFYING ENZYMES AT TARGET PROMOTERS WAS ANALYSED BY CHROMATIN IMMUNOPRECIPITATION IN RBV-TREATED PRIMARY HUMAN HEPATOCYTES AND IN PATIENTS' LIVER BIOPSIES. RESULTS: RBV DECREASES THE MRNA LEVELS OF SEVERAL ABNORMALLY PREACTIVATED ISGS IN PATIENTS WITH HCV, WHO ARE NON-RESPONDERS TO IFN THERAPY. RBV INCREASES G9A HISTONE METHYLTRANSFERASE RECRUITMENT AND HISTONE-H3 LYSINE-9 DIMETHYLATION/TRIMETHYLATION AT SELECTED ISG PROMOTERS IN VITRO AND IN VIVO. G9A PHARMACOLOGICAL BLOCKADE ABOLISHES RBV-INDUCED ISG DOWNREGULATION AND SEVERELY IMPAIRS RBV ABILITY TO POTENTIATE IFN ANTIVIRAL ACTION AND INDUCTION OF ISGS FOLLOWING HCV INFECTION OF PRIMARY HUMAN HEPATOCYTES. CONCLUSIONS: RBV-INDUCED EPIGENETIC CHANGES, LEADING TO DECREASED ISG EXPRESSION, RESTORE AN IFN-RESPONSIVE HEPATIC ENVIRONMENT IN PATIENTS WITH HCV, WHICH MAY ALSO PROVE USEFUL IN IFN-FREE REGIMENS.	2016	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
3  3686  27 INFLAMMATION-RELATED ABERRANT PATTERNS OF DNA METHYLATION: DETECTION AND ROLE IN EPIGENETIC DEREGULATION OF CANCER CELL TRANSCRIPTOME. IT IS NOW APPARENT THAT EPIGENETIC ABNORMALITIES, IN PARTICULAR ALTERED DNA METHYLATION, PLAY A CRUCIAL ROLE IN THE DEVELOPMENT AND PROGRESSION OF HUMAN CANCERS. DNA HYPERMETHYLATION AT PROMOTER CPG ISLANDS IS NOW RECOGNIZED AS A THIRD MECHANISM BY WHICH INACTIVATION OF TUMOR SUPPRESSOR GENES OCCURS. ABERRANT CPG ISLAND HYPERMETHYLATION IS ALSO FREQUENTLY OBSERVED IN CHRONIC INFLAMMATION AND PRECANCEROUS LESIONS, WHICH SUGGESTS THAT IT IS AN EARLY EVENT IN TUMORIGENESIS THAT COULD SERVE AS A USEFUL TUMOR MARKER. A VARIETY OF SCREENING TECHNIQUES HAVE BEEN DEVELOPED FOR GENOME-WIDE SCREENING OF METHYLATION STATUS. OF THOSE, TRANSCRIPTOME ANALYSIS COUPLED WITH PHARMACOLOGICAL UNMASKING HAS EMERGED AS A POWERFUL TOOL FOR REVEALING DNA METHYLATION PATTERNS IN CANCER CELLS AND IDENTIFYING NEW TUMOR MARKER CANDIDATES.	2009	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                
4   730  33 CANCER CELLS RESISTANT TO IMMUNE CHECKPOINT BLOCKADE ACQUIRE INTERFERON-ASSOCIATED EPIGENETIC MEMORY TO SUSTAIN T CELL DYSFUNCTION. PROLONGED INTERFERON (IFN) SIGNALING IN CANCER CELLS CAN PROMOTE RESISTANCE TO IMMUNE CHECKPOINT BLOCKADE (ICB). HOW CANCER CELLS RETAIN EFFECTS OF PROLONGED IFN STIMULATION TO COORDINATE RESISTANCE IS UNCLEAR. WE SHOW THAT, ACROSS HUMAN AND/OR MOUSE TUMORS, IMMUNE DYSFUNCTION IS ASSOCIATED WITH CANCER CELLS ACQUIRING EPIGENETIC FEATURES OF INFLAMMATORY MEMORY. HERE, INFLAMMATORY MEMORY DOMAINS, MANY OF WHICH ARE INITIATED BY CHRONIC IFN-GAMMA, ARE MAINTAINED BY SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION (STAT)1 AND IFN REGULATORY FACTOR (IRF)3 AND LINK HISTONE 3 LYSINE 4 MONOMETHYLATION (H3K4ME1)-MARKED CHROMATIN ACCESSIBILITY TO INCREASED EXPRESSION OF A SUBSET OF IFN-STIMULATED GENES (ISGS). THESE ISGS INCLUDE THE RNA SENSOR OAS1 THAT AMPLIFIES TYPE I IFN (IFN-I) AND IMMUNE INHIBITORY GENES. ABROGATING CANCER CELL IFN-I SIGNALING RESTORES ANTI-PROGRAMMED CELL DEATH PROTEIN 1 (PD1) RESPONSE BY INCREASING IFN-GAMMA IN IMMUNE CELLS, PROMOTING DENDRITIC CELL AND CD8(+) T CELL INTERACTIONS, AND EXPANDING T CELLS TOWARD EFFECTOR-LIKE STATES RATHER THAN EXHAUSTED STATES. THUS, CANCER CELLS ACQUIRE INFLAMMATORY MEMORY TO AUGMENT A SUBSET OF ISGS THAT PROMOTE AND PREDICT IFN-DRIVEN IMMUNE DYSFUNCTION.	2023	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      
5  4239  42 METHYLATION PROFILE OF HEPATITIS B VIRUS IS NOT INFLUENCED BY INTERFERON ALPHA IN HUMAN LIVER CANCER CELLS. INTERFERON (IFN) ALPHA IS USED FOR THE TREATMENT OF CHRONIC HEPATITIS B VIRUS (HBV) INFECTION, BUT THE MOLECULAR MECHANISMS UNDERLYING ITS ANTIVIRAL EFFECT HAVE NOT BEEN FULLY ELUCIDATED. EPIGENETIC MODIFICATIONS REGULATE THE TRANSCRIPTIONAL ACTIVITY OF COVALENTLY CLOSED CIRCULAR DNA (CCCDNA) IN CELLS WITH CHRONIC HBV INFECTION. IFN?ALPHA HAS BEEN SHOWN TO MODIFY CCCDNA?BOUND HISTONES, BUT IT IS NOT KNOWN WHETHER THE ANTI?HBV EFFECT OF IFN?ALPHA INVOLVES METHYLATION OF CCCDNA. THE PRESENT STUDY AIMED TO DETERMINE WHETHER IFN?ALPHA INDUCED METHYLATION OF HBV CCCDNA IN A CELL?BASED MODEL IN WHICH HEPG2 CELLS WERE DIRECTLY INFECTED WITH WILD?TYPE HBV VIRIONS. METHYLATION STATUS OF HBV CCCDNA WAS ASSESSED USING GLOBAL DNA METHYLATION ELISA ASSAY, METHYLATION?SPECIFIC PCR AND BISULFITE SEQUENCING. IFN?ALPHA SUPPRESSED HBV DNA AND RNA TRANSCRIPTS, BUT METHYLATION PROFILES WERE SIMILAR BETWEEN THE CONTROL AND IFN?ALPHA TREATED GROUPS. CHROMATIN IMMUNOPRECIPITATION RESULTS REVEALED BINDING OF DNA METHYLTRANSFERASES (DNMT) 3A AND DNMT3B TO HBV CCCDNA AND TREATMENT WITH IFN?ALPHA SUPPRESSED THE RECRUITMENT OF DNMT3B TO CCCDNA. TAKEN TOGETHER, THESE RESULTS SUGGEST THAT IFN?ALPHA DOES NOT INDUCE METHYLATION OF HBV CCCDNA. THEREFORE, IT WAS CONCLUDED THAT METHYLATION IS UNLIKELY TO CONTRIBUTE TO THE ANTI?HBV EFFECT OF IFN?ALPHA IN HEPG2 CELLS, AND THAT ALTERNATIVE MECHANISMS NEED TO BE SOUGHT TO ENHANCE CCCDNA METHYLATION AS A NOVEL THERAPY AGAINST HBV.	2021	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         
6  3779  47 INTERFERON ALPHA INDUCES MULTIPLE CELLULAR PROTEINS THAT COORDINATELY SUPPRESS HEPADNAVIRAL COVALENTLY CLOSED CIRCULAR DNA TRANSCRIPTION. COVALENTLY CLOSED CIRCULAR DNA (CCCDNA) OF HEPADNAVIRUSES EXISTS AS AN EPISOMAL MINICHROMOSOME IN THE NUCLEUS OF AN INFECTED HEPATOCYTE AND SERVES AS THE TEMPLATE FOR THE TRANSCRIPTION OF VIRAL MRNAS. IT HAD BEEN DEMONSTRATED BY OTHERS AND US THAT INTERFERON ALPHA (IFN-ALPHA) TREATMENT OF HEPATOCYTES INDUCED A PROLONGED SUPPRESSION OF HUMAN AND DUCK HEPATITIS B VIRUS CCCDNA TRANSCRIPTION, WHICH IS ASSOCIATED WITH THE REDUCTION OF CCCDNA-ASSOCIATED HISTONE MODIFICATIONS SPECIFYING ACTIVE TRANSCRIPTION (H3K9(AC) OR H3K27(AC)), BUT NOT THE HISTONE MODIFICATIONS MARKING CONSTITUTIVE (H3K9(ME3)) OR FACULTATIVE (H3K27(ME3)) HETEROCHROMATIN FORMATION. IN OUR EFFORTS TO IDENTIFY IFN-INDUCED CELLULAR PROTEINS THAT MEDIATE THE SUPPRESSION OF CCCDNA TRANSCRIPTION BY THE CYTOKINE, WE FOUND THAT DOWNREGULATING THE EXPRESSION OF SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 1 (STAT1), STRUCTURAL MAINTENANCE OF CHROMOSOMES FLEXIBLE HINGE DOMAIN CONTAINING 1 (SMCHD1), OR PROMYELOCYTIC LEUKEMIA (PML) PROTEIN INCREASED BASAL LEVEL OF CCCDNA TRANSCRIPTION ACTIVITY AND PARTIALLY ATTENUATED IFN-ALPHA SUPPRESSION OF CCCDNA TRANSCRIPTION. IN CONTRAST, ECTOPIC EXPRESSION OF STAT1, SMCHD1, OR PML SIGNIFICANTLY REDUCED CCCDNA TRANSCRIPTION ACTIVITY. SMCHD1 IS A NONCANONICAL SMC FAMILY PROTEIN AND IMPLICATED IN EPIGENETIC SILENCING OF GENE EXPRESSION. PML IS A COMPONENT OF NUCLEAR DOMAIN 10 (ND10) AND IS INVOLVED IN SUPPRESSING THE REPLICATION OF MANY DNA VIRUSES. MECHANISTIC ANALYSES DEMONSTRATED THAT STAT1, SMCHD1, AND PML WERE RECRUITED TO CCCDNA MINICHROMOSOMES AND PHENOCOPIED THE IFN-ALPHA-INDUCED POSTTRANSLATIONAL MODIFICATIONS OF CCCDNA-ASSOCIATED HISTONES. WE THUS CONCLUDE THAT STAT1, SMCHD1, AND PML MAY PARTLY MEDIATE THE SUPPRESSIVE EFFECT OF IFN-ALPHA ON HEPADNAVIRAL CCCDNA TRANSCRIPTION.IMPORTANCE PEGYLATED IFN-ALPHA IS THE ONLY THERAPEUTIC REGIMEN THAT CAN INDUCE A FUNCTIONAL CURE OF CHRONIC HEPATITIS B IN A SMALL, BUT SIGNIFICANT, FRACTION OF TREATED PATIENTS. UNDERSTANDING THE MECHANISMS UNDERLYING THE ANTIVIRAL FUNCTIONS OF IFN-ALPHA IN HEPADNAVIRAL INFECTION MAY REVEAL MOLECULAR TARGETS FOR DEVELOPMENT OF NOVEL ANTIVIRAL AGENTS TO IMPROVE THE THERAPEUTIC EFFICACY OF IFN-ALPHA. BY A LOSS-OF-FUNCTION GENETIC SCREENING OF INDIVIDUAL IFN-STIMULATED GENES (ISGS) ON HEPADNAVIRAL MRNAS TRANSCRIBED FROM CCCDNA, WE FOUND THAT DOWNREGULATING THE EXPRESSION OF STAT1, SMCHD1, OR PML SIGNIFICANTLY INCREASED THE LEVEL OF VIRAL RNAS WITHOUT ALTERING THE LEVEL OF CCCDNA. MECHANISTIC ANALYSES INDICATED THAT THOSE CELLULAR PROTEINS ARE RECRUITED TO CCCDNA MINICHROMOSOMES AND INDUCE THE POSTTRANSLATIONAL MODIFICATIONS OF CCCDNA-ASSOCIATED HISTONES SIMILAR TO THOSE INDUCED BY IFN-ALPHA TREATMENT. WE HAVE THUS IDENTIFIED THREE IFN-ALPHA-INDUCED CELLULAR PROTEINS THAT SUPPRESS CCCDNA TRANSCRIPTION AND MAY PARTLY MEDIATE IFN-ALPHA SILENCING OF HEPADNAVIRAL CCCDNA TRANSCRIPTION.	2020	

7  5678  36 SHORT HAIRPIN RNA INDUCES METHYLATION OF HEPATITIS B VIRUS COVALENTLY CLOSED CIRCULAR DNA IN HUMAN HEPATOMA CELLS. SMALL INTERFERING RNAS NOT ONLY MODULATE GENE EXPRESSION AT A POST-TRANSCRIPTIONAL LEVEL, BUT ALSO INDUCE TRANSCRIPTIONAL GENE SILENCING BY RNA INTERFERENCE-MEDIATED HETEROCHROMATIN FORMATION AND RNA-DIRECTED DNA METHYLATION (RDDM). HOWEVER, ALTHOUGH ESTABLISHED IN PLANTS, THERE HAVE BEEN CONTROVERSIES WHETHER RDDM OPERATES IN MAMMALS. HEPATITIS B VIRUS (HBV) COVALENTLY CLOSED CIRCULAR DNA (CCCDNA) SERVES AS A TEMPLATE FOR VIRAL RNA TRANSCRIPTION, AND TRANSCRIPTIONAL ACTIVITY OF HBV CCCDNA IS REGULATED BY METHYLATION IN PATIENTS WITH CHRONIC HBV INFECTION. IN THIS STUDY, WE STABLY EXPRESSED SHORT HAIRPIN RNA (SHRNA) AGAINST HBV IN HUMAN HEPATOMA CELLS TO DETERMINE WHETHER SHRNA INDUCES METHYLATION OF HBV CCCDNA. HEPAD38 CELLS WHICH PERMIT REPLICATION OF HBV UNDER CONTROL OF TETRACYCLINE-RESPONSIVE PROMOTER WERE TRANSDUCED WITH LENTIVIRAL VECTORS WHICH ENCODE SH-1580, A SHRNA AGAINST THE HEPATITIS B VIRAL PROTEIN HBX. BISULFITE SEQUENCING PCR ANALYSIS REVEALED THAT SH-1580 INDUCED CPG METHYLATIONS AT A HIGHER RATE COMPARED TO CONTROL (31.3% VS. 12.8%, P<0.05). THE SH-1580-INDUCED CPG METHYLATION WAS LOCALIZED NEAR THE TARGET SEQUENCE OF SH-1580 IN MORE THAN A HALF OF THE CLONES. METHYLATION-INDUCED TRANSCRIPTIONAL SUPPRESSION WAS CONFIRMED BY IN VITRO TRANSCRIPTION ASSAY. THESE RESULTS CONFIRM THE FEASIBILITY OF RDDM OF HBV CCCDNA IN HUMAN CELLS. LENTIVIRAL VECTOR-MEDIATED TRANSFER OF SHRNA MAY BE USED AS A TOOL FOR NOVEL TRANSCRIPTIONAL MODULATION BY EPIGENETIC MODIFICATION OF HBV CCCDNA.	2013	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  
8  3794  41 INTERLEUKIN-33 MEDIATES BOTH IMMUNE-RELATED AND NON-IMMUNE-RELATED INHIBITORY EFFECTS AGAINST HEPATITIS B VIRUS. CHRONIC INFECTION BY HEPATITIS B VIRUS (HBV) IS ASSOCIATED WITH HIGH RISKS OF LIVER FIBROSIS, CIRRHOSIS AND HEPATOCELLULAR CARCINOMA. HBV COVALENTLY CLOSED CIRCULAR DNA (CCCDNA) IN THE NUCLEUS OF INFECTED HEPATOCYTE SERVES AS TRANSCRIPTION TEMPLATE. NEITHER NATURAL RESOLUTION OF ACUTE INFECTION NOR CURRENT TREATMENT OPTIONS FOR CHRONIC INFECTION ARE BELIEVED TO CAUSE CCCDNA CLEARANCE. WE PREVIOUSLY SHOWED THAT INJECTION OF IL-33-EXPRESSING PLASMID FACILITATED CLEARANCE OF INTRAHEPATIC HBV DNA IN A MOUSE MODEL OF HBV PERSISTENCE. IN THIS WORK, HBV-TARGETING THERAPEUTIC EFFECTS OF IL-33 WERE FURTHER EXPLORED. MURINE IL-33 DELIVERED BY RECOMBINANT ADENO-ASSOCIATED VIRUS (AAV-MIL-33) INDUCED CLEARANCE OF BOTH SERUM HBV MARKERS AND INTRAHEPATIC HBV DNA IN TWO MOUSE MODELS OF HBV PERSISTENCE BASED ON REPLICON PLASMID AND RECOMBINANT CCCDNA (RCCCDNA) RESPECTIVELY. CLEARANCE WAS ASSOCIATED WITH SERUM ALT ELEVATIONS AND LIVER INFILTRATIONS BY CD4(+) AND CD8(+) T CELLS, INDICATING IL-33-INDUCED CELLULAR IMMUNE RESPONSES AGAINST HBV-HARBORING CELLS. ADOPTIVE TRANSFER OF SPLENOCYTES FROM AAV-MIL-33-CURED MICE WAS INDEED SUFFICIENT TO ENGENDER SIMILAR CLEARANCE IN RECIPIENT MICE. IN VITRO, INTRACELLULAR, INSTEAD OF EXTRACELLULAR, IL-33 WAS MAINLY RESPONSIBLE FOR REPRESSING VIRAL TRANSCRIPTION, PROTEIN PRODUCTION AND GENOME REPLICATION IN HUH7 CELLS TRANSFECTED WITH HBV REPLICON OR RCCCDNA. IL-33 WAS SHOWN TO BE RECRUITED ONTO RCCCDNA MINICHROMOSOME ACCOMPANIED BY LOSS OF TRANSCRIPTIONAL ACTIVATION EPIGENETIC MARKS. FINALLY, TRANSFECTION OF IL-33 INTO HBV-INFECTED HEPG2/NTCP CELLS RESULTED IN REDUCED TRANSCRIPTION, ANTIGEN EXPRESSION AND GENOME REPLICATION, SUGGESTING REPRESSION OF CANONICAL CCCDNA. THESE DATA DEMONSTRATED DIVERSE INHIBITORY EFFECTS ON HBV AND HBV-INFECTED CELLS MEDIATED BY IL-33, AND SUGGEST IL-33 AS AN INTERESTING THERAPEUTIC CANDIDATE.	2022	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            
9  6448  47 THERAPEUTIC SHUTDOWN OF HBV TRANSCRIPTS PROMOTES REAPPEARANCE OF THE SMC5/6 COMPLEX AND SILENCING OF THE VIRAL GENOME IN VIVO. OBJECTIVE: THERAPEUTIC STRATEGIES SILENCING AND REDUCING THE HEPATITIS B VIRUS (HBV) RESERVOIR, THE COVALENTLY CLOSED CIRCULAR DNA (CCCDNA), HAVE THE POTENTIAL TO CURE CHRONIC HBV INFECTION. WE AIMED TO INVESTIGATE THE IMPACT OF SMALL INTERFERRING RNA (SIRNA) TARGETING ALL HBV TRANSCRIPTS OR PEGYLATED INTERFERON-ALPHA (PEG-IFNALPHA) ON THE VIRAL REGULATORY HBX PROTEIN AND THE STRUCTURAL MAINTENANCE OF CHROMOSOME 5/6 COMPLEX (SMC5/6), A HOST FACTOR SUPPRESSING CCCDNA TRANSCRIPTION. IN PARTICULAR, WE ASSESSED WHETHER INTERVENTIONS LOWERING HBV TRANSCRIPTS CAN ACHIEVE AND MAINTAIN SILENCING OF CCCDNA TRANSCRIPTION IN VIVO. DESIGN: HBV-INFECTED HUMAN LIVER CHIMERIC MICE WERE TREATED WITH SIRNA OR PEG-IFNALPHA. VIROLOGICAL AND HOST CHANGES WERE ANALYSED AT THE END OF TREATMENT AND DURING THE REBOUND PHASE BY QUALITATIVE PCR, ELISA, IMMUNOBLOTTING AND CHROMATIN IMMUNOPRECIPITATION. RNA IN SITU HYBRIDISATION WAS COMBINED WITH IMMUNOFLUORESCENCE TO DETECT SMC6 AND HBV RNAS AT SINGLE CELL LEVEL. THE ENTRY INHIBITOR MYRCLUDEX-B WAS USED DURING THE REBOUND PHASE TO AVOID NEW INFECTION EVENTS. RESULTS: BOTH SIRNA AND PEG-IFNALPHA STRONGLY REDUCED ALL HBV MARKERS, INCLUDING HBX LEVELS, THUS ENABLING THE REAPPEARANCE OF SMC5/6 IN HEPATOCYTES THAT ACHIEVED HBV-RNA NEGATIVISATION AND SMC5/6 ASSOCIATION WITH THE CCCDNA. ONLY IFN REDUCED CCCDNA LOADS AND ENHANCED IFN-STIMULATED GENES. HOWEVER, THE ANTIVIRAL EFFECTS DID NOT PERSIST OFF TREATMENT AND SMC5/6 WAS AGAIN DEGRADED. REMARKABLY, THE BLOCKADE OF VIRAL ENTRY THAT STARTED AT THE END OF TREATMENT HINDERED RENEWED DEGRADATION OF SMC5/6. CONCLUSION: THESE RESULTS REVEAL THAT THERAPEUTICS ABROGATING ALL HBV TRANSCRIPTS INCLUDING HBX PROMOTE EPIGENETIC SUPPRESSION OF THE HBV MINICHROMOSOME, WHEREAS STRATEGIES PROTECTING THE HUMAN HEPATOCYTES FROM REINFECTION ARE NEEDED TO MAINTAIN CCCDNA SILENCING.	2022	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         
10   152  25 ABERRANT METHYLATION OF DAPK IN LONG-STANDING ULCERATIVE COLITIS AND ULCERATIVE COLITIS-ASSOCIATED CARCINOMA. DEATH-ASSOCIATED PROTEIN KINASE (DAPK) HAS PRO-APOPTOTIC FUNCTIONS AND PARTICIPATES IN VARIOUS APOPTOTIC SYSTEMS. DAPK ACTS AS A TUMOR SUPPRESSOR, AND ITS INACTIVATION BY PROMOTER HYPERMETHYLATION HAS BEEN FREQUENTLY OBSERVED IN VARIOUS HUMAN CANCERS. AS ALTERATIONS OF PRO-APOPTOTIC GENES MIGHT CAUSE INSTABILITY IN THE BALANCE OF CELL-TURNOVER DURING CHRONIC INFLAMMATORY PROCESSES, EPIGENETIC SILENCING OF DAPK MIGHT BE INVOLVED IN THE CARCINOGENESIS OF ULCERATIVE COLITIS-ASSOCIATED CARCINOMA (UCC). TO EVALUATE THE ROLE OF DAPK IN THE INFLAMMATION-DRIVEN CARCINOGENESIS OF ULCERATIVE COLITIS (UC), WE ANALYZED PROMOTER HYPERMETHYLATION AND PROTEIN EXPRESSION OF DAPK USING METHYLATION-SPECIFIC PCR AND IMMUNOHISTOCHEMISTRY IN 43 UCCS AND PAIRED UC-BACKGROUND MUCOSA, AS WELL AS IN UC-BACKGROUND MUCOSA OF 50 PATIENTS WITHOUT UCC. THE FREQUENCY OF METHYLATION OF DAPK IN UCCS WAS LOW (27.6%) COMPARED TO OVERALL NON-NEOPLASTIC UC-BACKGROUND MUCOSA (48.3%; P=0.02) AND SPORADIC COLORECTAL CARCINOMA (57.4%, P=0.019). THE DIFFERENCE IN THE METHYLATION FREQUENCY IN UC-BACKGROUND MUCOSA IN PATIENTS WITHOUT UCC (54.2%), COMPARED TO THOSE WITH UCC (40.0%), WAS NOT SIGNIFICANT (P=0.141). PROMOTER METHYLATION CORRELATED SIGNIFICANTLY WITH DECREASED DAPK PROTEIN EXPRESSION (P<0.001) AND SEVERITY OF INFLAMMATORY ACTIVITY (P=0.024). IN UNMETHYLATED UC-BACKGROUND MUCOSA, DAPK PROTEIN EXPRESSION INCREASED WITH ACTIVITY OF UC-ASSOCIATED INFLAMMATION, SUGGESTING A PROTECTIVE ROLE OF THE PRO-APOPTOTIC DAPK DURING THE CHRONIC INFLAMMATORY PROCESS OF UC. THUS, INACTIVATION OF DAPK BY PROMOTER HYPERMETHYLATION MIGHT BE CRUCIAL FOR ACCUMULATION OF DNA DAMAGE IN INFLAMED MUCOSA OF UC, AND MIGHT THEREFORE CONTRIBUTE TO THE INITIATION OF THE NEOPLASTIC PROCESS AND DEVELOPMENT OF UC-ASSOCIATED CARCINOMA.	2010	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
11  2837  45 FORKHEAD O TRANSCRIPTION FACTOR 4 RESTRICTS HBV COVALENTLY CLOSED CIRCULAR DNA TRANSCRIPTION AND HBV REPLICATION THROUGH GENETIC DOWNREGULATION OF HEPATOCYTE NUCLEAR FACTOR 4 ALPHA AND EPIGENETIC SUPPRESSION OF COVALENTLY CLOSED CIRCULAR DNA VIA INTERACTING WITH PROMYELOCYTIC LEUKEMIA PROTEIN. NUCLEAR LOCATED HEPATITIS B VIRUS (HBV) COVALENTLY CLOSED CIRCULAR DNA (CCCDNA) REMAINS THE KEY OBSTACLE TO CURE CHRONIC HEPATITIS B (CHB). IN OUR PREVIOUS INVESTIGATION, IT WAS FOUND THAT FOXO4 COULD INHIBIT HBV CORE PROMOTER ACTIVITY THROUGH DOWNREGULATING THE EXPRESSION OF HNF4ALPHA. HOWEVER, THE EXACT MECHANISMS WHEREBY FOXO4 INHIBITS HBV REPLICATION, ESPECIALLY ITS EFFECT ON CCCDNA, REMAIN UNCLEAR. HERE, OUR DATA FURTHER REVEALED THAT FOXO4 COULD EFFECTIVELY INHIBIT CCCDNA MEDIATED TRANSCRIPTION AND HBV REPLICATION WITHOUT AFFECTING CCCDNA LEVEL. MECHANISTIC STUDY SHOWED THAT FOXO4 COULD CAUSE EPIGENETIC SUPPRESSION OF CCCDNA. ALTHOUGH FOXO4-MEDIATED DOWNREGULATION OF HNF4ALPHA CONTRIBUTED TO INHIBITING HBV CORE PROMOTER ACTIVITY, IT HAD LITTLE EFFECT ON CCCDNA EPIGENETIC REGULATION. FURTHER, IT WAS FOUND THAT FOXO4 COULD COLOCALIZE WITHIN PROMYELOCYTIC LEUKEMIA PROTEIN (PML) NUCLEAR BODIES AND INTERACT WITH PML. OF NOTE, PML WAS REVEALED TO BE CRITICAL FOR FOXO4-MEDIATED INHIBITION OF CCCDNA EPIGENETIC MODIFICATION AND OF THE FOLLOWING CCCDNA TRANSCRIPTION AND HBV REPLICATION. FURTHERMORE, FOXO4 WAS FOUND TO BE DOWNREGULATED IN HBV-INFECTED HEPATOCYTES AND HUMAN LIVER TISSUES, AND IT WAS NEGATIVELY CORRELATED WITH CCCDNA TRANSCRIPTIONAL ACTIVITY IN CHB PATIENTS. TOGETHER, THESE FINDINGS HIGHLIGHT THE ROLE OF FOXO4 IN SUPPRESSING CCCDNA TRANSCRIPTION AND HBV REPLICATION VIA GENETIC DOWNREGULATION OF HNF4ALPHA AND EPIGENETIC SUPPRESSION OF CCCDNA THROUGH INTERACTING WITH PML. TARGETING FOXO4 MAY PRESENT AS A NEW THERAPEUTIC STRATEGY AGAINST CHRONIC HBV INFECTION. IMPORTANCE HBV CCCDNA IS A DETERMINING FACTOR FOR VIRAL PERSISTENCE AND THE MAIN OBSTACLE FOR A CURE OF CHRONIC HEPATITIS B. STRATEGIES THAT TARGET CCCDNA DIRECTLY ARE THEREFORE OF GREAT IMPORTANCE IN CONTROLLING PERSISTENT HBV INFECTION. IN PRESENT INVESTIGATION, WE FOUND THAT FOXO4 COULD EFFICIENTLY SUPPRESS CCCDNA TRANSCRIPTION AND HBV REPLICATION WITHOUT AFFECTING THE LEVEL OF CCCDNA ITSELF. FURTHER, OUR DATA REVEALED THAT FOXO4 MIGHT INHIBIT CCCDNA FUNCTION VIA A TWO-PART MECHANISM: ONE IS TO EPIGENETICALLY SUPPRESS CCCDNA TRANSCRIPTION VIA INTERACTING WITH PML, AND THE OTHER IS TO INHIBIT HBV CORE PROMOTER ACTIVITY VIA THE GENETIC DOWNREGULATION OF HNF4ALPHA. OF NOTE, HBV MIGHT DAMPEN THE EXPRESSION OF FOXO4 FOR ITS OWN PERSISTENT INFECTION. WE PROPOSE THAT MANIPULATION OF FOXO4 MAY PRESENT AS A POTENTIAL THERAPEUTIC STRATEGY AGAINST CHRONIC HBV INFECTION.	2022	
                                                                                                                                                                                                                                                                                  
12  3357  32 HISTONE H3 LYSINE 9 DI-METHYLATION AS AN EPIGENETIC SIGNATURE OF THE INTERFERON RESPONSE. EFFECTIVE ANTIVIRAL IMMUNITY DEPENDS ON THE ABILITY OF INFECTED CELLS OR CELLS TRIGGERED WITH VIRUS-DERIVED NUCLEIC ACIDS TO PRODUCE TYPE I INTERFERON (IFN), WHICH ACTIVATES TRANSCRIPTION OF NUMEROUS ANTIVIRAL GENES. HOWEVER, DISPROPORTIONATELY STRONG OR CHRONIC IFN EXPRESSION IS A COMMON CAUSE OF INFLAMMATORY AND AUTOIMMUNE DISEASES. WE DESCRIBE AN EPIGENETIC MECHANISM THAT DETERMINES CELL TYPE-SPECIFIC DIFFERENCES IN IFN AND IFN-STIMULATED GENE (ISG) EXPRESSION IN RESPONSE TO EXOGENOUS SIGNALS. WE IDENTIFY DI-METHYLATION OF HISTONE H3 AT LYSINE 9 (H3K9ME2) AS A SUPPRESSOR OF IFN AND IFN-INDUCIBLE ANTIVIRAL GENE EXPRESSION. WE SHOW THAT LEVELS OF H3K9ME2 AT IFN AND ISG CORRELATE INVERSELY WITH THE SCOPE AND AMPLITUDE OF IFN AND ISG EXPRESSION IN FIBROBLASTS AND DENDRITIC CELLS. ACCORDINGLY, GENETIC ABLATION OR PHARMACOLOGICAL INACTIVATION OF LYSINE METHYLTRANSFERASE G9A, WHICH IS ESSENTIAL FOR THE GENERATION OF H3K9ME2, RESULTED IN PHENOTYPIC CONVERSION OF FIBROBLASTS INTO HIGHLY POTENT IFN-PRODUCING CELLS AND RENDERED THESE CELLS RESISTANT TO PATHOGENIC RNA VIRUSES. IN SUMMARY, OUR STUDIES IMPLICATE H3K9ME2 AND ENZYMES CONTROLLING ITS ABUNDANCE AS KEY REGULATORS OF INNATE ANTIVIRAL IMMUNITY.	2012	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       
13  5955  44 TELBIVUDINE TREATMENT CORRECTS HBV-INDUCED EPIGENETIC ALTERATIONS IN LIVER CELLS OF PATIENTS WITH CHRONIC HEPATITIS B. HEPATITIS B VIRUS (HBV) ALTERS THE EXPRESSION OF HOST CELLULAR GENES TO SUPPORT ITS REPLICATION AND SURVIVAL AND TO PROMOTE THE LIVER CELL INJURY. HOWEVER, THE UNDERLYING MECHANISM REMAINED INCOMPLETELY UNDERSTOOD. IN THIS STUDY, WE INVESTIGATED HBV-INDUCED EPIGENETIC CHANGES IN HEPG2 CELLS BY PROFILING THE LANDSCAPES OF THE ACTIVE HISTONE MODIFICATION MARK H3K4ME3 AND REPRESSIVE MARK H3K27ME3 USING CHROMATIN IMMUNOPRECIPITATION-SEQUENCING. HBV CAUSED THE ALTERED HISTONE MODIFICATIONS AT THOUSANDS OF GENOMIC LOCI, WHICH ARE CRITICALLY INVOLVED IN HBV ENTRY, INFLAMMATION, FIBROSIS AND CARCINOGENESIS OF HOST CELLS. INTERESTINGLY, TREATMENT OF THE HBV-TRANSFORMED HEPG2 CELLS WITH THE ANTI-HBV DRUG TELBIVUDINE SUBSTANTIALLY RESTORED THE H3K4ME3 LEVEL TO THAT OF UNTRANSFORMED HEPG2 CELLS. MORE IMPORTANTLY, OUR ANALYSIS OF LIVER SAMPLES FROM CONTROL AND CHRONIC HEPATITIS B PATIENTS REVEALED THAT TREATMENT OF THE PATIENTS WITH TELBIVUDINE NOT ONLY CORRECTED THE TARGET GENE EXPRESSION BUT ALSO THE EPIGENETIC MODIFICATION OF CRITICAL GENES. IN ADDITION, THE EXPRESSION OF THE HISTONE METHYLTRANSFERASES SMYD3 AND EZH2 THAT REGULATE HISTONE H3-SPECIFIC METHYLATION SHOWED NO DIFFERENCE IN HEPG2 CELL WITH OR WITHOUT HBV EXISTENCE. THUS, OUR DATA SUGGEST THAT ABNORMAL HISTONE MODIFICATIONS MIGHT CRITICALLY INVOLVED IN HBV-MEDIATED LIVER PATHOGENESIS AND TELBIVUDINE THERAPY MIGHT BENEFIT PATIENTS WITH HBV-RELATED CHRONIC INFECTION, LIVER CIRRHOSIS AND EVEN HEPATIC CARCINOMA. SUMMARY: TELBIVUDINE SUBSTANTIALLY RESTORES IN VITRO AND IN VIVO HBV-CAUSED ABNORMAL EXPRESSIONS AND HISTONE H3K4ME3 AND H3K27ME3 MODIFICATIONS AT THOUSANDS OF GENOMIC LOCI THAT ARE INVOLVED IN THE PATHOGENESIS OF LIVER CELLS, REVEALING A NOVEL MECHANISM FOR HBV-MEDIATED LIVER DAMAGE.	2014	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             
14  4771  36 NUCLEAR SENSOR INTERFERON-INDUCIBLE PROTEIN 16 INHIBITS THE FUNCTION OF HEPATITIS B VIRUS COVALENTLY CLOSED CIRCULAR DNA BY INTEGRATING INNATE IMMUNE ACTIVATION AND EPIGENETIC SUPPRESSION. BACKGROUND AND AIMS: NUCLEAR-LOCATED COVALENTLY CLOSED CIRCULAR DNA (CCCDNA) OF HEPATITIS B VIRUS (HBV) IS A DETERMINING FACTOR FOR HBV PERSISTENCE AND THE KEY OBSTACLE FOR A CURE OF CHRONIC HEPATITIS B. HOWEVER, IT REMAINS UNCLEAR WHETHER AND HOW THE HOST IMMUNE SYSTEM SENSES HBV CCCDNA AND ITS BIOLOGICAL CONSEQUENCES. APPROACH AND RESULTS: HERE, WE DEMONSTRATED THAT INTERFERON-INDUCIBLE PROTEIN 16 (IFI16) COULD SERVE AS A UNIQUE INNATE SENSOR TO RECOGNIZE AND BIND TO HBV CCCDNA IN HEPATIC NUCLEI, LEADING TO THE INHIBITION OF CCCDNA TRANSCRIPTION AND HBV REPLICATION. MECHANISTICALLY, OUR DATA SHOWED THAT IFI16 PROMOTED THE EPIGENETIC SUPPRESSION OF HBV CCCDNA BY TARGETING AN INTERFERON-STIMULATED RESPONSE ELEMENT (ISRE) PRESENT IN CCCDNA. IT IS OF INTEREST THAT THIS ISRE WAS ALSO REVEALED TO PLAY AN IMPORTANT ROLE IN IFI16-ACTIVATED TYPE I INTERFERON RESPONSES. FURTHERMORE, OUR DATA REVEALED THAT HBV COULD DOWN-REGULATE THE EXPRESSION LEVEL OF IFI16 IN HEPATOCYTES, AND THERE WAS A NEGATIVE CORRELATION BETWEEN IFI16 AND HBV TRANSCRIPTS IN LIVER BIOPSIES, SUGGESTING THE POSSIBLE ROLE OF IFI16 IN SUPPRESSING CCCDNA FUNCTION UNDER PHYSIOLOGICAL CONDITIONS. CONCLUSIONS: THE NUCLEAR SENSOR IFI16 SUPPRESSES CCCDNA FUNCTION BY INTEGRATING INNATE IMMUNE ACTIVATION AND EPIGENETIC REGULATION BY TARGETING THE ISRE OF CCCDNA, AND IFI16 MAY PRESENT AS A THERAPEUTIC TARGET AGAINST HBV INFECTION.	2020	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    
15  3249  35 HEPATITIS B VIRUS HIJACKS CTHRC1 TO EVADE HOST IMMUNITY AND MAINTAIN REPLICATION. HEPATITIS B VIRUS (HBV) INFECTION CAUSES ACUTE AND CHRONIC LIVER DISEASES, BUT IS NOT DIRECTLY CYTOPATHIC. LIVER INJURY RESULTS FROM REPEATED ATTEMPTS OF THE CELLULAR IMMUNE RESPONSE SYSTEM TO CONTROL THE VIRAL INFECTION. HERE, WE INVESTIGATE THE ROLES OF CELLULAR FACTORS AND SIGNALING PATHWAYS INVOLVED IN THE REGULATION OF HBV REPLICATION TO REVEAL THE MECHANISM UNDERLYING HBV INFECTION AND PATHOGENESIS. WE SHOW THAT COLLAGEN TRIPLE HELIX REPEAT CONTAINING 1 (CTHRC1) EXPRESSION IS ELEVATED IN HBV-INFECTED PATIENTS AND IN HBV-TRANSFECTED CELLS THROUGH EPIGENETIC MODIFICATION AND TRANSCRIPTIONAL REGULATION. CTHRC1 FACILITATES HBV REPLICATION IN CULTURED CELLS AND BALB/C MICE BY ACTIVATING THE PKCALPHA/ERK/JNK/C-JUN CASCADE TO REPRESS THE IFN/JAK/STAT PATHWAY. HBV-ACTIVATED CTHRC1 DOWNREGULATES THE ACTIVITY OF TYPE I INTERFERON (IFN), THE PRODUCTION OF IFN-STIMULATED GENES (ISGS), AND THE PHOSPHORYLATION OF SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 1/2 (STAT1/2), WHEREAS IT UPREGULATES THE PHOSPHORYLATION AND UBIQUITINATION OF TYPE I IFN RECEPTORS (IFNARALPHA/BETA). THUS, OUR RESULTS SHOW THAT HBV USES A NOVEL MECHANISM TO HIJACK CELLULAR FACTORS AND SIGNAL CASCADES IN ORDER TO EVADE HOST ANTIVIRAL IMMUNITY AND MAINTAIN PERSISTENT INFECTION. WE ALSO DEMONSTRATE THAT CTHRC1 HAS A NOVEL ROLE IN VIRAL INFECTION.	2015	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    
16  3728  39 INHIBITION OF REPLICATION OF HEPATITIS B VIRUS USING TRANSCRIPTIONAL REPRESSORS THAT TARGET THE VIRAL DNA. BACKGROUND: CHRONIC INFECTION WITH HEPATITIS B VIRUS (HBV) IS A SERIOUS GLOBAL HEALTH PROBLEM. PERSISTENCE OF THE VIRUS OCCURS AS A RESULT OF STABILITY OF THE REPLICATION INTERMEDIATE COMPRISING COVALENTLY CLOSED CIRCULAR DNA (CCCDNA). DEVELOPMENT OF DRUGS THAT ARE CAPABLE OF DISABLING THIS CCCDNA IS VITAL. METHODS: TO INVESTIGATE AN EPIGENETIC APPROACH TO INACTIVATING VIRAL DNA, WE ENGINEERED TRANSCRIPTIONAL REPRESSORS THAT COMPRISE AN HBV DNA-BINDING DOMAIN OF TRANSCRIPTION ACTIVATOR LIKE EFFECTORS (TALES) AND A FUSED KRUPPEL ASSOCIATED BOX (KRAB). THESE REPRESSOR TALES (RTALES) TARGETED THE VIRAL SURFACE OPEN READING FRAME AND WERE PLACED UNDER TRANSCRIPTION CONTROL OF CONSTITUTIVELY ACTIVE OR LIVER-SPECIFIC PROMOTERS. RESULTS: EVALUATION IN CULTURED CELLS AND FOLLOWING HYDRODYNAMIC INJECTION OF MICE REVEALED THAT THE RTALES SIGNIFICANTLY INHIBITED PRODUCTION OF MARKERS OF HBV REPLICATION WITHOUT EVIDENCE OF HEPATOTOXICITY. INCREASED METHYLATION OF HBV DNA AT CPG ISLAND II SHOWED THAT THE RTALES CAUSED INTENDED EPIGENETIC MODIFICATION. CONCLUSIONS: EPIGENETIC MODIFICATION OF HBV DNA IS A NEW AND EFFECTIVE MEANS OF INACTIVATING THE VIRUS IN VIVO. THE APPROACH HAS THERAPEUTIC POTENTIAL AND AVOIDS POTENTIALLY PROBLEMATIC UNINTENDED MUTAGENESIS OF GENE EDITING.	2019	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  
17  3247  36 HEPATITIS B VIRUS BASAL CORE PROMOTER MUTATIONS SHOW LOWER REPLICATION FITNESS ASSOCIATED WITH CCCDNA ACETYLATION STATUS. IN CHRONIC HEPATITIS B VIRUS (HBV) INFECTION, VARIANTS WITH MUTATIONS IN THE BASAL CORE PROMOTER (BCP) AND PRECORE REGION PREDOMINATE AND ASSOCIATE WITH MORE SEVERE DISEASE FORMS. STUDIES ON THEIR EFFECT ON VIRAL REPLICATION REMAIN CONTROVERSIAL. INCREASING EVIDENCE SHOWS THAT EPIGENETIC MODIFICATIONS OF CCCDNA REGULATE HBV REPLICATION AND DISEASE OUTCOME. HERE WE DETERMINED THE TRANSCRIPTION AND VIRAL REPLICATION EFFICIENCY OF WELL-DEFINED BCP AND PRECORE MUTATIONS AND THEIR EFFECT ON CCCDNA EPIGENETIC CONTROL. HBV MONOMERS BEARING BCP MUTATIONS A1762T/G1764A AND A1762T/G1764A/C1766T, AND PRECORE MUTATIONS G1896A, G1899A AND G1896A/G1899A, WERE TRANSFECTED INTO HEPG2 CELLS USING A PLASMID-FREE APPROACH. VIRAL RNA TRANSCRIPTS WERE DETECTED BY NORTHERN BLOT HYBRIDIZATION AND RT PCR, DNA REPLICATIVE INTERMEDIATES BY SOUTHERN BLOTTING AND RT PCR, AND VIRAL RELEASE WAS MEASURED BY ELISA. ACETYLATION OF CCCDNA-BOUND HISTONES WAS ASSESSED BY CHROMATIN IMMUNOPRECIPITATION (CHIP) ASSAY AND METHYLATION OF CCCDNA BY BISULFITE SEQUENCING. BCP MUTATIONS RESULTED IN LOW VIRAL RELEASE, MRNA TRANSCRIPTION AND PGRNA/CCCDNA RATIOS THAT PARALLELED THE ACETYLATION OF CCCDNA-BOUND H4 HISTONE AND INVERSELY CORRELATED WITH THE HDAC1 RECRUITMENT ONTO CCCDNA. INDEPENDENTLY OF THE MUTATIONS, CCCDNA WAS A TARGET FOR METHYLATION, ACCOMPANIED BY THE UPREGULATION OF DNMT1 EXPRESSION AND DNMT1 RECRUITMENT ONTO CCCDNA. OUR RESULTS SUGGEST THAT BCP MUTATIONS DECREASE VIRAL REPLICATION CAPACITY POSSIBLY BY MODULATING THE ACETYLATION AND DEACETYLATION OF CCCDNA-BOUND HISTONES WHILE PRECORE MUTATIONS DO NOT HAVE A SIGNIFICANT EFFECT ON VIRAL REPLICATION. THESE DATA PROVIDE EVIDENCE THAT EPIGENETIC FACTORS CONTRIBUTE TO THE REGULATION OF HBV VIRAL REPLICATION.	2016	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         
18  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	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     
19  5335  30 QUANTIFICATION AND EPIGENETIC EVALUATION OF THE RESIDUAL POOL OF HEPATITIS B COVALENTLY CLOSED CIRCULAR DNA IN LONG-TERM NUCLEOSIDE ANALOGUE-TREATED PATIENTS. HEPATITIS B VIRUS (HBV) COVALENTLY CLOSED CIRCULAR (CCC)DNA IS THE KEY GENOMIC FORM RESPONSIBLE FOR VIRAL PERSISTENCE AND VIROLOGICAL RELAPSE AFTER TREATMENT WITHDRAWAL. THE ASSESSMENT OF RESIDUAL INTRAHEPATIC CCCDNA LEVELS AND ACTIVITY AFTER LONG-TERM NUCLEOS(T)IDE ANALOGUES THERAPY STILL REPRESENTS A TECHNICAL CHALLENGE. QUANTITATIVE (Q)PCR, ROLLING CIRCLE AMPLIFICATION (RCA) AND DROPLET DIGITAL (DD)PCR ASSAYS WERE USED TO QUANTIFY RESIDUAL INTRAHEPATIC CCCDNA IN LIVER BIOPSIES FROM 56 CHRONICALLY HBV INFECTED PATIENTS AFTER 3 TO 5 YEARS OF TELBIVUDINE TREATMENT. ACTIVITY OF RESIDUAL CCCDNA WAS EVALUATED BY QUANTIFYING 3.5 KB HBV RNA (PREC/PGRNA) AND BY ASSESSING CCCDNA-ASSOCIATED HISTONE TAILS POST-TRANSCRIPTIONAL MODIFICATIONS (PTMS) BY MICRO-CHROMATIN IMMUNOPRECIPITATION. LONG-TERM TELBIVUDINE TREATMENT RESULTED IN SERUM HBV DNA SUPPRESSION, WITH MOST OF THE PATIENTS REACHING UNDETECTABLE LEVELS. DESPITE 38 OUT OF 56 PATIENTS HAD UNDETECTABLE CCCDNA WHEN ASSESSED BY QPCR, RCA AND DDPCR ASSAYS DETECTED CCCDNA IN ALL-BUT-ONE NEGATIVE SAMPLES. LOW PREC/PGRNA LEVEL IN TELBIVUDINE-TREATED SAMPLES WAS ASSOCIATED WITH ENRICHMENT FOR CCCDNA HISTONE PTMS RELATED TO REPRESSED TRANSCRIPTION. NO DIFFERENCE IN CCCDNA LEVELS WAS FOUND ACCORDING TO SERUM VIRAL MARKERS EVOLUTION. THIS PANEL OF CCCDNA EVALUATION TECHNIQUES SHOULD PROVIDE AN ADDED VALUE FOR THE NEW PROOF-OF-CONCEPT CLINICAL TRIALS AIMING AT A FUNCTIONAL CURE OF CHRONIC HEPATITIS B.	2020	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           
20  3627  27 INACTIVATION OF LARS2, LOCATED AT THE COMMONLY DELETED REGION 3P21.3, BY BOTH EPIGENETIC AND GENETIC MECHANISMS IN NASOPHARYNGEAL CARCINOMA. ALLELIC LOSS OF CHROMOSOME 3P, INCLUDING THE 3P21.3 REGION, IS FOUND IN 95-100% OF PRIMARY NASOPHARYNGEAL CARCINOMA (NPC) BIOPSIES, SUGGESTING THAT THIS REGION SHOULD HARBOR SOME TUMOR SUPPRESSOR GENES (TSGS) CLOSELY RELATED TO NPC DEVELOPMENT. SEVERAL TSGS LOCATED AT 3P21.3, SUCH AS RASSF1A, LTF AND BLU, HAVE BEEN DEMONSTRATED TO BE INVOLVED IN NPC DEVELOPMENT. LARS2 (LEUCYL-TRNA SYNTHETASE 2, MITOCHONDRIAL) IS ANOTHER GENE LOCATED IN THE CHROMOSOME 3 COMMON ELIMINATED REGION-1 (C3CER1) AT 3P21.3. IN THIS STUDY, WE FOCUSSED ON THE EPIGENETIC AND GENETIC ALTERATIONS OF LARS2 IN NPC. THE MRNA EXPRESSION OF LARS2 WAS DETECTED IN 36 NPC AND 8 CHRONIC NASOPHARYNGITIS (NP) TISSUES BY SEMI-QUANTITATIVE REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION (RT-PCR) AND REAL-TIME RT-PCR. SUBSEQUENTLY, THE MUTATION, ALLELIC LOSS, AND METHYLATION STATUS OF LARS2 WERE ANALYSED BY POLYMERASE CHAIN REACTION-SINGLE-STRAND CONFORMATION POLYMORPHISM (PCR-SSCP), HOMOZYGOUS DELETION (HD) ANALYSIS AND METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION IN PRIMARY NPC TISSUES. NO EXPRESSION OR DOWNREGULATION OF LARS2 WAS OBSERVED IN 78% OF PRIMARY NPC TISSUES. NO MUTATIONS, ASSESSED BY PCR-SSCP AND DNA SEQUENCING, WERE FOUND IN THE PROMOTER REGION AND EXON 1 OF LARS2 IN NPC TISSUES, WHEREAS HD WAS DETECTED IN 28% OF NPC SPECIMENS AT THE LARS2 LOCUS. IN ADDITION, HYPERMETHYLATION OF LARS2 WAS FOUND IN 64% OF NPC SAMPLES BUT ONLY IN 12.5% OF NP BIOPSIES. OUR DATA INDICATE THAT INACTIVATION OF LARS2 BY BOTH GENETIC AND EPIGENETIC MECHANISMS MAY BE A COMMON AND IMPORTANT EVENT IN THE CARCINOGENESIS OF NPC.	2009