Discovery and mechanistic study of Imperatorin that inhibits HBsAg expression and cccDNA transcription.

Chronic hepatitis B virus (HBV) infection remains a significant global health challenge due to its link to severe conditions like HBV-related cirrhosis and hepatocellular carcinoma (HCC). Although current treatments effectively reduce viral levels, they have limited impact on certain HBV elements, namely hepatitis B surface antigen (HBsAg) and covalently closed circular DNA (cccDNA). This highlights the urgent need for innovative pharmaceutical and biological interventions that can disrupt HBsAg production originating from cccDNA. In this study, we identified a natural furanocoumarin compound, Imperatorin, which markedly inhibited the expression of HBsAg from cccDNA, by screening a library of natural compounds derived from Chinese herbal medicines using ELISA assay and qRT-PCR. The pharmacodynamics study of Imperatorin was explored on HBV infected HepG2-NTCP/PHHs and HBV-infected humanized mouse model. Proteome analysis was performed on HBV infected HepG2-NTCP cells following Imperatorin treatment. Molecular docking and bio-layer interferometry (BLI) were used for finding the target of Imperatorin. Our findings demonstrated Imperatorin remarkably reduced the level of HBsAg, HBV RNAs, HBV DNA and transcriptional activity of cccDNA both in vitro and in vivo. Additionally, Imperatorin effectively restrained the actions of HBV promoters responsible for cccDNA transcription. Mechanistic study revealed that Imperatorin directly binds to ERK and subsequently interfering with the activation of CAMP response element-binding protein (CREB), a crucial transcriptional factor for HBV and has been demonstrated to bind to the PreS2/S and X promoter regions of HBV. Importantly, the absence of ERK could nullify the antiviral impact triggered by Imperatorin. Collectively, the natural compound Imperatorin may be an effective candidate agent for inhibiting HBsAg production and cccDNA transcription by impeding the activities of HBV promoters through ERK-CREB axis.

Circular single-stranded DNA as a programmable vector for gene regulation in cell-free protein expression systems.

Cell-free protein expression (CFE) systems have emerged as a critical platform for synthetic biology research. The vectors for protein expression in CFE systems mainly rely on double-stranded DNA and single-stranded RNA for transcription and translation processing. Here, we introduce a programmable vector - circular single-stranded DNA (CssDNA), which is shown to be processed by DNA and RNA polymerases for gene expression in a yeast-based CFE system. CssDNA is already widely employed in DNA nanotechnology due to its addressability and programmability. To apply above methods in the context of synthetic biology, CssDNA can not only be engineered for gene regulation via the different pathways of sense CssDNA and antisense CssDNA, but also be constructed into several gene regulatory logic gates in CFE systems. Our findings advance the understanding of how CssDNA can be utilized in gene expression and gene regulation, and thus enrich the synthetic biology toolbox.

Extrachromosomal telomere DNA derived from excessive strand displacements.

Alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism mediated by break-induced replication, evident in approximately 15% of human cancers. A characteristic feature of ALT cancers is the presence of C-circles, circular single-stranded telomeric DNAs composed of C-rich sequences. Despite the fact that extrachromosomal C-rich single-stranded DNAs (ssDNAs), including C-circles, are unique to ALT cells, their generation process remains undefined. Here, we introduce a method to detect single-stranded telomeric DNA, called 4SET (Strand-Specific Southern-blot for Single-stranded Extrachromosomal Telomeres) assay. Utilizing 4SET, we are able to capture C-rich single-stranded DNAs that are near 200 to 1500 nucleotides in size. Both linear C-rich ssDNAs and C-circles are abundant in the fractions of cytoplasm and nucleoplasm, which supports the idea that linear and circular C-rich ssDNAs are generated concurrently. We also found that C-rich ssDNAs originate during Okazaki fragment processing during lagging strand DNA synthesis. The generation of C-rich ssDNA requires CST-PP (CTC1/STN1/TEN1-PRIMASE-Polymerase alpha) complex-mediated priming of the C-strand DNA synthesis and subsequent excessive strand displacement of the C-rich strand mediated by the DNA Polymerase delta and the BLM helicase. Our work proposes a model for the generation of C-rich ssDNAs and C-circles during ALT-mediated telomere elongation.

An allosteric inhibitor of sirtuin 2 blocks hepatitis B virus covalently closed circular DNA establishment and its transcriptional activity.

296 million people worldwide are predisposed to developing severe end-stage liver diseases due to chronic hepatitis B virus (HBV) infection. HBV forms covalently closed circular DNA (cccDNA) molecules that persist as episomal DNA in the nucleus of infected hepatocytes and drive viral replication. Occasionally, the HBV genome becomes integrated into host chromosomal DNA, a process that is believed to significantly contribute to circulating HBsAg levels and HCC development. Neither cccDNA accumulation nor expression from integrated HBV DNA are directly targeted by current antiviral treatments. In this study, we investigated the antiviral properties of a newly described allosteric modulator, FLS-359, that targets sirtuin 2 (SIRT2), an NAD+-dependent deacylase. Our results demonstrate that SIRT2 modulation by FLS-359 and by other tool compounds inhibits cccDNA synthesis following de novo infection of primary human hepatocytes and HepG2 (C3A)-NTCP cells, and FLS-359 substantially reduces cccDNA recycling in HepAD38 cells. While pre-existing cccDNA is not eradicated by short-term treatment with FLS-359, its transcriptional activity is substantially impaired, likely through inhibition of viral promoter activities. Consistent with the inhibition of viral transcription, HBsAg production by HepG2.2.15 cells, which contain integrated HBV genomes, is also suppressed by FLS-359. Our study provides further insights on SIRT2 regulation of HBV infection and supports the development of potent SIRT2 inhibitors as HBV antivirals.

Arsenic trioxide impacts hepatitis B virus core nuclear localization and efficiently interferes with HBV infection.

The key to a curative treatment of hepatitis B virus (HBV) infection is the eradication of the intranuclear episomal covalently closed circular DNA (cccDNA), the stable persistence reservoir of HBV. Currently, established therapies can only limit HBV replication but fail to tackle the cccDNA. Thus, novel therapeutic approaches toward curative treatment are urgently needed. Recent publications indicated a strong association between the HBV core protein SUMOylation and the association with promyelocytic leukemia nuclear bodies (PML-NBs) on relaxed circular DNA to cccDNA conversion. We propose that interference with the cellular SUMOylation system and PML-NB integrity using arsenic trioxide provides a useful tool in the treatment of HBV infection. Our study showed a significant reduction in HBV-infected cells, core protein levels, HBV mRNA, and total DNA. Additionally, a reduction, albeit to a limited extent, of HBV cccDNA could be observed. Furthermore, this interference was also applied for the treatment of an established HBV infection, characterized by a stably present nuclear pool of cccDNA. Arsenic trioxide (ATO) treatment not only changed the amount of expressed HBV core protein but also induced a distinct relocalization to an extranuclear phenotype during infection. Moreover, ATO treatment resulted in the redistribution of transfected HBV core protein away from PML-NBs, a phenotype similar to that previously observed with SUMOylation-deficient HBV core. Taken together, these findings revealed the inhibition of HBV replication by ATO treatment during several steps of the viral replication cycle, including viral entry into the nucleus as well as cccDNA formation and maintenance. We propose ATO as a novel prospective treatment option for further pre-clinical and clinical studies against HBV infection. IMPORTANCE: The main challenge for the achievement of a functional cure for hepatitis B virus (HBV) is the covalently closed circular DNA (cccDNA), the highly stable persistence reservoir of HBV, which is maintained by further rounds of infection with newly generated progeny viruses or by intracellular recycling of mature nucleocapsids. Eradication of the cccDNA is considered to be the holy grail for HBV curative treatment; however, current therapeutic approaches fail to directly tackle this HBV persistence reservoir. The molecular effect of arsenic trioxide (ATO) on HBV infection, protein expression, and cccDNA formation and maintenance, however, has not been characterized and understood until now. In this study, we reveal ATO treatment as a novel and innovative therapeutic approach against HBV infections, repressing viral gene expression and replication as well as the stable cccDNA pool at low micromolar concentrations by affecting the cellular function of promyelocytic leukemia nuclear bodies.

Co-Transcriptional Regulation of HBV Replication: RNA Quality Also Matters.

Chronic hepatitis B (CHB) virus infection is a major public health burden and the leading cause of hepatocellular carcinoma. Despite the efficacy of current treatments, hepatitis B virus (HBV) cannot be fully eradicated due to the persistence of its minichromosome, or covalently closed circular DNA (cccDNA). The HBV community is investing large human and financial resources to develop new therapeutic strategies that either silence or ideally degrade cccDNA, to cure HBV completely or functionally. cccDNA transcription is considered to be the key step for HBV replication. Transcription not only influences the levels of viral RNA produced, but also directly impacts their quality, generating multiple variants. Growing evidence advocates for the role of the co-transcriptional regulation of HBV RNAs during CHB and viral replication, paving the way for the development of novel therapies targeting these processes. This review focuses on the mechanisms controlling the different co-transcriptional processes that HBV RNAs undergo, and their contribution to both viral replication and HBV-induced liver pathogenesis.

Furanocoumarins promote proteasomal degradation of viral HBx protein and down-regulate cccDNA transcription and replication of hepatitis B virus.

Nucleot(s)ide analogues, the current antiviral treatments against chronic hepatitis B (CHB) infection, are non-curative due to their inability to eliminate covalently closed circular DNA (cccDNA) from the infected hepatocytes. Preclinical studies have shown that coumarin derivatives can effectively reduce the HBV DNA replication. We evaluated the antiviral efficacy of thirty new coumarin derivatives in cell culture models for studying HBV. Furanocoumarins Fc-20 and Fc-31 suppressed the levels of pre-genomic RNA as well as cccDNA, and reduced the secretion of virions, HBsAg and HBeAg. The antiviral efficacies of Fc-20 and Fc31 improved further when used in combination with the hepatitis B antiviral drug Entecavir. There was a marked reduction in the intracellular HBx level in the presence of these furanocoumarins due to proteasomal degradation resulting in the down-regulation of HBx-dependent viral genes. Importantly, both Fc-20 and Fc-31 were non-cytotoxic to cells even at high concentrations. Further, our molecular docking studies confirmed a moderate to high affinity interaction between furanocoumarins and viral HBx via residues Ala3, Arg26 and Lys140. These data suggest that furanocoumarins could be developed as a new therapeutic for CHB infection.

PreS1BP mediates inhibition of Hepatitis B virus replication by promoting HBx protein degradation.

BACKGROUND: PreS1-binding protein (PreS1BP), recognized as a nucleolar protein and tumor suppressor, influences the replication of various viruses, including vesicular stomatitis virus (VSV) and herpes simplex virus type 1 (HSV-1). Its role in hepatitis B virus (HBV) replication and the underlying mechanisms, however, remain elusive. METHODS: We investigated PreS1BP expression levels in an HBV-replicating cell and animal model and analyzed the impact of its overexpression on viral replication metrics. HBV DNA, covalently closed circular DNA (cccDNA), hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg), and HBV RNA levels were assessed in HBV-expressing stable cell lines under varying PreS1BP conditions. Furthermore, co-immunoprecipitation and ubiquitination assays were used to detect PreS1BP- hepatitis B virus X protein (HBx) interactions and HBx stability modulated by PreS1BP. RESULTS: Our study revealed a marked decrease in PreS1BP expression in the presence of active HBV replication. Functional assays showed that PreS1BP overexpression significantly inhibited HBV replication and transcription, evidenced by the reduction in HBV DNA, cccDNA, HBsAg, HBcAg, and HBV RNA levels. At the molecular level, PreS1BP facilitated the degradation of HBx in a dose-dependent fashion, whereas siRNA-mediated knockdown of PreS1BP led to an increase in HBx levels. Subsequent investigations uncovered that PreS1BP accelerated HBx protein degradation via K63-linked ubiquitination in a ubiquitin-proteasome system-dependent manner. Co-immunoprecipitation assays further established that PreS1BP enhances the recruitment of the proteasome 20S subunit alpha 3 (PSMA3) for interaction with HBx, thereby fostering its degradation. CONCLUSIONS: These findings unveil a previously unidentified mechanism wherein PreS1BP mediates HBx protein degradation through the ubiquitin-proteasome system, consequentially inhibiting HBV replication. This insight positions PreS1BP as a promising therapeutic target for future HBV interventions. Further studies are warranted to explore the clinical applicability of modulating PreS1BP in HBV therapy.

Chromatin binding protein HMGN1 promotes HBV cccDNA transcription and replication by regulating the phosphorylation of histone 3.

BACKGROUND AND AIMS: Direct elimination of cccDNA remains a formidable obstacle due to the persistent and stable presence of cccDNA in hepatocyte nuclei. The silencing of cccDNA transcription enduringly is one of alternative strategies in the treatment of hepatitis B. Protein binding to cccDNA plays an important role in its transcriptional regulation; thus, the identification of key factors involved in this process is of great importance. APPROACHES AND RESULTS: In the present study, high mobility group nucleosome binding domain 1 (HMGN1) was screened out based on our biotin-avidin enrichment system. First, chromatin immunoprecipitation and fluorescent in situ hybridization assays confirmed the binding of HMGN1 with cccDNA in the nucleus. Second, functional experiments in HBV-infected cells showed that the promoting effect of HMGN1 on HBV transcription and replication depended on the functional region of the nucleosomal binding domain, while transfection of the HMGN1 mutant showed no influence on HBV compared with the vector. Third, further mechanistic exploration revealed that the silencing of HMGN1 increased the level of phosphorylase CLK2 and promoted H3 phosphorylation causing the reduced accessibility of cccDNA. Moreover, silenced HMGN1 was mimicked in HBV (r) cccDNA mouse model of HBV infection in vivo. The results showed that silencing HMGN1 inhibited HBV replication in vivo. CONCLUSIONS: In summary, our study identified that a host protein can bind to cccDNA and promote its transcription, providing a candidate strategy for anti-HBV targeting to interfere with the transcriptional activity of cccDNA microchromosomes.

Engineered extracellular vesicles for delivering functional Cas9/gRNA to eliminate hepatitis B virus cccDNA and integration.

The persistence of HBV covalently closed circular DNA (cccDNA) and HBV integration into the host genome in infected hepatocytes pose significant challenges to the cure of chronic HBV infection. Although CRISPR/Cas9-mediated genome editing shows promise for targeted clearance of viral genomes, a safe and efficient delivery method is currently lacking. Here, we developed a novel approach by combining light-induced heterodimerization and protein acylation to enhance the loading efficiency of Cas9 protein into extracellular vesicles (EVs). Moreover, vesicular stomatitis virus-glycoprotein (VSV-G) was incorporated onto the EVs membrane, significantly facilitating the endosomal escape of Cas9 protein and increasing its gene editing activity in recipient cells. Our results demonstrated that engineered EVs containing Cas9/gRNA and VSV-G can effectively reduce viral antigens and cccDNA levels in the HBV-replicating and infected cell models. Notably, we also confirmed the antiviral activity and high safety of the engineered EVs in the HBV-replicating mouse model generated by hydrodynamic injection and the HBV transgenic mouse model. In conclusion, engineered EVs could successfully mediate functional CRISPR/Cas9 delivery both in vitro and in vivo, leading to the clearance of episomal cccDNA and integrated viral DNA fragments, and providing a novel therapeutic approach for curing chronic HBV infection.

G-quadruplexes control hepatitis B virus replication by promoting cccDNA transcription and phase separation in hepatocytes.

Phase separation regulates fundamental processes in gene expression and is mediated by the local concentration of proteins and nucleic acids, as well as nucleic acid secondary structures such as G-quadruplexes (G4s). These structures play fundamental roles in both host gene expression and in viral replication due to their peculiar localisation in regulatory sequences. Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is an episomal minichromosome whose persistence is at the basis of chronic infection. Identifying the mechanisms controlling its transcriptional activity is indispensable to develop new therapeutic strategies against chronic hepatitis B. The aim of this study was to determine whether G4s are formed in cccDNA and regulate viral replication. Combining biochemistry and functional studies, we demonstrate that cccDNA indeed contains ten G4s structures. Furthermore, mutations disrupting two G4s located in the enhancer I HBV regulatory region altered cccDNA transcription and viral replication. Finally, we showed for the first time that cccDNA undergoes phase separation in a G4-dependent manner to promote its transcription in infected hepatocytes. Altogether, our data give new insight in the transcriptional regulation of the HBV minichromosome that might pave the way for the identification of novel targets to destabilize or silence cccDNA.

Gambogic acid inhibits HBx-mediated hepatitis B virus replication by targeting the DTX1-Notch signaling pathway.

BACKGROUND & AIMS: Current antiviral drugs, including nucleoside analogs and interferon, fail to eliminate the HBV covalently closed circular DNA (cccDNA), which serves as a transcript template in infected hepatocytes. Silencing the HBV X protein, which plays a crucial role in cccDNA transcription, is a promising approach to inhibit HBV replication. Therefore, the identification of novel compounds that can inhibit HBx-mediated cccDNA transcription is critical. METHODS: Initially, a compound library consisting of 715 monomers derived from traditional Chinese medicines known for their liver-protecting properties was established. Then, MTT assays were used to determine the cytotoxicity of each compound. The effect of candidates on Flag-HBx expression was examined by real-time PCR and western blotting in Flag-HBx transfected HepG2-NTCP cells. Ultimately, the antiviral effect of gambogic acid (GA) on HBV was observed in HBV-infected HepG2-NTCP cells. Mechanistically, the functional role of DTX1 in GA-induced HBV inhibition was examined using RNA-seq. Finally, the antiviral effect of GA was estimated in vivo. RESULTS: Gambogic acid (GA), a natural bioactive compound with a myriad of biological activities, markedly reduced Flag-HBx expression. Potent and dose-dependent reductions in extracellular HBV RNAs, HBV DNA, HBsAg, HBeAg and HBc protein were discovered three days after GA treatment in HBV-infected cells, accompanied by the absence of significant cytotoxicity. Furthermore, our research revealed that GA exhibited a dose-dependent inhibition of HBx expression, which is a pleiotropic protein required for HBV infection in vivo. We explored the mechanisms underlying GA-mediated inhibition of HBV and confirmed that this inhibition is accomplished by upregulating the expression of the DTX1 gene and boosting the Notch signaling pathway. Finally, the inhibitory effect of GA on HBV replication was tested in vivo using a mouse model of hepatitis B virus recombinant cccDNA. CONCLUSIONS: Herein, we discovered GA, which is a natural bioactive compound that targets HBx to inhibit hepatitis B virus replication by activating the DTX1-Notch signaling pathway.

A computational spatial whole-Cell model for hepatitis B viral infection and drug interactions.

Despite a vaccine, hepatitis B virus (HBV) remains a world-wide source of infections and deaths. We develop a whole-cell computational platform combining spatial and kinetic models describing the infection cycle of HBV in a hepatocyte host. We simulate key parts of the infection cycle with this whole-cell platform for 10 min of biological time, to predict infection progression, map out virus-host and virus-drug interactions. We find that starting from an established infection, decreasing the copy number of the viral envelope proteins shifts the dominant infection pathway from capsid secretion to re-importing the capsids into the nucleus, resulting in more nuclear-localized viral covalently closed circular DNA (cccDNA) and boosting transcription. This scenario can mimic the consequence of drugs designed to manipulate viral gene expression. Mutating capsid proteins facilitates capsid destabilization and disassembly at nuclear pore complexes, resulting in an increase in cccDNA copy number. However, excessive destabilization leads to premature cytoplasmic disassembly and does not increase the cccDNA counts. Finally, our simulations can predict the best drug dosage and its administration timing to reduce the cccDNA counts. Our adaptable computational platform can be parameterized to study other viruses and identify the most central viral pathways that can be targeted by drugs.

Recent Advances in the Development of Sulfamoyl-Based Hepatitis B Virus Nucleocapsid Assembly Modulators.

Hepatitis B virus (HBV) is the primary contributor to severe liver ailments, encompassing conditions such as cirrhosis and hepatocellular carcinoma. Globally, 257 million people are affected by HBV annually and 887,000 deaths are attributed to it, representing a substantial health burden. Regrettably, none of the existing therapies for chronic hepatitis B (CHB) have achieved satisfactory clinical cure rates. This issue stems from the existence of covalently closed circular DNA (cccDNA), which is difficult to eliminate from the nucleus of infected hepatocytes. HBV genetic material is composed of partially double-stranded DNA that forms complexes with viral polymerase inside an icosahedral capsid composed of a dimeric core protein. The HBV core protein, consisting of 183 to 185 amino acids, plays integral roles in multiple essential functions within the HBV replication process. In this review, we describe the effects of sulfamoyl-based carboxamide capsid assembly modulators (CAMs) on capsid assembly, which can suppress HBV replication and disrupt the production of new cccDNA. We present research on classical, first-generation sulfamoyl benzocarboxamide CAMs, elucidating their structural composition and antiviral efficacy. Additionally, we explore newly identified sulfamoyl-based CAMs, including sulfamoyl bicyclic carboxamides, sulfamoyl aromatic heterocyclic carboxamides, sulfamoyl aliphatic heterocyclic carboxamides, cyclic sulfonamides, and non-carboxamide sulfomoyl-based CAMs. We believe that certain molecules derived from sulfamoyl groups have the potential to be developed into essential components of a well-suited combination therapy, ultimately yielding superior clinical efficacy outcomes in the future.

Nucleolin binds to and regulates transcription of hepatitis B virus covalently closed circular DNA minichromosome.

Hepatitis B virus (HBV) remains a major public health threat with nearly 300 million people chronically infected worldwide who are at a high risk of developing hepatocellular carcinoma. Current therapies are effective in suppressing HBV replication but rarely lead to cure. Current therapies do not affect the HBV covalently closed circular DNA (cccDNA), which serves as the template for viral transcription and replication and is highly stable in infected cells to ensure viral persistence. In this study, we aim to identify and elucidate the functional role of cccDNA-associated host factors using affinity purification and protein mass spectrometry in HBV-infected cells. Nucleolin was identified as a key cccDNA-binding protein and shown to play an important role in HBV cccDNA transcription, likely via epigenetic regulation. Targeting nucleolin to silence cccDNA transcription in infected hepatocytes may be a promising therapeutic strategy for a functional cure of HBV.

Targeting Hepatitis B Virus DNA Using Designer Gene Editors.

Chronic hepatitis B virus (HBV) infection is a serious disease that currently has no cure. Key forms of HBV include covalently closed circular DNA, which mediates chronic persistence, and integrated DNA, which contributes to immune evasion and carcinogenesis. These forms are not targeted by current therapies; however, gene editing technologies have emerged as promising tools for disrupting HBV DNA. Gene editor-induced double-stranded breaks at precise locations within the HBV genome can induce effects ranging from inactivation of target genes to complete degradation of the target genome. Although promising, several challenges remain in efficacy and safety that require solutions.

Molecular insights into Spindlin1-HBx interplay and its impact on HBV transcription from cccDNA minichromosome.

Molecular interplay between host epigenetic factors and viral proteins constitutes an intriguing mechanism for sustaining hepatitis B virus (HBV) life cycle and its chronic infection. HBV encodes a regulatory protein, HBx, which activates transcription and replication of HBV genome organized as covalently closed circular (ccc) DNA minichromosome. Here we illustrate how HBx accomplishes its task by hijacking Spindlin1, an epigenetic reader comprising three consecutive Tudor domains. Our biochemical and structural studies have revealed that the highly conserved N-terminal 2-21 segment of HBx (HBx2-21) associates intimately with Tudor 3 of Spindlin1, enhancing histone H3 "K4me3-K9me3" readout by Tudors 2 and 1. Functionally, Spindlin1-HBx engagement promotes gene expression from the chromatinized cccDNA, accompanied by an epigenetic switch from an H3K9me3-enriched repressive state to an H3K4me3-marked active state, as well as a conformational switch of HBx that may occur in coordination with other HBx-binding factors, such as DDB1. Despite a proposed transrepression activity of HBx2-21, our study reveals a key role of Spindlin1 in derepressing this conserved motif, thereby promoting HBV transcription from its chromatinized genome.

Retrotransposons hijack alt-EJ for DNA replication and eccDNA biogenesis.

Retrotransposons are highly enriched in the animal genome1-3. The activation of retrotransposons can rewrite host DNA information and fundamentally impact host biology1-3. Although developmental activation of retrotransposons can offer benefits for the host, such as against virus infection, uncontrolled activation promotes disease or potentially drives ageing1-5. After activation, retrotransposons use their mRNA as templates to synthesize double-stranded DNA for making new insertions in the host genome1-3,6. Although the reverse transcriptase that they encode can synthesize the first-strand DNA1-3,6, how the second-strand DNA is generated remains largely unclear. Here we report that retrotransposons hijack the alternative end-joining (alt-EJ) DNA repair process of the host for a circularization step to synthesize their second-strand DNA. We used Nanopore sequencing to examine the fates of replicated retrotransposon DNA, and found that 10% of them achieve new insertions, whereas 90% exist as extrachromosomal circular DNA (eccDNA). Using eccDNA production as a readout, further genetic screens identified factors from alt-EJ as essential for retrotransposon replication. alt-EJ drives the second-strand synthesis of the long terminal repeat retrotransposon DNA through a circularization process and is therefore necessary for eccDNA production and new insertions. Together, our study reveals that alt-EJ is essential in driving the propagation of parasitic genomic retroelements. Our study uncovers a conserved function of this understudied DNA repair process, and provides a new perspective to understand-and potentially control-the retrotransposon life cycle.

SLF2 Interacts with the SMC5/6 Complex to Direct Hepatitis B Virus Episomal DNA to Promyelocytic Leukemia Bodies for Transcriptional Repression.

Hepatitis B virus (HBV) chronically infects approximately 300 million people worldwide, and permanently repressing transcription of covalently closed circular DNA (cccDNA), the episomal viral DNA reservoir, is an attractive approach toward curing HBV. However, the mechanism underlying cccDNA transcription is only partially understood. In this study, by illuminating cccDNA of wild-type HBV (HBV-WT) and transcriptionally inactive HBV that bears a deficient HBV X gene (HBV-ΔX), we found that the HBV-ΔX cccDNA more frequently colocalizes with promyelocytic leukemia (PML) bodies than that of HBV-WT cccDNA. A small interfering RNA (siRNA) screen targeting 91 PML body-related proteins identified SMC5-SMC6 localization factor 2 (SLF2) as a host restriction factor of cccDNA transcription, and subsequent studies showed that SLF2 mediates HBV cccDNA entrapment in PML bodies by interacting with the SMC5/6 complex. We further showed that the region of SLF2 comprising residues 590 to 710 interacts with and recruits the SMC5/6 complex to PML bodies, and the C-terminal domain of SLF2 containing this region is necessary for repression of cccDNA transcription. Our findings shed new light on cellular mechanisms that inhibit HBV infection and lend further support for targeting the HBx pathway to repress HBV activity. IMPORTANCE Chronic HBV infection remains a major public health problem worldwide. Current antiviral treatments rarely cure the infection, as they cannot clear the viral reservoir, cccDNA, in the nucleus. Therefore, permanently silencing HBV cccDNA transcription represents a promising approach for a cure of HBV infection. Our study provides new insights into the cellular mechanisms that restrict HBV infection, revealing the role of SLF2 in directing HBV cccDNA to PML bodies for transcriptional repression. These findings have important implications for the development of antiviral therapies against HBV.

Targeting hepatitis B virus cccDNA levels: Recent progress in seeking small molecule drug candidates.

Hepatitis B virus (HBV) infection is a major global health problem that puts people at high risk of death from cirrhosis and liver cancer. The presence of covalently closed circular DNA (cccDNA) in infected cells is considered to be the main obstacle to curing chronic hepatitis B. At present, the cccDNA cannot be completely eliminated by standard treatments. There is an urgent need to develop drugs or therapies that can reduce HBV cccDNA levels in infected cells. We summarize the discovery and optimization of small molecules that target cccDNA synthesis and degradation. These compounds are cccDNA synthesis inhibitors, cccDNA reducers, core protein allosteric modulators, ribonuclease H inhibitors, cccDNA transcriptional modulators, HBx inhibitors and other small molecules that reduce cccDNA levels.