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.

Nonproductive Hepatitis B Virus Covalently Closed Circular DNA Generates HBx-Related Transcripts from the HBx/Enhancer I Region and Acquires Reactivation by Superinfection in Single Cells.

Hepatitis B virus (HBV) infection remains a public health problem worldwide. Persistent HBV infection relies on active transcription of the covalently closed circular DNA (cccDNA) in hepatocytes, which is less understood at the single-cell level. In this study, we isolated primary human hepatocytes from liver-humanized FRG mice infected with HBV and examined cccDNA transcripts in single cells based on 5' end sequencing. Our 5' transcriptome sequencing (RNA-seq) analysis unambiguously assigns different viral transcripts with overlapping 3' sequences and quantitatively measures viral transcripts for structural genes (3.5 kb, 2.4 kb, and 2.1 kb) and the nonstructural X gene (0.7 kb and related) in single cells. We found that an infected cell either can generate all viral transcripts, signifying active transcription, or presents only transcripts from the X gene and its associated enhancer I domain and no structural gene transcripts. Results from cell infection assays with recombinant HBV show that nonproductive transcription of cccDNA can be activated by incoming virus through superinfection. Moreover, upon HBV infection, cccDNA apparently can be transcribed in the absence of HBx and produces HBx, needed for productive transcription of other viral genes. These results shed new light on cccDNA transcription at the single-cell level and provide insights useful for improving the treatment strategy against chronic HBV infection. IMPORTANCE Hepatitis B virus (HBV) infection can be effectively suppressed but rarely cured by available drugs. Chronic HBV infection is based on persistence of covalently closed circular DNA (cccDNA) and continuous infection and reinfection with HBV in the liver. Understanding transcriptional regulation of cccDNA will help to achieve permanent transcriptional silencing, i.e., functional cure of HBV. In our study, we found that an infected cell either can generate all viral transcripts, signifying active transcription, or presents only transcripts from the X gene and its associated enhancer I domain and no structural gene transcripts. The nonproductive transcription of cccDNA can be activated by incoming virus through superinfection. Upon an infection, cccDNA apparently can be transcribed in the absence of HBx to produce HBx, necessary for subsequent transcription of other HBV genes. Our studies shed new light on the mechanism of HBV infection and may have implications for a functional cure regimen for HBV.

DNA Polymerase κ Is a Key Cellular Factor for the Formation of Covalently Closed Circular DNA of Hepatitis B Virus.

Hepatitis B virus (HBV) infection of hepatocytes begins by binding to its cellular receptor sodium taurocholate cotransporting polypeptide (NTCP), followed by the internalization of viral nucleocapsid into the cytoplasm. The viral relaxed circular (rc) DNA genome in nucleocapsid is transported into the nucleus and converted into covalently closed circular (ccc) DNA to serve as a viral persistence reservoir that is refractory to current antiviral therapies. Host DNA repair enzymes have been speculated to catalyze the conversion of rcDNA to cccDNA, however, the DNA polymerase(s) that fills the gap in the plus strand of rcDNA remains to be determined. Here we conducted targeted genetic screening in combination with chemical inhibition to identify the cellular DNA polymerase(s) responsible for cccDNA formation, and exploited recombinant HBV with capsid coding deficiency which infects HepG2-NTCP cells with similar efficiency of wild-type HBV to assure cccDNA synthesis is exclusively from de novo HBV infection. We found that DNA polymerase κ (POLK), a Y-family DNA polymerase with maximum activity in non-dividing cells, substantially contributes to cccDNA formation during de novo HBV infection. Depleting gene expression of POLK in HepG2-NTCP cells by either siRNA knockdown or CRISPR/Cas9 knockout inhibited the conversion of rcDNA into cccDNA, while the diminished cccDNA formation in, and hence the viral infection of, the knockout cells could be effectively rescued by ectopic expression of POLK. These studies revealed that POLK is a crucial host factor required for cccDNA formation during a de novo HBV infection and suggest that POLK may be a potential target for developing antivirals against HBV.

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.