Potential capacity of interferon-α to eliminate covalently closed circular DNA (cccDNA) in hepatocytes infected with hepatitis B virus

For approximately 60 years, it has been known that hepatitis B is caused by a viral infection, and the hepatitis B virus (HBV) has been definitively identified as the causative factor [1]. Over time, an increasing details have been discovered in regards to hepatitis B, and is now well known that HBV is a hepadnavirus. More specifically, HBV is a hepatotropic DNA virus approximately 42 nm in diameter with particles consisting of surface proteins and core proteins [2] surrounding a partially double-stranded, relaxed, and circular 3.2 kb genomic DNA component (called rcDNA) [3] (additional details can be found in other reviews [1, 4]). During its natural life cycle, HBV enters hepatocytes by attaching to surface proteins, such as hepatocyte-associated heparan sulfate proteoglycans (HSPG) and sodium taurocholate cotransporting polypeptide (NTCP) [5]. Next, the core particle (nucleocapsid) of HBV is released into the cytoplasm [6]. The genomic rcDNA of HBV is released from the nucleocapsid and, through the nuclear pore complex, enters into the nucleus of the hepatocyte [7]. Once inside the nucleus, the gap in the rcDNA is repaired by the DNA repair machinery of the host cell to generate the covalently closed circular DNA (cccDNA) [6‐8]. Importantly, this cccDNA is responsible for difficulty in curing the HBV infections, which can lead to chronic liver inflammation, cirrhosis, and even hepatocellular carcinoma [6, 9, 10].

Once formed in the cell nucleus, cccDNA is very difficult to get eliminated from infected cells and can persist continuously until the death of the host cell [11]. cccDNA can form a dynamic pool within the nuclei, where it supports its persistence and encodes its structural proteins (including surface proteins, core proteins), polymerases and the X protein [6]. According to the guidelines from the European Association for the Study of the Liver, the American Association for the Study of Liver Diseases, and the Chinese Society of Hepatology and the Chinese Society of Infectious Diseases, interferon (IFN-α) and nucleotide analogues (NAs) are recommended, as needed, for the treatment of HBV infection in patients [12‐14]. In addition to IFN (pegylated interferon-alpha, [PegIFNα]), the following are also used for the inhibition of viral replication: entecavir (ETV), tenofovir disoproxil fumarate (TDF), adefovir (ADF), lamivudine (LAM) and telbivudine (LDT) are used for the inhibition of viral replication [12‐14]. Although NAs have been shown to be efficient for decreasing the viral protein and DNA loads in the serum of HBV-infected patients and effectively hinder the progression of chronic HBV infection, cccDNA is the most difficult obstacle preventing a complete cure of HBV since it is not effectively and directly eliminated by current drugs [15‐18]. Until now, IFN-α was the only potential candidate against persistent cccDNA in the nuclei of the hepatocytes in patients with chronic HBV infections [11, 19‐21].

Based on basic research indications, IFN-α might be one of the most promising treatments for the elimination of HBV cccDNA. However, clinical evidence has shown that IFN-α treatment has limited efficacy against chronic infections in HBV-positive patients [22]. Unfortunately, there is insufficient information to clarify why the IFN-α mechanisms of action are ineffective for the treatment of HBV. Herein, the details of IFN-α on cccDNA that persists in the hepatocytes of HBV-infected patients are discussed in hopes of uncovering even a small insight that would lead to an effective treatment or even a cure for chronic HBV infections.

It is necessary to understand the details of the HBV life cycle to fully comprehend the mechanisms of an anti-HBV treatment (Fig. 1). As a member of Hepadnaviridae family of viruses, HBV has a 42 nm diameter (Dane particle) and spherical structure, and it consists of viral surface antigens (HBsAgs), including large, middle and small surface glycoproteins, and a lipid envelope. These components help the virus enter into hepatocytes via binding to receptors and fusing with the cell membrane [23]. Within the Dane particle (virion), under the outer proteolipid envelope, there is an icosahedral nucleocapsid core composed of HBV core antigen (HBcAgs) that surrounds both the DNA polymerases as well as the circular, partially double stranded DNA [24]. Upon infection, binding of the small surface antigen (SHBs) protein to the HSPG and the binding of the large surface antigen (LHBs) to NTCP are essential and sufficient for HBV attachment to a hepatocyte [5, 25]. Through protein-mediated endocytosis, HBV particles enter hepatocytes, and the fusion of the HBV envelope with the membrane of an endosome triggers the release of the capsid into the cytoplasm [26]. Following an interaction with microtubules, the viral capsid is then transported into the nucleus through the nuclear pore complex (NPC) [27]. The polymerase or the capsid protein contains a nuclear localization signal (NLS) that enables the rcDNA to localize to the nucleus with the help of importin α and importin β [28]. Once in the nucleoplasm, the rcDNA is repaired by the associated factors of the host cells in the final process of completing the cccDNA. After the short RNA primer and polymerase protein are removed, the minichromosome persists long-term in the nucleus [29]. cccDNA is a template in the nucleus that can be transcribed by the host RNA polymerase II into four types of HBV transcripts: pregenomic RNA (pgRNA, 3.5 kb), pre-S1 RNA (2.4 kb), pre-S2/S RNA (2.1 kb) and the X gene (0.7 kb). These transcripts are then translated into HBV core proteins and a viral polymerase, large surface proteins, middle and small surface proteins and the HBx protein in the cytoplasm, respectively [30]. The formation of a complex consisting of pgRNA and a polymerase initiates nucleocapsid assembly using core HBV protein dimers as building blocks, and during this process, the P protein itself primes the reverse transcription of the pgRNA to synthesize the minus strand of HBV rcDNA. Subsequently, the plus-strand of the rcDNA is synthesized by the P protein in a DNA-dependent manner [31]. The current anti-HBV drugs, including NAs (lamivudine, adefovir, telbivudine, entecavir, and tenofovir), all target this process that primes, initiates, or elongates new synthesis [31]. With the RNase H activity of the P protein, pgRNA can be degraded during the formation of minus-strand [32]. The nucleocapsid containing the rcDNA acquires a lipid bilayer with HBV surface antigens to form the viral envelope, and the viruses are trafficked and secreted outside the hepatocytes, possibly with the help of the endosomal sorting complex required for transport (ESCRT) protein [33]. Although this is a brief depiction of the HBV life cycle, most of the current drugs effectively target the reverse transcription step; however, new drugs with novel targets are desired for clinical application.

During the HBV life cycle, viral rcDNA is translocated to the nucleus of the host cell, where the cccDNA persists after a complicated process in which the viral polymerase is removed from the 5′-end of the negative DNA strand, a short RNA oligomer is used as a primer for DNA synthesis of the sense strand, the redundant sequence of the viral DNA is removed, and the cccDNA is formed after a ligation performed by the cellular replication machinery of the host [11, 34]. cccDNA in the nucleus is a constant transcriptional template for pgRNA, which is subsequently reverse transcribed into viral genomic DNA packages in the nucleocapsid for HBV assembly [35]. In addition, the mRNA of the surface, core and e-antigens, as well as the X protein are all transcribed from the cccDNA in the nucleus [6].

The initial nuclear cccDNA originates from the viral infection; however, the major sources of cccDNA in the nuclear pool are the newly reverse-transcribed rcDNA in the nucleocapsids [36]. Further, the size of the cccDNA pool, which is formed at the early stage of infection, ranges from 30 to 50 copies of cccDNA per cell [37, 38]. Within the pool, the half-life of the cccDNA varies between different cell types; the half-life is longer than 30 days in stable hepatocytes and fewer than 5 days in unstable hepatocytes [39, 40].

The stable structure of cccDNA in the nucleus is the minichromosome, also called the episome, which is extrachromosomal DNA [41, 42]. Using the duck liver virus model and HepG2.2.15 (HepG2 cells with stable HBV expression), two separate research groups from the USA and Germany discovered histone proteins that coprecipitated with the viral genome and a 180 bp fragment of the HBV genome was detected when the samples were enzymatically digested [41, 42]. In eukaryotic cells, the predominant length of the chromatin fragments was 200 bp, and compared with the DNA of host cells, the digested HBV genomic fraction was subsequently shorter [43]. With respect to the arrangement, a HBV genomic minichromosome was visualized under an electron microscope and was found to resemble beads-on-a-string [42]. Similar to those of the host cells, histone proteins are essential components of the minichromosome and can bind tightly to cccDNA. With the help of HBV core proteins, the minichromosome is supercoiled with histones, where histone H3 and H2b are predominant, while H2A, H4 and H1 are less commone [11, 44]. Similar to standard histone function, these histones are involved in the regulation of viral cccDNA gene expression [45]. Histones H3 and H4 can be acetylated or deacetylated, resulting in the suppression or enhanced expression of HBV genes [45].