Functional cure of hepatitis B requires silencing covalently closed circular and integrated hepatitis B virus DNA

Hepatitis B virus (HBV) is a small DNA virus that infects the liver. Its entry into hepatocytes is facilitated by the sodium-taurocholate–cotransporting polypeptide (NTCP), a bile acid receptor, which contributes to its hepatotropism (1). After entry, the nucleocapsid containing the partially double-stranded, relaxed circular DNA genome (rcDNA) of the complete virion is transported to the hepatocyte nucleus where host cellular enzymes repair the rcDNA to form the covalently closed circular DNA (cccDNA) (Figure 1A). The cccDNA is a highly stable, supercoiled, episomal form of the viral DNA. It serves as the template for transcription of all viral mRNAs, including the pregenomic RNA (pgRNA). In the cytoplasm, the viral core protein self-assembles with the pgRNA and viral polymerase to form nucleocapsids. Viral replication occurs within the nucleocapsid through reverse transcription of the pgRNA. The mature viral capsids containing rcDNA are then enveloped with the surface proteins in the endoplasmic reticulum and secreted from the infected cell as complete virions or transported back to the nucleus to replenish the cccDNA pool.

Illustration of the HBV life cycle and genomic organization.Figure 1

Illustration of the HBV life cycle and genomic organization. (A) HBV enters the hepatocyte via NTCP, a bile acid receptor. Following entry and uncoating, the nucleocapsid containing a partially double-stranded rcDNA translocates to the hepatocyte nucleus where host cellular enzymes convert the rcDNA into cccDNA. The cccDNA is a highly stable, supercoiled, episomal form of the viral DNA that serves as the template for transcription of all viral mRNAs, including the pgRNA. In the cytoplasm, the viral core protein self-assembles with the pgRNA and viral polymerase to form new nucleocapsids. Viral replication occurs within the nucleocapsid through reverse transcription of the pgRNA. The mature viral capsids containing rcDNA have two fates; they can be enveloped with the surface proteins in the endoplasmic reticulum and secreted from the infected cell as complete virions or they can be transported back to the nucleus to replenish the cccDNA pool. NAs inhibit genome replication by blocking reverse transcription of pgRNA, resulting in a rapid decrease in HBV DNA levels. Rarely, the minus-strand HBV DNA produces dslDNA instead of rcDNA. dslDNA can integrate into the host DNA as iDNA and can produce HBsAg. iDNA can continue to produce HBsAg even if cccDNA transcription is silenced and no longer produces HBsAg. Grudda et al. (15) developed a ddPCR assay that can differentiate HBsAg originating from cccDNA from that originating from iDNA. (B) The viral genome encodes four open reading frames that yield four mRNA transcripts with common 3′ ends. Notably, iDNAs lack the 3′ ends, which served as the basis for distinguishing the source of the HBsAg transcripts in the ddPCR assay. Four viral mRNAs — 3.5 kb pgRNA and 2.4 kb, 2.1 kb, and 0.7 kb mRNAs — yield precore (preC), core and polymerase, large, middle, and small HBsAg and X proteins, respectively. DR1, direct repeat 1; DR2, direct repeat 2.

Subviral filamentous and spherical particles, comprising surface proteins but devoid of viral DNA, are produced in quantities that exceed complete virions by 1,000- to 100,000-fold. Rarely, during genome replication, there is defective primer translocation to the newly formed minus-strand DNA, leading to the formation of a double-stranded linear DNA (dslDNA) genome instead of the rcDNA genome. These dslDNA forms may integrate randomly into the host genome by utilizing sites of host cell DNA breaks (2). Recently, targeted long-read sequencing revealed that the full-length genome may be integrated but this integrated DNA (iDNA) does not support viral replication because it lacks the precore and core transcripts. Most of the transcribed integrants contain HBV promoter sequences that drive S transcript expression, resulting in production of hepatitis B surface antigen (HBsAg) (3).

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