To establish chronic disease, the hepatitis B virus (HBV) must have its genome of relaxed circular DNA (rcDNA) turned into covalently closed circular DNA (cccDNA). Exactly how rcDNA becomes cccDNA has been unclear, but new details about the transformation have been uncovered by Princeton University researchers. According to the Princeton team, the rcDNA genome, which contains four lesions, undergoes repair by five host proteins.

The new findings appeared March 11 in Nature Communications, in an article titled, “Hepatitis B virus cccDNA is formed through distinct repair processes of each strand.” The article suggests that steps in the repair processes could be blocked by new, targeted drugs, preventing much of the suffering caused by hepatitis B.

HBV hasn’t caused any nationwide lockdowns or stock market crashes. It is slow to spread from person to person, and it is rarely immediately fatal. Nonetheless, it is incredibly damaging because it can establish lifelong chronic infection with profound consequences for its victims.

“An estimated two billion people have been exposed to HBV, of whom 250–400 million are chronically infected,” said Alexander Ploss, associate professor of molecular biology at Princeton University, and senior author on the Nature Communications article. “Currently, there is no cure for chronic HBV infection, and patients need to be on a lifelong antiviral regimen. Approximately 887,000 individuals die each year from HBV-related liver diseases or liver cancer.”

Ploss and his team are striving to understand HBV’s life cycle in hopes of finding a way to prevent the virus from establishing damaging chronic infections. In the current study, they focused on an early stage of the life cycle—the formation of cccDNA from rcDNA after HBV carries rcDNA into the host cell.

A key feature of HBV rcDNA is that each of its two strands contains a gap in its nucleotide sequence. One strand, called the plus strand, has a gap that is considerably larger than and offset from the gap on the other, minus strand. Cells perceive gaps in DNA as damage that needs to be filled in and repaired. The cellular proteins that carry out DNA repair can’t tell the difference between viral DNA and cellular DNA, so they set to work “repairing” rcDNA as soon as it arrives in the nucleus. This repair process converts rcDNA into an intact circle of double-stranded DNA (that is, cccDNA) that can be maintained in the cell’s nucleus.

To understand how this repair process takes place, Lei Wei, a postdoctoral fellow in the Ploss lab, generated recombinant rcDNA (RrcDNA) substrates with defined lesions on either the plus or the minus strand—psl-RrcDNA and msl-RrcDNA, respectively. By experimenting with these substrates, the Ploss team determine that the minus- and plus-strand lesions of HBV rcDNA require different sets of human repair factors.

“We demonstrate that the plus-strand repair resembles DNA lagging strand synthesis, and requires proliferating cell nuclear antigen (PCNA), the replication factor C (RFC) complex, DNA polymerase delta (POLδ), flap endonuclease 1 (FEN-1), and DNA ligase 1 (LIG1),” the authors of the Nature Communications article wrote. “Only FEN-1 and LIG1 are required for the repair of the minus strand.”

As its name implies, rcDNA is a loop of DNA. The sequence of nucleotides on one strand encodes the instructions for making a protein while the other strand is its mirror image. Whereas human cellular DNA contains over 20,000 genes, HBV’s DNA genome only contains four. None of the viral proteins made from these genes is required for the conversion of rcDNA to cccDNA. Instead, the virus coopts cellular proteins to accomplish this and other steps of viral replication.

The experiments showed that conversion of the plus strand into a continuous circle happens rapidly and requires all five of the cellular proteins working in concert. In contrast, repair of the minus strand requires only two of the five proteins (FEN-1 and LIG1) but is slower because there is a viral protein attached to one end of the minus strand that must be removed before the nucleotide gap can be sealed.

Researchers at Princeton have dissected the steps taken by cellular proteins to convert Hepatitis B virus (HBV) relaxed circular DNA (rcDNA) into closed circular DNA (cccDNA), a process that takes place in the nuclei of infected cells and is critical to the establishment of chronic HBV infection. The repair steps of the plus strand (upper panel) and the minus strand (lower panel) are shown. A viral protein called Pol is linked to the minus strand, while an RNA primer (red line) is linked to the plus strand. [Lei Wei, Princeton University]

Wei and Ploss also demonstrated that their work could inform the development of drugs to treat hepatitis B. Specifically, using an in vitro system, the researchers showed that aphidicolin and cyclin-dependent kinase inhibitor p21 peptide specifically inhibit the repair of the rcDNA plus-strand.

The researchers are hopeful that future studies will identify drugs that work in the human body. Ploss stated, “Our findings, biochemical approaches, and the novel reagents that we generated and engineered, open the door to providing an in-depth understanding of how this major human virus establishes persistence in host cells.”