RNA Gymnastics: How Does Hepatitis Delta Virus Replicate?

(Posted on Monday, May 15, 2023)

This story is part of our series on viroids and virusoids, small infectious RNAs. The story is also the second installment in a series on hepatitis D virus, a virusoid-like pathogen that causes serious human disease. You may read the others on Forbes or www.williamhaseltine.com. Now we turn to the structure and replication of the infectious hepatitis D RNA.

Hepatitis D “virus” is a virus in name only. Underneath the hood, it more closely resembles viroids and virusoids: it is a small circular RNA that, due to high sequence complementarity, folds in on itself to form a rod-like structure. Like other virusoids, hepatitis D depends on a helper virus to infect cells and to migrate from host to host. Usually this help comes from hepatitis B virus, although recent findings suggest that other viruses may also be able to contribute, raising the possibility that hepatitis D virus may contribute to disease other than fulminant hepatitis.

Hepatitis D Genome 

The hepatitis D virus genome consists of roughly 1670 to 1700 nucleotides, depending on the genotype. Currently, there are eight known genotypes, spread across the globe in geographic clusters (Figure 1). There are further minor variants within each genotype. Changes in the genome contribute to noticeable differences in pathogenesis, with some genotypes causing more serious disease than others.

FIGURE 1. (Left) Global distribution of hepatitis D virus (HDV) genotypes (Right) Phylogenetic network of HDV genome sequences. SOURCE: Abdominal Key

Around 74% of the nucleotides in the hepatitis D virus genome are complementary, meaning they bind to one another. This compresses the circular genome into a rod-like form (Figure 2). Both the genomic and the antigenomic strands of hepatitis D virus encode ribozymes; these are active sequences of RNA that both cleave and rejoin the RNA during replication, as is the case for all viroids and virusoids (Figure 2).

FIGURE 2. Schematic representation of the sequences and secondary structures of the genomic and antigenomic hepatitis D ribozymes. SOURCE: ACCESS Health International (Adapted from Fields Virology: DNA Viruses, 7th Edition)

Strikingly, both complementary genomic strands have large open reading frames that have the potential to encode proteins. The antigenomic strand of the hepatitis D virus open reading frame encodes two distinct proteins: the small hepatitis D virus antigen (S-HDAg) and the large hepatitis D virus antigen (L-HDAg) (Figure 3). These play a critical role in replication. There is no report of the even larger open reading frame of the genomic strand, which spans 352 amino acids, in the literature.

FIGURE 3. (Top) Schematic of the rod-like shape of the hepatitis D virus (HDV) genome/antigenome. (Bottom) Overview of the genome and antigenome of HDV, with ribozyme cleavage sites and open reading frame (ORF) clearly marked. SOURCE: ACCESS Health International (Adapted from: Magnius et al. 2018, https://doi.org/10.1099/jgv.0.001150)

Messenger RNA Synthesis

Hepatitis D virus is carried between hosts and into cells using the hepatitis B virus coat proteins. Once inside the cell, the hepatitis D genome wends its way into the nucleus via nuclear localization signals (NLS) embedded in the hepatitis D antigens—there, replication begins. With the exception of the final assembly stage of the infectious particle —at which point the hepatitis B surface proteins are reintroduced— the replication cycle of  hepatitis D virus is thought to be independent of hepatitis B. To exit the cell, hepatitis D virus once again makes use of the hepatitis B virus coat proteins (Figure 4).

FIGURE 4. Schematic representation of the hepatitis Delta virus (HDV) lifecycle. Notice how cell entry and cell exit make use of hepatitis B virus surface antigens. SOURCE: Yurdaydin et al. 2017 https://doi.org/10.1016/S0168-8278(17)30828-0

Three kinds of hepatitis D RNA are involved in replication: genomic RNA, antigenomic RNA, and messenger RNA (mRNA). The genomic strand acts as a template for the production of the other two.

Upon entry into the nucleus, the first step of hepatitis D replication is the synthesis of mRNAs. Down the line, these will be used to produce the antigen proteins. Unlike most RNA viruses, hepatitis D does not encode an RNA-dependent RNA polymerase (RdRp). This is an enzyme that allows viruses to produce multiple copies of their RNA from an original RNA template. Instead, hepatitis D virus hijacks the cellular DNA-dependent RNA polymerase II (Pol-II), normally used to produce messenger RNAs from genomic DNA. Exactly how hepatitis D virus manages to appropriate the host polymerases remains unclear, but it’s likely that its rod-like double-stranded structure confuses the cell into mistaking it for a strand of DNA. This is another respect in which hepatitis D resembles viroids and virusoids, which depend on the same strategy.

The newly synthesized HDV mRNAs are then exported back into the cytoplasm, where they are translated into HDV antigen proteins (Figure 5) before being transported back into the nucleus.

FIGURE 5. Overview of hepatitis D virus (HDV) mRNA synthesis. Abbreviations: 5’, five-prime end; pA, poly-A tail; Pol-II, DNA-dependent RNA-polymerase II; L-HDAg, large hepatitis D antigen; S-HDAg, small hepatitis D antigen. SOURCE: ACCESS Health International

HDV RNA Replication

With mRNA synthesis in full swing, the next step of the replication process can begin: amplification of genomic RNA. For this, the genomic RNA must first travel to the nucleolus, a special subcompartment of the nucleus. The specifics of this migration are not yet understood, but it is thought that the newly synthesized antigens play a role in triggering and assisting the journey.

Once in the nucleolus, hepatitis D relies on a rolling-circle replication pathway (Figure 6A). The key mechanism behind this form of replication is the creation of a large chain of RNAs using the original circular RNA as a template. This chain, called an oligomer, is made up of many unit-length RNAs tethered together, but each is the mirror image of the original RNA. Such “mirror-image” RNA is referred to as antigenomic; it is the inverse version of the genome. Unlike mRNA synthesis, creation of hepatitis D antigenomic RNAs relies on DNA-dependent RNA-polymerase I (Pol-I), which can only be found in the nucleolus.

As soon as the long antigenomic chain has been produced, the hepatitis D ribozymes spring into action, cleaving the long chain into unit-length RNAs. The antigenomic unit-length RNAs, currently in linear form, still need to be turned back into circular RNA. This process, known as ligation, happens either through the assistance of yet-unknown host enzymes or through the ribozymes themselves, which self-ligate the RNA back into circular form.

The antigenomic circular RNAs can then be used as a template to produce new genomic RNAs in a second round of rolling-circle replication; the mirror image is reflected once more, bringing it back to its original state (Figure 6B).

FIGURE 6. Schematic representation of hepatitis D virus RNA replication. (A) Rolling-circle replication of antigenomic HDV RNA in the nucleolus, (B) Rolling-circle replication of genomic HDV RNA in the nucleus, with antigenomic RNA as template. SOURCE: ACCESS Health International

Following synthesis of HDV antigens and amplification of HDV RNA, all that is left is for the various pieces to be assembled and packaged in a protein coat containing the hepatitis B virus surface antigens. The finished viral particle is then ready to exit the host cell and infect neighboring cells.

The subsequent article in this series will provide an in-depth look at the hepatitis D virus antigens and their various functions throughout the viral life cycle.