Denying the wolf access to sheep’s clothing

Abstract

Hepatitis delta virus (HDV) is a cause of acute and chronic liver disease for which no effective therapy is currently available. Previous research has demonstrated that prenylation of the large HDV antigen is essential for viral assembly. A new report describes a novel small animal model for HDV replication and demonstrates that prenylation inhibitors are highly effective at clearing viremia and thus have potential relevance for the treatment of chronic delta hepatitis.

Delta hepatitis is the least frequent but most severe form of viral hepatitis. Acute infection with hepatitis delta virus (HDV) is often severe and has a fatality rate of at least 5%, even in children. Chronic delta hepatitis leads to cirrhosis in at least two thirds of persons and can present as end-stage liver disease in early adulthood (1). Hepatitis D also has the distinction of being the form of viral hepatitis that is most resistant to current antiviral therapies (2).

Virology of HDV

The unique clinical features of delta hepatitis are matched by the unique qualities of its viral agent. HDV is the smallest known human pathogen and uses a unique replicative strategy (3). The HDV genome is merely 1.7 kb in length and consists of a single-stranded circular molecule of RNA of negative polarity that is held in a rigid rod-like form by extensive intramolecular base pairing (4). The antigenome of HDV RNA has a single open reading frame that encodes hepatitis delta antigen. Importantly, HDV requires the presence of another (helper) virus for productive replication and spread (5). The helper virus is the hepatitis B virus (HBV), a very significant human pathogen in its own right. Being linked to HBV and only occurring in persons who have hepatitis B surface antigen (HBsAg) in serum, HDV is spread in the same ways as hepatitis B, through parenteral or sexual exposure to blood or body fluids. Up to 5% of the world’s population is infected with HBV, and probably 5% of these have HDV superinfection (6). Delta hepatitis has a worldwide distribution, but areas of dramatically high endemicity associated with severe clinical outcomes have been identified in the Amazon basin, Central Africa, and parts of Eastern Europe. Prevention of hepatitis B will also prevent hepatitis D. In a triumph of public health, increasing vaccination rates for hepatitis B are contributing to the decline in HDV incidence. Nevertheless, there are still millions of persons with chronic HDV infection, and until hepatitis B is fully eradicated, new cases of delta hepatitis will occur.

HDV circulates in serum as a 35- to 40-nm particle containing viral RNA and HDV antigen, encapsidated by HBsAg (7, 8). The envelope protein of HBV allows for the specific binding and uptake of virions into hepatocytes. Inside the cell, the viral genome is released and replicated by a double-rolling circle mechanism (4). Replication of HDV, like that of other viruses, requires multiple enzymatic activities. Although many viruses encode the majority of their replicative and processing enzymes, HDV apparently does not. The sole enzymatic activity HDV possesses is a ribozyme (similar to that in plant satellite viruses) that autocleaves the circular RNA, producing a linear molecule (9). Other enzyme activities are apparently provided by the host cell.

Editing of HDV RNA and prenylation of the large HDV antigen

HDV antigen is produced in infected cells in two sizes, small (S) and large (L), the L form having an additional 19–20 amino acids on its carboxyl terminus (10). It was initially unclear how a single open reading frame encoded two different-sized polypeptides. In a series of elegant studies, Casey and coworkers demonstrated that HDV RNA is edited by a cellular enzyme (double-stranded RNA adenosine deaminase) that change A to I on the antigenomic (+) strand, which with replication causes a change in the UAG stop codon to UGG, which encodes tryptophan (Figure 1). Without the initial stop codon, a larger delta antigen is created that is terminated 19–20 codons downstream by a second stop codon. Virtually all strains of HDV use this editing mechanism.

Figure 1.

Replication of HDV. Viral particles consist of HDV RNA (black lines) and HDV antigen (blue) encapsidated by HBsAg (red). The HBsAg allows for binding and uptake of the virion into hepatocytes. Inside the cell, the RNA genome (negative polarity) is released and undergoes replication by the double-rolling circle pattern (double arrows). The antigenomic RNA (positive polarity) serves as the template for production of more genomic RNA but can also be edited by intracellular host dsRNA adenosine deaminase, changing A to I so that with production of genomic RNA, C rather than U will be transcribed (right side of the figure). The editing changes the UAG stop codon on the positive strand to a UGG, which is read as tryptophan, resulting in a protein with an additional 19 amino acids, the large delta antigen (large δ) that inhibits replication. Small delta antigen (small δ), the product of the unedited transcript (left side of the figure), binds to HDV RNA and promotes replication. Large delta antigen has a prenylation site that creates a hydrophobic molecule that binds both to HDV RNA and to HBsAg, leading to viral assembly and release. Thus, inhibition of prenylation will interfere with viral assembly and release but should affect replication minimally, if at all.

In complementary studies, Glenn and coworkers demonstrated that the L HDV antigen had a prominent isoprenylation site near the added carboxyl terminus (11). Addition of a farnesyl molecule to the site creates a lipophilic area, allowing for membrane association and virion assembly. The association of HBsAg and prenylated L HDV antigen thus promotes packaging of the virus and export. These two lines of research pointed to how HDV replication depends on cellular enzymes that the virus redirects toward production of viral antigen and virions. HDV also clearly uses other cellular enzymes, not all of which are well defined but perhaps include a helicase to unwind the intramolecular base pairing of the HDV RNA circular genome and a polymerase for production of viral RNA. These findings are important because knowledge of the viral life cycle can point to targets for antiviral therapy. The difficulty (and unique feature) of delta hepatitis is that most of the revealed targets are normal cellular proteins. Thus, HDV hides inside the envelope of HBV as well as behind normal cellular enzymes: the wolf in sheep’s clothing.

Inhibition of HDV by inhibition of prenylation

In this issue of the JCI, Bordier et al. (12) extend the previous studies of prenylation of the L HDAg and show how this knowledge can be used to develop therapies for delta hepatitis. Since prenylation is critical for virion formation, prenylation inhibitors might be effective in blocking viral replication. The hurdle for demonstrating activity of prenylation inhibitors (many of which were developed as anticancer agents) (13) was the lack of availability of a practical model for studying the viral life cycle. To this end, the authors developed a murine model using hydrodynamic transfection of HDV cDNA into an established HBV transgenic mouse. They reasoned that if HBsAg was provided, introduction of HDV RNA might result in HDV viremia. This was indeed the case. The authors then showed that viremia was blocked by prenylation inhibition.

At present, therapy of delta hepatitis is difficult and unsatisfactory. The nucleoside analogues that block HBV replication have little or no effect against HDV replication or the disease that it causes. IFN-α inhibits HDV replication, but therapy usually requires high doses given for a prolonged period, which are often poorly tolerated (2).

The work by Bordier et al. (12) can be viewed as a new phase of therapeutics for HDV. In the typical first stage, known and established antiviral drugs are taken off the shelf and tried. Such was the case with studies of IFN-α and the nucleoside analogue lamivudine for HDV (2, 14). In the second stage, fundamental knowledge about the virus is applied, and drugs are used on the basis of such knowledge. With prenylation inhibitors, hepatitis D may enter the second stage of drug discovery, an excellent example of translational research. For the third phase, drugs are specifically designed and synthesized on the basis of the structure and life cycle of the virus. Delta hepatitis may be the least common form of viral hepatitis, but in many ways it is highly deserving of focused drug discovery.