Activation of Heterologous Gene Expression by the Large Isoform of Hepatitis Delta Antigen

Hepatitis delta virus (HDV) encodes two isoforms of its principal gene product, hepatitis delta antigen (HDAg). These two forms play distinctive and complementary roles in viral replication. Here we report that the large (LHDAg), but not the small (SHDAg), isoform of HDAg has the capacity to activate the expression of cotransfected genes driven by a variety of promoters, including the pre-S, S, and C promoters of hepatitis B virus. Mutational analysis of the C-terminal 19 amino acids unique to LHDAg shows that changing prolines to alanines in the two PXXP motifs in this region specifically ablates the activation function without abolishing another activity of LHDAg, namely, its ability to inhibit HDV RNA synthesis. However, C-terminal truncations that also disrupt these PXXP motifs only slightly diminished the activation function, indicating that the proline mutations were not acting by inactivating potential SH3 interactions that could be mediated by these motifs. Mutation of the isoprenylated cysteine to serine decreases but does not abolish the activation activity, and overexpression of SHDAg does not interfere with the transactivation function of LHDAg. Although the mechanism and biological significance of this activity of LHDAg remain unknown, the presence of this activity serves as yet another marker that functionally distinguishes this protein from the closely related isoform SHDAg.Hepatitis delta virus (HDV) is an RNA virus that requires coinfection with hepatitis B virus (HBV) to complete its life cycle. The helper function supplied by HBV is limited to the provision of envelope proteins (hepatitis B surface antigens) for the completion of HDV assembly (28, 29, 31). HDV RNA replication is independent of its HBV helper (19). In fact, the presence of HDV suppresses HBV replication in vivo (30, 39). Nonetheless, clinical studies have shown that HDV infection can be associated with more severe hepatitis than HBV alone and is often implicated in cases of fulminant hepatitis (4, 32).The genome of HDV is a circular, single-stranded RNA of about 1,700 nucleotides (nt), of which approximately 70% are self-complementary (for a review, see references 20 and 21). This self-complemetarity allows the genome to form an unbranched rod-like structure. A unique functional protein, hepatitis delta antigen (HDAg), is encoded by the genome (3, 38), and two isoforms of this protein are produced during infection. The canonical small form of HDAg (SHDAg) is 195 amino acids (aa) long; it harbors an N-terminal coiled-coil domain responsible for oligomerization (37), a central domain responsible for binding to the RNA genome (7, 23), a nuclear localization signal (2, 7), and a C-terminal glycine- and proline-rich region with an uncertain function. This form of HDAg is essential for viral RNA replication, although it is not itself a polymerase. Host RNA polymerase II is thought to supply the polymerase function for replication (15, 26). During viral replication, an RNA editing event occurs at the UAG termination codon of SHDAg, allowing readthrough of another 19 aa (Fig. ​(Fig.1)1) to generate the large isoform of the protein, LHDAg (25). Since LHDAg contains all of the domains of SHDAg, it too can form multimers with itself and with the SHDAg isoform, bind HDV RNA (as a homo- or heteromultimer), and be localized to the nucleus. Open in a separate windowFIG. 1Sequence of the 19 aa unique to the C terminus of LHDAg. The PXXP motifs are underlined. Below are shown the amino acid changes present in the mutants employed in this study. The positions of the termination codons introduced into the truncation mutants are indicated by asterisks.Despite these similarities, the two HDAgs have very distinct functions (22) and play complementary roles in HDV replication, which takes place largely in the nuclei of infected cells (34). While SHDAg activates HDV RNA replication, LHDAg is a trans-dominant inhibitor of this process (8). By contrast, LHDAg, but not SHDAg, is capable of interacting with the HBV envelope proteins to mediate envelopment of the HDV ribonucleoprotein in viral assembly (6). This interaction has been shown to require farnesylation of a cysteine residue found in the C-terminal 19 aa unique to LHDAg (27, 16). Furthermore, it has been shown recently that only LHDAg is phosphorylated in cells (1).In this report, we describe yet another activity of LHDAg that further differentiates it from the related isoform SHDAg, i.e., the ability to activate gene expression in trans.