In vivo expression of a new hepatitis

Hepatitis B virus (HBV) is a small, enveloped DNA virus that causes acute and chronic liver infections (1). The viral genome contains 4 overlapping open reading frames encoding for capsid, surface, polymerase, and X proteins (1). Three major unspliced transcripts, of 3.5 kb, 2.4 kb, and 2.1 kb, and a less abundant 0.8-kb transcript with a common polyadenylation site, all code for viral proteins identified to date. The 3.5-kb pregenomic RNA also serves as a template for viral replication through a reverse transcription mechanism. In addition, spliced HBV transcripts have been identified in human liver tissues and in HBV-transfected hepatoma cell lines (2–8). Two major HBV-spliced forms of the pregenomic RNA, each with a size of 2.2 kb, have been found in vitro to represent about 30% of the pregenomic RNAs (5). Spliced RNAs have also been detected in ducks and woodchucks infected with duck hepatitis B virus or woodchuck hepatitis virus, respectively. However, the splicing pattern differed among these hepadnaviruses (9–11). We and others have detected defective HBV particles containing a 2.2-kb subgenomic HBV DNA, complementary to a singly spliced HBV RNA, in most livers and sera from the chronically infected patients tested (4, 12, 13). These defective HBV particles are secreted through transcomplementation with a helper wild-type virus, which allows for the packaging and reverse transcription of the singly spliced RNA (4). This raises the possibility that these defective HBV particles and their expressed proteins may be implicated in viral persistence. The singly spliced RNA had previously been shown to encode for capsid and X proteins (12). In addition, it contains a novel open reading frame overlapping the splice junction, which potentially encodes for an HBV splice-generated protein (HBSP). This protein, with 93 amino acids, begins at the polymerase start codon and includes the first 46 amino acids of the polymerase, followed by the 47 amino acids corresponding to a new HBV amino acid sequence generated by the frameshift (Figure 1a). In this report we demonstrate the in vivo expression of this new protein.