Heterogeneity and Common Features of Defective Hepatitis B Virus Genomes Derived from Spliced Pregenomic RNA

Abstract

Defective hepatitis B virus (HBV) genomes derived from packaging and reverse transcription of spliced RNA pregenomes were reported to be associated with progression to chronic infection. Since only two types with similarly spliced regions were characterized so far we reasoned that additional "spliced" genome variants may exist. Therefore, we isolated a large number of defective HBV genomes from sera of seven chronic carriers by full-length PCR. Forty-eight were found to contain deletions caused by splicing as identified by cloning, subgenomic PCR, and sequencing. In total, 11 types of spliced genomes derived from excision of 10 different introns were present in various combinations in each serum. This diversity resulted from alternative usage of five splice donor and four acceptor sites present in most but not all HBV genotypes. All spliced genomes shared sequence elements essential for replication as well as for transcription of the pre-C and pregenome/C mRNAs and the X mRNA. Moreover, all contained the coding regions for the X protein and for precore/core or precore/ core fusion proteins but lacked the pre-S/S gene promoters. These data demonstrate substantial and HBV genotype-dependent diversity of spliced genomes from which a variety of aberrant precore/core fusion proteins and normal X protein but no functional envelope and P proteins could be expressed. These genomes and the encoded proteins may play a role in the viral life cycle, persistence, and pathogenesis.

Hepatitis B Virus Nucleocapsids Formed by Carboxy-Terminally Mutated Core Proteins Contain Spliced Viral Genomes but Lack Full-Size DNA

“…Additional low-molecular-weight DNA species were detected in all cases, most likely representing minor splice variants as described before (9,23).…”

“…Specific encapsidation of pgRNA occurs via binding of P protein to a 5Ј-proximal RNA stem-loop structure, termed ε. Subgenomic RNAs lack the 5Ј ε and are excluded from encapsidation. Notably, however, spliced RNA species containing all sequence elements necessary for packaging and for reverse transcription have been observed in liver tissue and in transfected cells (9,23,(26)(27)(28)(29). Viral DNA synthesis is a highly complex process involving three translocation events of P to priming sites located at the respective 5Ј and 3Ј ends of the template (17).…”

Hepatitis B virus-related hepatocellular carcinoma: paradigms for viral-related human carcinogenesis

“…However, the role of these spliced RNAs in viral replication has not yet been established. A previous study showed that these spliced RNA might be encapsidated and retrotranscribed and give rise to defective particles (Terre´et al, 1991;Gu¨nther et al, 1997). We showed that, in vivo, at least one of these defective particles was detected in the sera of patients with chronic hepatitis or acute hepatitis developing towards chronicity, but not in patients who had recovered from their acute hepatitis (Rosmorduc et al, 1995) (Figure 5).…”

“…Defective viral particles and hepatisis B virus spliced protein Data in the literature have shown that HBV pregenomic RNA, the matrix for viral replication, may undergo simple or multiple splicing (Gu¨nther et al, 1997). These RNAs are differentiated by the site of splicing and may represent up to 30% of total HBV pregenomic RNA.…”

Doubly Spliced RNA of Hepatitis B Virus Suppresses Viral Transcription via TATA-Binding Protein and Induces Stress Granule Assembly

“…In addition, numerous spliced mRNA variants derived from the 3.5-kb precore mRNA and pgRNA have been identified in HBV-transfected hepatoma cells, HBV transgenic mice, and the liver tissues and virions isolated from serum of chronic hepatitis B patients (15)(16)(17)(18)(19). Recent studies have shown that the expression of proteins encoded by certain HBV spliced transcripts is associated with viral replication, liver fibrosis, and immune surveillance (20)(21)(22).…”