A new hepatitis B virus (HBV) transcript of about 2.2 kilobases was identified in HBV DNA-transfected human hepatoma cells. The 5' terminus of this viral RNA appears to map at one or more of the precore initiation sites, contains a deletion of 1,223 bases corresponding to the last codon of the core gene to the middle of the surface antigen gene, and terminates at the 3' polyadenylation site used by the other known HBV RNAs. The junction region of the deleted sequences showed the conserved splice donor and acceptor GT-AG sequences. Moreover, when a mutant HBV DNA in which the splice acceptor site was changed from AG to CG was transfected into human hepatoma cells, no 2.2-kilobase RNA was detected, further suggesting that this RNA represents a spliced transcript. The core gene, although an amino acid shorter, still encoded a functional viral core protein in complementation experiments. Sequence analysis of the cDNA of the 2.2-kilobase RNA suggests that this transcript can potentially encode a new protein that comprises the reverse transcriptase domain of HBV. However, genetic analysis using a transient DNA transfection system suggests that the gene product(s) of this transcript is not essential for viral replication. The function of this transcript remains to be studied.
The hepatitis B virus transcripts in human hepatoma and its adjacent nontumorous liver were examined with probes specific to hepatitis B virus surface antigen, core antigen, X region and pre-S region. The study shows that the patterns of hepatitis B virus transcripts for tumorous tissue and the counterpart in nontumorous liver differ. In most of the tissues examined, the surface antigen gene is transcribed. Most of these transcripts, besides having surface antigen sequences, also have an X region; some also include a pre-S region. The transcripts that hybridized to a core-specific probe were a pair of poly(A+) RNA, 3.5 and 2.2 kilobases in size, present in two of the nontumorous hepatocytes where the virus was actively replicating. The 3.5-kilobase transcript not only hybridized to the core probe, but was able to be hybridized to other hepatitis B virus subgenomic probes and might represent the RNA pregenome involving hepatitis B virus DNA replication. Whereas most of the transcripts hybridizable to hepatitis B virus probe are in the size range of 2.1 to 2.7 kilobases, some transcripts other than the pregenomic RNA appear to be greater than 3.2 kilobases in size and may represent the hybrid RNAs of viral and host sequences.
Hepatitis B virus (HBV) polymerase (P) gene is translated from the bicistronic pregenomic RNA with the core (C) gene in the first cistron. The P ORF is preceded by the C AUG and three AUG codons within the C region, where a minicistron of 7 amino acids can potentially be translated. Our results indicate that the efficiency of the P gene translation initiation was about 10 % of that of the C gene when both genes were fused in-frame to a lacZ reporter in an mRNA similar in structure to the pregenomic RNA. By mutational analysis, about 74 % of the translation initiation of HBV P gene was shown to be by ribosomes that reinitiated after terminating translation of this minicistron, while the rest was by two mechanisms : one by ribosomes leaky scanning through every upstream AUG and the other by
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