| Summary: | 碩士 === 國立陽明大學 === 微生物暨免疫學研究所 === 91 === The hepatitis B virus (HBV) 3.5-kb pregenomic RNA serves as template for both viral DNA replication and viral core (C) and polymerase (P) protein synthesis. Translation initiation of the P ORF which resided downstream of the overlapping out-of-frame C ORF has been shown to depend mainly on a mechanism of termination-reinitiation of an upstream minicistron, in addition to two minor mechanisms: one by ribosomes leaky scanning through upstream AUGs and another by ribosomal backwards scanning after finishing the translation of the C ORF.
In HBV infected or transfected liver cells, a 2.2-kb singly-spliced RNA, a major splicing transcripts of HBV, is generated from the 3.5-kb RNA. There are three open reading frames (ORFs) in this RNA: a C ORF encoding core protein with an amino acid shorter at the C-terminus, a P10-ORF encoding a truncated terminal protein and a P42 ORF encoding protein comprising the RNase H domain and partial reverse transcriptase domain of the P protein. Translation of these ORFs presumably also depends on leaky scanning mechanism. However, in contrast to that in the bicistronic 3.5-kb RNA, the scanning ribosomes have to face two downstream P10 and P42 ORFs in the 2.2-kb RNA. In this study, a genetic approach using lacZ as a reporter was taken to study translation regulation in the tricistronic 2.2-kb RNA. Our results show that translation initiation of the P10 ORF mainly utilizes the termination-reinitiation mechanism as that of the P ORF. Nevertheless, ribosomal backwards scanning that contributes about 18% of the P translation is not utilized in the P10 expression; instead, such ribosomes are employed in the initiation of the P42 ORF. Our results also show that efficiencies of translation initiation of the P10 and P42 ORFs are similar. Furthermore, if initiation codon of P10 ORF was changed to an optimal context, initiation of the P42 ORF drops to about 20% of the initial level. This suggests that initiation of the P42 ORF depends mainly on ribosomes bypassing P10 initiation codon because of its poor context.
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