Translation of duck hepatitis B virus reverse transcriptase by ribosomal shunting.
The duck hepatitis B virus (DHBV) polymerase (P) is translated by de novo initiation from a downstream open reading frame (ORF) that partially overlaps the core (C) ORF on the bicistronic pregenomic RNA (pgRNA). The DHBV P AUG is in a poor context for translational initiation and is preceded by 14 AUGs that could intercept scanning ribosomes, yet P translation is unanticipatedly rapid. Therefore, we assessed C and P translation in the context of the pgRNA. Mutating the upstream C ORF revealed that P translation was inversely related to C translation, primarily due to occlusion of P translation by ribosomes translating C. Translation of the pgRNA was found to be cap dependent, because inserting a stem-loop (BamHI-SL) that blocked >90% of scanning ribosomes at the 5' end of the pgRNA greatly inhibited C and P synthesis. Neither mutating AUGs between the C and P start sites in contexts similar to that of the P AUG nor blocking ribosomal scanning by inserting the BamHI-SL between the C and P start codons greatly altered P translation, indicating that most ribosomes that translate P do not scan through these sequences. Finally, optimizing the P AUG context did not increase P translation. Therefore, the majority of the ribosomes that translate P are shunted from a donor region near the 5' end of the pgRNA to an acceptor site at or near the P AUG, and the shunt acceptor sequences may augment initiation at the P AUG.
Department of Molecular Microbiology and Immunology, St. Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104, USA.
Nandini
N
Sen
Feng
F
Cao
John E
JE
Tavis
D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D015611:Hepatitis B Virus, Duck / Q000201:enzymology*; D016366:Open Reading Frames; D014176:Protein Biosynthesis; D012194:RNA-Directed DNA Polymerase / Q000235:genetics*; D012270:Ribosomes / Q000378:metabolism*
D012194:RNA-Directed DNA Polymerase; D012321:DNA-Directed RNA Polymerases