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Journal of Virology, November 2004, p. 11751-11757, Vol. 78, No. 21
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.21.11751-11757.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Translation of Duck Hepatitis B Virus Reverse Transcriptase by Ribosomal Shunting

Nandini Sen,^1, Feng Cao,¹ and John E. Tavis^1,2*

Department of Molecular Microbiology and Immunology,¹ St. Louis University Liver Center, St. Louis University School of Medicine, St. Louis, Missouri²

Received 4 March 2004/ Accepted 24 June 2004

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.

Present address: Department of Medicine, SUNY Stony Brook, Stony Brook, NY 11794.

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