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Journal of Virology, July 2006, p. 6478-6486, Vol. 80, No. 13
0022-538X/06/$08.00+0     doi:10.1128/JVI.02650-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

RNA-Templated Replication of Hepatitis Delta Virus: Genomic and Antigenomic RNAs Associate with Different Nuclear Bodies

Yi-Jia Li,1 Thomas Macnaughton,1 Lu Gao,2 and Michael M. C. Lai1,3*

Department of Molecular Microbiology and Immunology, University of Southern California, Keck School of Medicine, Los Angeles, California,1 Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, California,2 Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan3

Received 19 December 2005/ Accepted 17 February 2006

Lacking an RNA-dependent RNA polymerase, hepatitis delta virus (HDV), which contains a circular RNA of 1.7 kilobases, is nonetheless able to replicate its RNA by use of cellular transcription machineries. Previously, we have shown that the replications of genomic- and antigenomic-strand HDV RNAs have different sensitivities to {alpha}-amanitin, suggesting that these two strands are synthesized in different transcription machineries in the cells, but the nature of these transcription machineries is not clear. In this study, we performed metabolic labeling and immunofluorescence staining of newly synthesized HDV RNA with bromouridine after HDV RNA transfection into hepatocytes and confirmed that HDV RNA synthesis had both {alpha}-amanitin-sensitive and -resistant components. The antigenomic RNA labeling was {alpha}-amanitin resistant and localized to the nucleolus. The genomic RNA labeling was {alpha}-amanitin sensitive and more diffusely localized in the nucleoplasm. Most of the genomic RNA labeling appeared to colocalize with the PML nuclear bodies. Furthermore, promyelocytic leukemia protein, RNA polymerase II (Pol II), and the Pol I-associated transcription factor SL1 could be precipitated together with hepatitis delta antigen, suggesting the association of HDV replication complex with the Pol I and Pol II transcription machineries. This conclusion was further confirmed by an in vitro replication assay. These findings provide additional evidence that HDV RNA synthesis occurs in the Pol I and Pol II transcription machineries, thus extending the capability of the cellular DNA-dependent RNA polymerases to utilizing RNA as templates.

* Corresponding author. Mailing address: Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, 2011 Zonal Ave., Los Angeles, CA 90033-1054. Phone: (323) 442-1748. Fax: (323) 442-1721. E-mail: michlai{at}usc.edu.

Journal of Virology, July 2006, p. 6478-6486, Vol. 80, No. 13
0022-538X/06/$08.00+0     doi:10.1128/JVI.02650-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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