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Journal of Virology, April 2002, p. 3920-3927, Vol. 76, No. 8
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.8.3920-3927.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Rolling Circle Replication of Hepatitis Delta Virus RNA Is Carried Out by Two Different Cellular RNA Polymerases

Thomas B. Macnaughton,1 Stephanie T. Shi,1 Lucy E. Modahl,1 and Michael M. C. Lai1,2*

Department of Molecular Microbiology and Immunology,1 Howard Hughes Medical Institute, Keck School of Medicine, University of Southern California, Los Angeles, California 90033-10542

Received 2 October 2001/ Accepted 4 January 2002

Hepatitis delta virus (HDV) contains a viroid-like circular RNA that is presumed to replicate via a rolling circle replication mechanism mediated by cellular RNA polymerases. However, the exact mechanism of rolling circle replication for HDV RNA and viroids is not clear. Using our recently described cDNA-free transfection system (L. E. Modahl and M. M. Lai, J. Virol. 72:5449-5456, 1998), we have succeeded in detecting HDV RNA replication by metabolic labeling with [32P]orthophosphate in vivo and obtained direct evidence that HDV RNA replication generates high-molecular-weight multimeric species of HDV RNA, which are processed into monomeric and dimeric forms. Thus, these multimeric RNAs are the true intermediates of HDV RNA replication. We also found that HDV RNA synthesis is highly temperature sensitive, occurring most efficiently at 37 to 40°C and becoming virtually undetectable at temperatures below 30°C. Moreover, genomic HDV RNA synthesis was found to occur at a rate roughly 30-fold higher than that of antigenomic RNA synthesis. Finally, in lysolecithin-permeabilized cells, the synthesis of full-length antigenomic HDV RNA was completely resistant to high concentrations (100 µg/ml) of {alpha}-amanitin. In contrast, synthesis of genomic HDV RNA was totally inhibited by {alpha}-amanitin at concentrations as low as 2.5 µg/ml. Thus, these results suggest that genomic and antigenomic HDV RNA syntheses are performed by two different host cell enzymes. This observation, combined with our previous finding that hepatitis delta antigen mRNA synthesis is likely performed by RNA polymerase II, suggests that the different HDV RNA species are synthesized by different cellular transcriptional machineries.

* Corresponding author. Mailing address: Department of Molecular Microbiology and Immunology, University of Southern California, 2011 Zonal Ave., Los Angeles, CA 90033-1054. Phone: (323) 442-1748. Fax: (323) 342-9555. E-mail: michlai{at}hsc.usc.edu.

Journal of Virology, April 2002, p. 3920-3927, Vol. 76, No. 8
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.8.3920-3927.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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