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Journal of Virology, May 2006, p. 4336-4343, Vol. 80, No. 9
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.9.4336-4343.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Repair and Polyadenylation of a Naturally Occurring Hepatitis C Virus 3' Nontranslated Region-Shorter Variant in Selectable Replicon Cell Lines

Hans C. van Leeuwen,* Jolanda M. P. Liefhebber, and Willy J. M. Spaan

Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, 2300 RC Leiden, The Netherlands

Received 19 December 2005/ Accepted 1 February 2006

The 3' nontranslated region (NTR) of the hepatitis C virus (HCV) genome is highly conserved and contains specific cis-acting RNA motifs that are essential in directing the viral replication machinery to initiate at the correct 3' end of the viral genome. Since the ends of viral genomes may be damaged by cellular RNases, preventing the initiation of viral RNA replication, stable RNA hairpin structures in the 3' NTR may also be essential in host defense against exoribonucleases. During 3'-terminal sequence analysis of serum samples of a patient with chronic hepatitis related to an HCV1b infection, a number of clones were obtained that were several nucleotides shorter at the extreme 3' end of the genome. These shorter 3' ends were engineered in selectable HCV replicons in order to enable the study of RNA replication in cell culture. When in vitro-transcribed subgenomic RNAs, containing shorter 3' ends, were introduced into Huh-7 cells, a few selectable colonies were obtained, and the 3' terminus of these subgenomic RNAs was sequenced. Interestingly, most genomes recovered from these colonies had regained the wild-type 3' ends, showing that HCV, like several other positive-stranded RNA viruses, has developed a strategy to repair deleted 3' end nucleotides. Furthermore, we found several genomes in these replicon colonies that contained a poly(A) tail and a short linker sequence preceding the poly(A) tail. After recloning and subsequent passage in Huh-7 cells, these poly(A) tails persisted and varied in length. In addition, the connecting linker became highly diverse in sequence and length, suggesting that these tails are actively replicated. The possible terminal repair mechanisms, including roles for the poly(A) tail addition, are discussed.

* Corresponding author. Mailing address: Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Albinusdreef 2, 2300 RC Leiden, The Netherlands. Phone: 31-71-5263649. Fax: 31-71-5266761. E-mail: H.C.van_Leeuwen{at}lumc.nl.

Journal of Virology, May 2006, p. 4336-4343, Vol. 80, No. 9
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.9.4336-4343.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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