Selection of different 5¢ untranslated region hepatitis C virus variants during post-transfusion and post-transplantation infection J. F. Gallegos-Orozco, 1,* J. I. Arenas, 1,* H. E. Vargas, 1 K. V. Kibler, 1 J. K. Wilkinson, 1,4 M. Nowicki, 2 M. Radkowski, 3 J. Nasseri, 4 J. Rakela 1 and T. Laskus 1,4 1 Division of Transplantation Medicine, Mayo Clinic, Scottsdale, AZ; 2 Maternal-Child Virology Research Laboratory, University of Southern California, Los Angeles, CA, USA; 3 Medical University of Warsaw, Warsaw, Poland; and 4 St Joseph’s Hospital and Medical Center, Phoenix, AZ, USA Received May 2005; accepted for publication September 2005 SUMMARY. BACKGROUND: Hepatitis C virus (HCV) translation is initiated in a cap-independent mannerby an internal ribosome entry site (IRES) located within the 5¢ untranslated region (5¢UTR). Sequence changes in this region could affect translationefficiencyand presumablyviral replication. AIM: To determine translation efficiency of 5¢UTR variants developingduringpost-transfusionhepatitisC in two immunocompetent subjects and in two immunosuppressed liver recipientswith recurrentHCV. methods: Sequential samples were screened for 5¢UTR changes by single-strand conformationpolymorphism followedby cloning and sequencingwheneverband pattern suggested sequence changes. 5¢UTR variants were tested for IRES activity using a bicistronic dual luciferase expression plasmid transfected into HepG2 and Huh7 cell-lines. results:In the transfused patients, translation efficiency of 5¢UTR variants from early post-transfusion samples was 5.1- to 13.7-fold higher than that of predominant variants found in late follow-up sam- ples.Post-transplant variants in the other two patients had 2.6- to 5.9-fold highertranslation efficiency than those presentonly in pretransplant samples. conclusion:In the immunocompetent host there may be selection oflow translation efficiency HCV variants over the courseof infection.However,in immunosuppressed subjectsthe opposite seems to be true as low translation efficiency vari- ants are superseded by high translation efficiency variants. Keywords: internal ribosomal entry site, reverse transcriptase polymerasechain reaction,single-strandconformation polymorphism, translation efficiency, viral hepatitis. INTRODUCTION HepatitisC virus (HCV) genome translation starts at an internal ribosomal entry site (IRES) element, which comprises most of the 5¢ untranslated region (5¢UTR) and extends 12– 30 nucleotides downstream of the initiation codon [1,2]. The importance of the 5¢UTR in viral replication and translation is reflected by the fact that it is the most conserved region in the HCV genome, with a >85% homology between different viral strains [3,4]. Although IRES elements from different HCV genotypes and strains manifest differing translation efficiency in vitro [5–7] it is unclear at present whether these variations affect clinical outcome [8]. In the currentstudy we describe changes in the 5¢UTR developing during the natural course of transfusion-acquired hepatitis C, as wellas in recurrent HCV infection after liver transplantation, and provideevidencethat they affect translational efficiency of viral IRES in vitro. The selection of 5¢UTR variants seemed to follow two opposite patterns: in transfusion-acquired infectionlow translation efficiency variants prevailed over the high replicating variants, while in immunosuppressed liver transplant recipients, low transla- tion efficiency variants were supplanted by high-translation efficiency variants. Switch between high and low translation efficiency variants could represent a novel mechanism aimed at adjusting viral replication to current host conditions and may be affected by the host immune status. *These authors contributed equally to the present work. Abbreviations: 5¢UTR, 5¢ untranslated region; ALT, alanine ami- notransferase; HCV, hepatitis C virus; IRES, internal ribosome entry site; RT-PCR, reverse transcriptase polymerase chain reaction; SSCP, single-strand conformation polymorphism. Correspondence: Juan F. Gallegos-Orozco MD, Mayo Clinic, 13400 E. Shea Blvd., SCJRB 3rd floor, Scottsdale, AZ 85259, USA. E-mail: gallegos.juan@mayo.edu Journal of Viral Hepatitis, 2006, 13, 489–498 doi:10.1111/j.1365-2893.2006.00724.x 2006 The Authors Journal compilation 2006 Blackwell Publishing Ltd.