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Journal of Virology, March 2007, p. 3018-3026, Vol. 81, No. 6
0022-538X/07/$08.00+0     doi:10.1128/JVI.02259-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Initiation at the Third In-Frame AUG Codon of Open Reading Frame 3 of the Hepatitis E Virus Is Essential for Viral Infectivity In Vivo

Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24601,¹ Department of Veterinary Diagnostic and Productive Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa 50011²

Received 15 October 2006/ Accepted 21 December 2006

To determine the initiation strategy of the hepatitis E virus (HEV) open reading frame 3 (ORF3), we constructed five HEV mutants with desired mutations in the ORF1 and ORF2 junction region and tested their levels of in vivo infectivity in pigs. A mutant with a C-terminally truncated ORF3 is noninfectious in pigs, indicating that an intact ORF3 is required for in vivo infectivity. Mutations with substitutions in the first in-frame AUG in the junction region or with the same T insertion at the corresponding position of HEV genotype 4 did not affect the virus infectivity or rescue, although mutations with combinations of the two affected virus recovery efficiency, and a single mutation at the third in-frame AUG completely abolished virus infectivity in vivo, indicating that the third in-frame AUG in the junction region is required for virus infection and is likely the authentic initiation site for ORF3. A conserved double stem-loop RNA structure, which may be important for HEV replication, was identified in the junction region. This represents the first report of using a unique homologous pig model system to study the molecular mechanism of HEV replication and to systematically and definitively identify the authentic ORF3 initiation site.

Published ahead of print on 3 January 2007.

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