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Journal of Virology, February 2008, p. 1185-1194, Vol. 82, No. 3
0022-538X/08/$08.00+0     doi:10.1128/JVI.01219-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Mutations within Potential Glycosylation Sites in the Capsid Protein of Hepatitis E Virus Prevent the Formation of Infectious Virus Particles

Judith Graff,¹ Yi-Hua Zhou,¹ Udana Torian,¹ Hanh Nguyen,¹ Marisa St. Claire,³ Claro Yu,² Robert H. Purcell,² and Suzanne U. Emerson^1*

Molecular Hepatitis,¹ Hepatitis Viruses Sections, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892,² Bioqual, Incorporated, 2501 Research Boulevard, Rockville, Maryland 20850³

Received 4 June 2007/ Accepted 11 November 2007

Hepatitis E virus is a nonenveloped RNA virus. However, the single capsid protein resembles a typical glycoprotein in that it contains a signal sequence and potential glycosylation sites that are utilized when recombinant capsid protein is overexpressed in cell culture. In order to determine whether these unexpected observations were biologically relevant or were artifacts of overexpression, we analyzed capsid protein produced during a normal viral replication cycle. In vitro transcripts from an infectious cDNA clone mutated to eliminate potential glycosylation sites were transfected into cultured Huh-7 cells and into the livers of rhesus macaques. The mutations did not detectably affect genome replication or capsid protein synthesis in cell culture. However, none of the mutants infected rhesus macaques. Velocity sedimentation analyses of transfected cell lysates revealed that mutation of the first two glycosylation sites prevented virion assembly, whereas mutation of the third site permitted particle formation and RNA encapsidation, but the particles were not infectious. However, conservative mutations that did not destroy glycosylation motifs also prevented infection. Overall, the data suggested that the mutations were lethal because they perturbed protein structure rather than because they eliminated glycosylation.

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* Corresponding author. Mailing address: Molecular Hepatitis Section, LID, NIAID, National Institutes of Health, Room 6537, Building 50, 50 South Drive, MSC 8009, Bethesda, MD 20892-8009. Phone: (301) 496-2787. Fax: (301) 402-0524. E-mail: semerson{at}niaid.nih.gov

Published ahead of print on 21 November 2007.

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Journal of Virology, February 2008, p. 1185-1194, Vol. 82, No. 3
0022-538X/08/$08.00+0     doi:10.1128/JVI.01219-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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