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Journal of Virology, February 2005, p. 1871-1887, Vol. 79, No. 3
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.3.1871-1887.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Exposure of RNA Templates and Encapsidation of Spliced Viral RNA Are Influenced by the Arginine-Rich Domain of Human Hepatitis B Virus Core Antigen (HBcAg 165-173)

Sophie Le Pogam,{dagger} Pong Kian Chua, Margaret Newman, and Chiaho Shih*

Department of Pathology and Department of Microbiology and Immunology, WHO Collaborating Center for Tropical Diseases, and Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, Texas

Received 8 July 2004/ Accepted 1 October 2004

Previously, human hepatitis B virus (HBV) mutant 164, which has a truncation at the C terminus of the HBV core antigen (HBcAg), was speculated to secrete immature genomes. For this study, we further characterized mutant 164 by different approaches. In addition to the 3.5-kb pregenomic RNA (pgRNA), the mutant preferentially encapsidated the 2.2-kb or shorter species of spliced RNA, which can be reverse transcribed into double-stranded DNA before virion secretion. We observed that mutant 164 produced less 2.2-kb spliced RNA than the wild type. Furthermore, it appeared to produce at least two different populations of capsids: one encapsidated a nuclease-sensitive 3.5-kb pgRNA while the other encapsidated a nuclease-resistant 2.2-kb spliced RNA. In contrast, the wild-type core-associated RNA appeared to be resistant to nuclease. When arginines and serines were systematically restored at the truncated C terminus, the core-associated DNA and nuclease-resistant RNA gradually increased in both size and signal intensity. Full protection of encapsidated pgRNA from nuclease was observed for HBcAg 1-171. A full-length positive-strand DNA phenotype requires positive charges at amino acids 172 and 173. Phosphorylation at serine 170 is required for optimal RNA encapsidation and a full-length positive-strand DNA phenotype. RNAs encapsidated in Escherichia coli by capsids of HBcAg 154, 164, and 167, but not HBcAg 183, exhibited nuclease sensitivity; however, capsid instability after nuclease treatment was observed only for HBcAg 164 and 167. A new hypothesis is proposed here to highlight the importance of a balanced charge density for capsid stability and intracapsid anchoring of RNA templates.

* Corresponding author. Mailing address: Department of Pathology and Department of Microbiology and Immunology, WHO Collaborating Center for Tropical Diseases, and Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, TX 77555-0609. Phone: (409) 772-2563. Fax: (409) 747-2429. E-mail: cshih{at}utmb.edu.

{dagger} Present address: Roche Palo Alto, Palo Alto, CA 94304.

Journal of Virology, February 2005, p. 1871-1887, Vol. 79, No. 3
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.3.1871-1887.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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