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Structural Organization of Pregenomic RNA and the Carboxy-Terminal Domain of the Capsid Protein of Hepatitis B Virus

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PLoS Pathogens

Public Library of Science

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      Abstract

      The Hepatitis B Virus (HBV) double-stranded DNA genome is reverse transcribed from its RNA pregenome (pgRNA) within the virus core (or capsid). Phosphorylation of the arginine-rich carboxy-terminal domain (CTD) of the HBV capsid protein (Cp183) is essential for pgRNA encapsidation and reverse transcription. However, the structure of the CTD remains poorly defined. Here we report sub-nanometer resolution cryo-EM structures of in vitro assembled empty and pgRNA-filled Cp183 capsids in unphosphorylated and phosphorylation-mimic states. In empty capsids, we found unexpected evidence of surface accessible CTD density partially occluding pores in the capsid surface. We also observed that CTD organization changed substantively as a function of phosphorylation. In RNA-filled capsids, unphosphorylated CTDs favored thick ropes of RNA, while the phosphorylation-mimic favored a mesh of thin, high-density strands suggestive of single stranded RNA. These results demonstrate that the CTD can regulate nucleic acid structure, supporting the hypothesis that the HBV capsid has a functional role as a nucleic acid chaperone.

      Author Summary

      Many single stranded RNA virus encapsidate their genome through positively-charged domains of their capsid proteins. Hepatitis B virus (HBV) is a double stranded DNA virus which packages a single-stranded RNA pregenome (pgRNA) that is reverse transcribed within the capsid. RNA packaging requires a phosphorylated form of the HBV capsid protein's RNA-binding carboxy-terminal domain (CTD). Although the capsid has been well studied, the internal structures, the CTDs and the packaged RNA, are poorly characterized. By using in vitro reassembly, we have generated empty and pgRNA-filled capsids using phosphorylation-mimic and unphosphorylated forms of the capsid protein. Using cryo-EM image reconstruction, we have been able to show the structure of encapsidated pgRNA and, independently, the CTD in the absence of RNA to visualize early stages of the HBV assembly. We showed that the structural organization of the CTD changes as a function of the phosphorylation. Changes in CTD structure affect the structure of the encapsidated pgRNA, changing it from thin segments of density in the phosphorylated state, suggestive of single-stranded RNA, to thick rope-like structures consistent with duplex nucleic acid in the unphosphorylated state.

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      Most cited references 55

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          The lysine-rich H1 histone family in mammals includes eleven different subtypes, and thus it is the most divergent class of histone proteins. The central globular H1 domain asymmetrically interacts with DNA at the exit or entry end of the nucleosomal core DNA, and the C-terminal domain has a major impact on the linker DNA conformation and chromatin condensation. H1 histones are thus involved in the formation of higher order chromatin structures, and they modulate the accessibility of regulatory proteins, chromatin remodeling factors and histone modification enzymes to their target sites. The major posttranslational modification of H1 histones is phosphorylation, which reaches a peak during G2 and mitosis. Phosphorylation is, however, also involved in the control of DNA replication and it contributes to the regulation of gene expression. Disruption of linker histone genes, initially performed in order to delineate subtype-specific functions, revealed that disruption of one or two H1 subtype genes is quantitatively compensated by an increased expression of other subtypes. This suggests a functional redundancy among H1 subtypes. However, the inactivation of three subtypes and the reduction of the H1 moiety in half finally resulted in a phenotypic effect. On the other hand, studies on the role of particular subtypes at specific developmental stages in lower eukaryotes, but also in vertebrates suggest that specific subtypes of H1 participate in particular systems of gene regulation.
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            Author and article information

            Affiliations
            Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, United States of America
            Institut Pasteur, France
            Author notes

            The authors have declared that no competing interests exist.

            Conceived and designed the experiments: JCW AZ. Performed the experiments: JCW MSD. Analyzed the data: JCW AZ. Contributed reagents/materials/analysis tools: MSD. Wrote the paper: JCW AZ.

            Contributors
            Role: Editor
            Journal
            PLoS Pathog
            PLoS Pathog
            plos
            plospath
            PLoS Pathogens
            Public Library of Science (San Francisco, USA )
            1553-7366
            1553-7374
            September 2012
            September 2012
            20 September 2012
            : 8
            : 9
            3447754
            23028319
            PPATHOGENS-D-12-00785
            10.1371/journal.ppat.1002919
            (Editor)

            This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

            Counts
            Pages: 10
            Funding
            This work was supported by NIH R01-AI077688 to AZ. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
            Categories
            Research Article
            Biology
            Biochemistry
            Macromolecular Assemblies
            Microbiology
            Virology

            Infectious disease & Microbiology

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