Blog
About

  • Record: found
  • Abstract: found
  • Article: found
Is Open Access

A VLP Library of C-Terminally Truncated Hepatitis B Core Proteins: Correlation of RNA Encapsidation with a Th1/Th2 Switch in the Immune Responses of Mice

Read this article at

Bookmark
      There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

      Abstract

      An efficient pBR327- and Ptrp-based E. coli expression system was used to generate a large-scale library of virus like particles (VLP) formed by recombinant hepatitis B virus (HBV) core (HBc) protein derivatives. To construct the library, the gene of HBc protein of the genotype D/subtype ayw2 virus was gradually truncated from the 3`-end and twenty-two HBc variants (with truncation up to 139 aa) were expressed at high levels. The proteins were purified by salt precipitation and gel filtration. Background RNA binding was observed for VLPs formed by HBc1-149, which lacked all C-terminal Arg blocks, and the addition of three Arg residues (HBc1-152) only slightly increased RNA binding. The presence of two Arg blocks (proteins HBc1-162 and HBc1-163) resulted in approximately half of the typical level of RNA binding, and the presence of three blocks (protein HBc1-171) led to approximately 85% of the typical level of binding. Only a small increase in the level of RNA binding was found for the HBc1-175 VLPs, which contained all four Arg blocks but lacked the last 8 aa of the full-length HBc protein. VLPs containing high levels of RNA had higher antigenicity according to an ELISA with anti-HBc mAbs than the VLPs formed by HBc variants without C-terminal Arg blocks and lacking RNA. The results indicate that the VLPs were stabilised by nucleic acids. The immunogenicity in BALB/c mice was comparable for VLPs formed by different HBc proteins, but a clear switch from a Th1 response to a Th2 response occurred after the loss of encapsidated RNA. We did not observe significant differences in lymphocyte proliferation in vitro for the tested VLP variants; however, the loss of RNA encapsidation correlated with a decreased level of IFN-γ induction, which is a measure of the potential CTL activity of immunogens.

      Related collections

      Most cited references 52

      • Record: found
      • Abstract: found
      • Article: not found

      The crystal structure of the human hepatitis B virus capsid.

      Hepatitis B is a small enveloped DNA virus that poses a major hazard to human health. The crystal structure of the T = 4 capsid has been solved at 3.3 A resolution, revealing a largely helical protein fold that is unusual for icosahedral viruses. The monomer fold is stabilized by a hydrophobic core that is highly conserved among human viral variants. Association of two amphipathic alpha-helical hairpins results in formation of a dimer with a four-helix bundle as the major central feature. The capsid is assembled from dimers via interactions involving a highly conserved region near the C terminus of the truncated protein used for crystallization. The major immunodominant region lies at the tips of the alpha-helical hairpins that form spikes on the capsid surface.
        Bookmark
        • Record: found
        • Abstract: found
        • Article: not found

        Three-dimensional structure of hepatitis B virus core particles determined by electron cryomicroscopy.

        Human hepatitis B virus core protein expressed in E. coli assembles into two sizes of particle. We have determined their three-dimensional structures by electron cryomicroscopy and image processing. The large and small particles correspond to triangulation number T = 4 and T = 3 dimer clustered packings, containing 240 and 180 protein subunits, respectively. The local packing of subunits is very similar in the two sizes of particle and shows holes or channels through the shell. The native viral core particle packages RNA and is active in reverse transcription to DNA. The holes we observe may provide access for the necessary small molecules. Shells assembled from the intact core protein contain additional material, probably RNA, which appears as an icosahedrally ordered inner shell in the three-dimensional map.
          Bookmark
          • Record: found
          • Abstract: found
          • Article: not found

          The arginine-rich domain of the hepatitis B virus core protein is required for pregenome encapsidation and productive viral positive-strand DNA synthesis but not for virus assembly.

          Assembly of replication-competent hepatitis B virus (HBV) nucleocapsids requires the interaction of the core protein, the P protein, and the RNA pregenome. The core protein contains an arginine-rich C-terminal domain which is dispensable for particle formation in heterologous expression systems. Using transient expression in HuH7 cells of a series of C-terminally truncated core proteins, I examined the functional role of this basic region in the context of a complete HBV genome. All variants containing at least the 144 N-terminal amino acids were assembly competent, but efficient pregenome encapsidation was observed only with variants consisting of 164 or more amino acids. These data indicate that one function of the arginine-rich region is to provide the interactions between core protein and RNA pregenome. However, in cores from the variant ending with amino acid 164, the production of complete positive-strand DNA was drastically reduced. Moreover, almost all positive-strand DNA originated from in situ priming, whereas in wild-type particles, this type of priming not supporting the formation of relaxed circular DNA (RC-DNA) accounted for about one half of the positive strands. Further C-terminal residues to position 173 restored RC-DNA formation, and the corresponding variant did not differ from the full-length core protein in all assays used. The observation that RNA encapsidation and formation of RC-DNA can be genetically separated suggests that the core protein, via its basic C-terminal region, also acts as an essential auxiliary component in HBV replication, possibly like a histone, or like a single-stranded-DNA-binding protein. In contrast to their importance for HBV replication, sequences beyond amino acid 164 were not required for the formation of enveloped virions. Since particles from variant 164 did not contain mature DNA genomes, a genome maturation signal is apparently not required for HBV nucleocapsid envelopment.
            Bookmark

            Author and article information

            Affiliations
            Protein Engineering Department, Latvian Biomedical Research and Study Centre, Riga, Latvia
            Institut National de la Santé et de la Recherche Médicale, France
            Author notes

            Competing Interests: The authors have declared that no competing interests exist.

            Conceived and designed the experiments: I. Sominskaya PP DS. Performed the experiments: I. Sominskaya DS IB MM IB IA I. Stahovska DD VO. Analyzed the data: IP IB JJ. Wrote the manuscript: I. Sominskaya IP AD PP.

            Contributors
            Role: Editor
            Journal
            PLoS One
            PLoS ONE
            plos
            plosone
            PLoS ONE
            Public Library of Science (San Francisco, USA )
            1932-6203
            2013
            23 September 2013
            : 8
            : 9
            24086668 3781094 PONE-D-13-17613 10.1371/journal.pone.0075938

            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.

            Funding
            This work was supported by grants ERAF Nr. (2010/0224/2DP/2.1.1.1.0/10/APIA/VIAA/164 and 2010/0211/2DP/2.1.1.1.0/10/APIA/VIAA/048.) The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
            Categories
            Research Article

            Uncategorized

            Comments

            Comment on this article

            Similar content 77

            Cited by 9

            Most referenced authors 306