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      A SELEX-Screened Aptamer of Human Hepatitis B Virus RNA Encapsidation Signal Suppresses Viral Replication

      research-article
      1 , 2 , 2 , 1 , *
      PLoS ONE
      Public Library of Science

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          Abstract

          Background

          The specific interaction between hepatitis B virus (HBV) polymerase (P protein) and the ε RNA stem-loop on pregenomic (pg) RNA is crucial for viral replication. It triggers both pgRNA packaging and reverse transcription and thus represents an attractive antiviral target. RNA decoys mimicking ε in P protein binding but not supporting replication might represent novel HBV inhibitors. However, because generation of recombinant enzymatically active HBV polymerase is notoriously difficult, such decoys have as yet not been identified.

          Methodology/Principal Findings

          Here we used a SELEX approach, based on a new in vitro reconstitution system exploiting a recombinant truncated HBV P protein (miniP), to identify potential ε decoys in two large ε RNA pools with randomized upper stem. Selection of strongly P protein binding RNAs correlated with an unexpected strong enrichment of A residues. Two aptamers, S6 and S9, displayed particularly high affinity and specificity for miniP in vitro, yet did not support viral replication when part of a complete HBV genome. Introducing S9 RNA into transiently HBV producing HepG2 cells strongly suppressed pgRNA packaging and DNA synthesis, indicating the S9 RNA can indeed act as an ε decoy that competitively inhibits P protein binding to the authentic ε signal on pgRNA.

          Conclusions/Significance

          This study demonstrates the first successful identification of human HBV ε aptamers by an in vitro SELEX approach. Effective suppression of HBV replication by the S9 aptamer provides proof-of-principle for the ability of ε decoy RNAs to interfere with viral P-ε complex formation and suggests that S9-like RNAs may further be developed into useful therapeutics against chronic hepatitis B.

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          Most cited references45

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          Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.

          L Gold, C Tuerk (1990)
          High-affinity nucleic acid ligands for a protein were isolated by a procedure that depends on alternate cycles of ligand selection from pools of variant sequences and amplification of the bound species. Multiple rounds exponentially enrich the population for the highest affinity species that can be clonally isolated and characterized. In particular one eight-base region of an RNA that interacts with the T4 DNA polymerase was chosen and randomized. Two different sequences were selected by this procedure from the calculated pool of 65,536 species. One is the wild-type sequence found in the bacteriophage mRNA; one is varied from wild type at four positions. The binding constants of these two RNA's to T4 DNA polymerase are equivalent. These protocols with minimal modification can yield high-affinity ligands for any protein that binds nucleic acids as part of its function; high-affinity ligands could conceivably be developed for any target molecule.
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            Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused.

            Although it is usually possible to achieve a favorable yield of a recombinant protein in Escherichia coli, obtaining the protein in a soluble, biologically active form continues to be a major challenge. Sometimes this problem can be overcome by fusing an aggregation-prone polypeptide to a highly soluble partner. To study this phenomenon in greater detail, we compared the ability of three soluble fusion partners--maltose-binding protein (MBP), glutathione S-transferase (GST), and thioredoxin (TRX)--to inhibit the aggregation of six diverse proteins that normally accumulate in an insoluble form. Remarkably, we found that MBP is a far more effective solubilizing agent than the other two fusion partners. Moreover, we demonstrated that in some cases fusion to MBP can promote the proper folding of the attached protein into its biologically active conformation. Thus, MBP seems to be capable of functioning as a general molecular chaperone in the context of a fusion protein. A model is proposed to explain how MBP promotes the solubility and influences the folding of its fusion partners.
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              Hepatocellular carcinoma and hepatitis B virus. A prospective study of 22 707 men in Taiwan.

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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2011
                18 November 2011
                : 6
                : 11
                : e27862
                Affiliations
                [1 ]State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
                [2 ]University Hospital Freiburg, Internal Medicine II/Molecular Biology, Freiburg, Germany
                Yonsei University, Republic of Korea
                Author notes

                Conceived and designed the experiments: HF KH. Performed the experiments: HF. Analyzed the data: HF MN KH. Contributed reagents/materials/analysis tools: JB MN. Wrote the paper: HF MN KH.

                Article
                PONE-D-11-15446
                10.1371/journal.pone.0027862
                3220704
                22125633
                9864b0da-9955-493d-a5b4-de2d9ff1bd63
                Feng et al. 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.
                History
                : 8 August 2011
                : 26 October 2011
                Page count
                Pages: 10
                Categories
                Research Article
                Biology
                Microbiology
                Virology
                Viral Classification
                DNA viruses
                Viral Replication
                Viral Nucleic Acid
                Viral Packaging
                Viral Replication Complex
                Antivirals
                Viral Enzymes
                Medicine
                Gastroenterology and Hepatology
                Liver Diseases
                Infectious Hepatitis
                Hepatitis B
                Infectious Diseases
                Viral Diseases
                Hepatitis
                Hepatitis B

                Uncategorized
                Uncategorized

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