a selex-screened aptamer of human hepatitis b virus rna encapsidation signal suppresses viral replication一个selex-screened人类乙型肝炎病毒核糖核酸适配子衣壳化信号抑制病毒复制.pdfVIP

A SELEX-Screened Aptamer of Human Hepatitis B Virus RNA Encapsidation Signal Suppresses Viral Replication 1 ¨ 2 2 1 Hui Feng , Jurgen Beck , Michael Nassal , Kang-hong Hu * 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 Abstract Background: The specific interaction between hepatitis B virus (HBV) polymerase (P protein) and the e 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 e 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 e decoys in two large e 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 e decoy that competitively inhibits P protein binding to the authentic e signal on pgRNA. Conclusions/Significance: This study demonstrates the first successful identifica