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A SELEX-screened aptamer of human hepatitis B virus RNA encapsidation signal suppresses viral replication.

ABSTRACT: BACKGROUND: The specific interaction between hepatitis B virus (HBV) polymerase (P protein) and the ? 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 ? 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 ? decoys in two large ? 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 ? decoy that competitively inhibits P protein binding to the authentic ? signal on pgRNA. CONCLUSIONS/SIGNIFICANCE: This study demonstrates the first successful identification of human HBV ? aptamers by an in vitro SELEX approach. Effective suppression of HBV replication by the S9 aptamer provides proof-of-principle for the ability of ? decoy RNAs to interfere with viral P-? complex formation and suggests that S9-like RNAs may further be developed into useful therapeutics against chronic hepatitis B.

SUBMITTER: Feng H

PROVIDER: S-EPMC3220704 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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A SELEX-screened aptamer of human hepatitis B virus RNA encapsidation signal suppresses viral replication.

Feng Hui H Beck Jürgen J Nassal Michael M Hu Kang-Hong KH

PloS one 20111118 11

<h4>Background</h4>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 di ...[more]

PMID: 22125633

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Project description:To elucidate the linkage between replication and encapsidation in Picornavirales, we have taken advantage of the bipartite nature of a plant-infecting member of this order, cowpea mosaic virus (CPMV), to decouple the two processes. RNA-free virus-like particles (empty virus-like particles [eVLPs]) can be generated by transiently coexpressing the RNA-2-encoded coat protein precursor (VP60) with the RNA-1-encoded 24,000-molecular-weight (24K) protease, in the absence of the replication machinery (K. Saunders, F. Sainsbury, and G. P. Lomonossoff, Virology 393:329-337, 2009, https://doi.org/10.1016/j.virol.2009.08.023). We have made use of the ability to produce assembled capsids of CPMV in the absence of replication to examine the putative linkage between RNA replication and packaging in the Picornavirales We have created a series of mutant RNA-1 and RNA-2 molecules and have assessed the effects of the mutations on both the replication and packaging of the viral RNAs. We demonstrate that mutations that affect replication have a concomitant impact on encapsidation and that RNA-1-mediated replication is required for encapsidation of both RNA-1 and RNA-2. This close coupling between replication and encapsidation provides a means for the specific packaging of viral RNAs. Moreover, we demonstrate that this feature of CPMV can be used to specifically encapsidate custom RNA by placing a sequence of choice between the RNA-2 sequences required for replication.IMPORTANCE The mechanism whereby members of the order Picornavirales specifically package their genomic RNAs is poorly understood. Research with monopartite members of the order, such as poliovirus, indicated that packaging is linked to replication, although the presence of "packaging signals" along the length of the viral RNA has also been suggested. Thanks to the bipartite nature of the CPMV genome, which allows the manipulation of RNA-1 without modifying RNA-2, we show here that this specificity is due to a functional link between the two processes of viral replication and encapsidation. This has important implications for our understanding of the fundamental molecular biology of Picornavirales and opens the door to novel research and therapeutic applications in the field of custom RNA packaging and delivery technologies.

Dataset Information

A SELEX-screened aptamer of human hepatitis B virus RNA encapsidation signal suppresses viral replication.

Publications

A SELEX-screened aptamer of human hepatitis B virus RNA encapsidation signal suppresses viral replication.

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