Book of Abstracts: Albany 2003
June 17-21 2003
Encapsidation Signal of the Hepatitis B Virus, A Possible Target for a Drug Design Strategy. Structural Studies by NMR Spectroscopy.
The HBV is the viral agent responsible for hepatitis B in humans. Its replication takes place in liver cells and can cause chronic infections that could evolve into hepato-cellular carcinoma. This hepadnavirus displays the smallest genome among all known human virus (3.2Kda). Strikingly, its replication cycle proceeds via a temporary pregenomic RNA step (pgRNA), involving a viral reverse transcriptase (P protein). The whole circular DNA genome is very efficient in that it has overlapping open reading frames (ORF's) with superimposition of regulatory regions. Some redundant and highly conserved sequences, such as the encapsidation signal (epsilon e) or the primer binding site (PBS), are involved in packaging and/or DNA priming. Using sequence alignments and computer-generated prediction folding, secondary structure of independent Epsilon sequence had been proposed (1) and the fold of the apical stem-loop of Epsilon had been subsequently confirmed by NMR studies (2). Nevertheless research on HBV replication faces a major obstacle: the HBV polymerase is not able to perform any DNA-priming when expressed by in vitro translation, so replication studies rely on genetic analysis. In order to address the mechanistic questions of encapsidation/priming, studies have been performed on the Duck ?version? of HBV (DHBV):the reconstitution of such a replication system has been successfully developed for the DHBV strain (3). Thus, as transcription process had been better investigated for DHBV (chemical and enzymatic probing of Epsilon-P protein interactions, mutagenesis) and because of the high similarity to the HBV biology, we started structural study on the DHBV Epsilon sequence in order to establish the 3D structural properties of this central element of the virus replication.
Our structural investigation by NMR involves the use of a complex labelling scheme (13C, 15N, 2H - nucleotides) that relies on selective deuteration. Due to the complexity and size of the RNA system (37nt), residual dipolar coupling (RDC?s) are exploited to attempt to refine the local structure but furthermore to define the global orientation of the different structural elements that compose Epsilon. Particular efforts have been engaged to extract numerous of accurate and structurally valuable RDC?s within residues defining the Epsilon loop element (CC, CH and HH couplings). Thus, beyond the biological question addressed here, we emphasize on the great interest of selective labelling pattern combined with selective deuteration which appears here not only of great help for the NMR assignment purpose but particularly worthwhile as it allows us to access a large set of RDC?s that wouldn?t be accessible for such RNA size.
1Department of Biophysical Chemistry