Book of Abstracts: Albany 2005

category image Volume 22
No. 6
June 2005

Structure of the Epsilon Encapsidation Signal of Human Hepatitis B Virus

We have determined the structure of the epsilon encapsidation signal of human Hepatitis B virus (HBV) using NMR spectroscopy. Epsilon is a highly conserved structured 61 nucleotide (nt) RNA motif located in the 5?-UTR of the pregenomic RNA of HBV. Epsilon plays an essential role in the HBV life cycle. It is recognized by and bound to the viral polymerase or reverse transcriptase (RT), which initiates the process of reverse transcription of the viral pregenomic RNA, essential for virus replication.

Epsilon consists of three helical domains connected by 6 and 1 nt bulges, respectively, and capped by a pseuso triloop. The apical stems are connected via the 1 nt bulge and capped with the pseudo triloop. After recognition of and initial binding to this apical stem-loop motif, the RT synthesizes a 4 nt primer using part of the 6 nt-bulge as a template. Our structure shows that epsilon has an elongated global structure in which helices are bent approximately 30° relative to each other. The 6 nt bulge is dynamic but overall has a hook-like appearance.

In the investigation of epsilon, we have used the divide and conquer approach in which the large RNA molecule of interest is cut into smaller pieces, each with a size conducive for state-of-the-art NMR examination. In addition, we have used new isotope labelling (13C/15N/selective 2D-labelling) of nucleotides. This labelling scheme allows us to obtain spectra of the complete 61 nt RNA with little overlap.

The structure of epsilon enables us to propose a model for the initial recognition and binding process between epsilon and HBV polymerase leading up to the primer synthesis.

Michael Petersen1,*
Sara Flodell2
Frederic Girard3
Janusz Zdunek2
Jurgen Schleucher2
Karin Kidd-Ljunggren4
Sybren Wijmenga3

1Department of Chemistry
University of Southern Denmark
Odense, Denmark
2Department of Medical Biochemistry and Biophysics
Umea University
Umea, Sweden
3Biophysical Chemistry
University of Nijmegen
Nijmegen, The Netherlands
4Department of Infectious Diseases
Lund University
Lund, Sweden

Phone: +45 65 50 25 30
Fax: +45 66 15 87 80
Email: mip@chem.sdu.dk