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Structure and Dynamics of the Hepatitis B Virus Encapsidation Signal Revealed by NMR Spectroscopy
Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes the study of the three-dimensional structure and dynamics of the hepatitis B virus (HBV) encapsidation signal, epsilon, by means of nuclear magnetic resonance (NMR) and mutational data. HBV replicates by reverse transcription of an RNA pregenome into the viral DNA genome, which becomes enclosed in viral particles (encapsidation). Epsilon is a stem-loop structure within the RNA pregenome and both the primary sequence and secondary structure of epsilon are strongly conserved, in agreement with its essential function of propagating HBV. Epsilon is therefore a potential target for drug design. Studying the structure of epsilon requires development of new methods in the field of structural biology, as it is such a large RNA. Knowing the structure of epsilon will help to better understand the encapsidation mechanism and priming step of reverse transcription. This will help us in the search for antiviral drugs that block epsilon and prevent the viral reverse transcriptase from binding.

NMR spectroscopy is a method that provides detailed structural and dynamical data in solution under natural conditions. However, the size of the molecules that can be studied with NMR is limited. NMR spectra become more and more difficult to interpret as the size of the molecule increases. To circumvent this problem, large RNA molecules can be divided into smaller parts and only the parts essential for NMR studies are selected. The information obtained from these smaller fragments can then be used to determine the structure of the larger molecule. Furthermore, a new method of enzymatically synthesizing nucleoside triphosphates with isotopes suitable for NMR has made it possible to specifically label the RNA molecules. Using this method it is possible to derive highly detailed molecular structures of RNA up to a size of 150 nucleotides. The method of selective isotope labelling was applied to different parts of HBV epsilon. Three RNA fragments of 27 (apical loop), 36 (internal bulge) and 61 (whole epsilon) nucleotides (nt) were synthesized in the unlabelled form. The 27-nt and 36-nt RNAs were also synthesized with (13C, 15N, 1', 3', 4', 5', 5"-2H5)-labelled uridines. The 61-nt sequence was (13C, 15N)-guanidine labelled. This labelling allowed unambiguous assignment of otherwise inaccessible parameters. The unlabelled and labelled RNA sequences provided the necessary data for structure derivation of the whole epsilon.

The apical loop of epsilon forms a pseudo-triloop motif. There is only one conformation of the loop that fulfils all the restraints, including experimental chemical shifts. However, the loop adopts several structures that fulfil the experimental distance, torsion angle and residual dipolar coupling restraints. This may reflect true flexibility. Indeed, relaxation studies on the unlabelled and labelled 27-nt sequences show that the residues that show multiple conformations are flexible. This can be an important feature for the recognition and subsequent binding of epsilon to the viral polymerase.

The information gained on the HBV encapsidation signal is useful in our understanding of the initiation of replication of the virus. This can in turn contribute to the search for drugs against HBV.

Place, publisher, year, edition, pages
Umeå: Medicinsk biokemi och biofysik , 2004. , p. 43
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 913
Keywords [en]
HBV, RNA, isotope labelling, NMR, structure determination
Research subject
Medical Biochemistry
Identifiers
URN: urn:nbn:se:umu:diva-316ISBN: 91-7305-719-3 (print)OAI: oai:DiVA.org:umu-316DiVA, id: diva2:143072
Public defence
2004-10-08, KB3A9, KBC, 13:00 (English)
Opponent
Available from: 2004-09-16 Created: 2004-09-16 Last updated: 2018-03-15Bibliographically approved
List of papers
1. Structure elucidation of the hepatitis B virus encapsidation signal by NMR on selectively labeled RNAs.
Open this publication in new window or tab >>Structure elucidation of the hepatitis B virus encapsidation signal by NMR on selectively labeled RNAs.
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2002 (English)In: Journal of Biomolecular Structure and Dynamics, ISSN 0739-1102, E-ISSN 1538-0254, Vol. 19, no 4, p. 627-636Article in journal (Other academic) Published
Abstract [en]

Hepatitis B virus (HBV) HBV is DNA virus with a unique replication strategy, which involves reverse transcription of its pregenomic RNA. Essential for this reverse transcription are the 5'- and 3'-ends of its pregenomic RNA (5'-RT-RNA and 3'-RT-RNA, respectively) which form conserved bulged stem-loop structures. The 5'-RT-RNA consists of a 67 nucleotide bulged stem-loop structure, epsilon, which constitutes the signal for encapsidation of the pregenomic RNA and subsequent reverse transcription. The reverse transcriptase (RT) initially binds to the completely conserved apical loop of epsilon and a 4-nucleotide primer is synthesized from the adjacent 6-nucleotide bulge. Structural studies of epsilon can provide important parameters required for the design of RNA targeted anti- viral drugs directed against Hepatitis B virus. NMR studies of large RNA systems (> ca. 50 nucleotides) require novel approaches, e.g., different labeling schemes and reduction of the system into separate structural building blocks. Recently, a new method of synthesizing (13)C/(15)N/(2)H labeled nucleotides has been developed based on converting specifically labeled glucose and bases into nucleotides by using enzymes from the pentose phosphate pathway and nucleotide and salvage pathways. These NTPs give a large freedom in designing different labeling patterns in in vitro synthesized RNAs under study for NMR. This opens up the way for NMR studies of RNAs that are considerably above the present size limit (up to 150 nucleotides). Here this new technique is applied for structural studies on 27, 36 and 61 nucleotides long RNA fragments, mimicking different regions of epsilon.

Keywords
Base Sequence, Capsid/*chemistry, DNA Replication, Hepatitis B virus/*chemistry/genetics/physiology, Magnetic Resonance Spectroscopy, Molecular Sequence Data, Nucleic Acid Conformation, RNA; Viral/*chemistry/genetics, RNA-Directed DNA Polymerase/chemistry/*metabolism
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-10297 (URN)11843624 (PubMedID)
Available from: 2008-08-11 Created: 2008-08-11 Last updated: 2018-06-09Bibliographically approved
2. The apical stem-loop of the hepatitis B virus encapsidation signal folds into a stable tri-loop with two underlying pyrimidine bulges.
Open this publication in new window or tab >>The apical stem-loop of the hepatitis B virus encapsidation signal folds into a stable tri-loop with two underlying pyrimidine bulges.
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2002 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 30, no 21, p. 4803-4811Article in journal (Refereed) Published
Abstract [en]

Reverse transcription of hepatitis B virus (HBV) pregenomic RNA is essential for virus replication. In the first step of this process, HBV reverse transcriptase binds to the highly conserved encapsidation signal, epsilon (epsilon), situated near the 5' end of the pregenome. epsilon has been predicted to form a bulged stem-loop with the apical stem capped by a hexa- loop. After the initial binding to this apical stem- loop, the reverse transcriptase synthesizes a 4 nt primer using the bulge as a template. Here we present mutational and structural data from NMR on the apical stem-loop of epsilon. Application of new isotope-labeling techniques (13C/15N/2H-U-labeling) allowed resolution of many resonance overlaps and an extensive structural data set could be derived. The NMR data show that, instead of the predicted hexa-loop, the apical stem is capped by a stable UGU tri-loop closed by a C-G base pair, followed by a bulged out C. The apical stem contains therefore two unpaired pyrimidines (C1882 and U1889), rather than one as was predicted, spaced by 6 nt. C1882, the 3' neighbour to the G of the loop-closing C-G base pair, is completely bulged out, while U1889 is at least partially intercalated into the stem. Analysis of 205 of our own HBV sequences and 1026 strains from the literature, covering all genotypes, reveals a high degree of conservation of epsilon. In particular, the residues essential for this fold are either totally conserved or show rare non-disruptive mutations. These data strongly indicate that this fold is essential for recognition by the reverse transcriptase.

Keywords
Base Pairing, Base Sequence, Genome; Viral, Genotype, Hepatitis B virus/classification/enzymology/*genetics, Humans, Mutation, Nuclear Magnetic Resonance; Biomolecular, Nucleic Acid Conformation, Pyrimidines/*metabolism, RNA; Viral/*chemistry/genetics/*metabolism, RNA-Directed DNA Polymerase/metabolism, Substrate Specificity, Uridine/metabolism
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-10293 (URN)12409471 (PubMedID)
Available from: 2008-08-11 Created: 2008-08-11 Last updated: 2018-06-09Bibliographically approved
3. Solution structure of the apical stem-loop of the human hepatitis B virus encapsidation signal.
Open this publication in new window or tab >>Solution structure of the apical stem-loop of the human hepatitis B virus encapsidation signal.
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2006 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 34, no 16, p. 4449-4457Article in journal (Refereed) Published
Abstract [en]

Hepatitis B virus (HBV) replication is initiated by HBV RT binding to the highly conserved encapsidation signal, epsilon, at the 5' end of the RNA pregenome. Epsilon contains an apical stem-loop, whose residues are either totally conserved or show rare non-disruptive mutations. Here we present the structure of the apical stem-loop based on NOE, RDC and (1)H chemical shift NMR data. The (1)H chemical shifts proved to be crucial to define the loop conformation. The loop sequence 5'-CUGUGC-3' folds into a UGU triloop with a CG closing base pair and a bulged out C and hence forms a pseudo-triloop, a proposed protein recognition motif. In the UGU loop conformations most consistent with experimental data, the guanine nucleobase is located on the minor groove face and the two uracil bases on the major groove face. The underlying helix is disrupted by a conserved non-paired U bulge. This U bulge adopts multiple conformations, with the nucleobase being located either in the major groove or partially intercalated in the helix from the minor groove side, and bends the helical stem. The pseudo-triloop motif, together with the U bulge, may represent important anchor points for the initial recognition of epsilon by the viral RT.

Keywords
5' Untranslated Regions/chemistry, Hepatitis B virus/*genetics, Models; Molecular, Nuclear Magnetic Resonance; Biomolecular, Nucleic Acid Conformation, RNA; Viral/*chemistry, Solutions, Virus Replication
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-6614 (URN)10.1093/nar/gkl582 (DOI)16945960 (PubMedID)
Available from: 2007-12-16 Created: 2007-12-16 Last updated: 2018-06-09Bibliographically approved
4. Mobile nucleotides mediate binding of HBV reverse transcriptase to its RNA target
Open this publication in new window or tab >>Mobile nucleotides mediate binding of HBV reverse transcriptase to its RNA target
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(English)Manuscript (preprint) (Other academic)
National Category
Natural Sciences
Identifiers
urn:nbn:se:umu:diva-4103 (URN)
Available from: 2004-09-16 Created: 2004-09-16 Last updated: 2018-06-09Bibliographically approved

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