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The apical stem-loop of the hepatitis B virus encapsidation signal folds into a stable tri-loop with two underlying pyrimidine bulges

Flodell, S; Schleucher, J; Cromsigt, J; Ippel, H; Kidd-Ljunggren, Karin LU and Wijmenga, S (2002) In Nucleic Acids Research 30(21). p.4803-4811
Abstract
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 (C-13/N-15/H-2-U-labeling) allowed resolution of many resonance overlaps and an extensive structural... (More)
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 (C-13/N-15/H-2-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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Nucleic Acids Research
volume
30
issue
21
pages
4803 - 4811
publisher
Oxford University Press
external identifiers
  • wos:000179038100025
  • pmid:12409471
  • scopus:0036849272
ISSN
1362-4962
DOI
10.1093/nar/gkf603
language
English
LU publication?
yes
id
796ac5e8-2423-4986-9c68-7e2224c72db2 (old id 324166)
date added to LUP
2007-08-17 13:01:57
date last changed
2017-08-13 03:28:45
@article{796ac5e8-2423-4986-9c68-7e2224c72db2,
  abstract     = {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 (C-13/N-15/H-2-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.},
  author       = {Flodell, S and Schleucher, J and Cromsigt, J and Ippel, H and Kidd-Ljunggren, Karin and Wijmenga, S},
  issn         = {1362-4962},
  language     = {eng},
  number       = {21},
  pages        = {4803--4811},
  publisher    = {Oxford University Press},
  series       = {Nucleic Acids Research},
  title        = {The apical stem-loop of the hepatitis B virus encapsidation signal folds into a stable tri-loop with two underlying pyrimidine bulges},
  url          = {http://dx.doi.org/10.1093/nar/gkf603},
  volume       = {30},
  year         = {2002},
}