The nucleic acid data:
IRESite Id: 54 Version: 10
Originaly submitted by: Václav Vopálenský Submission date: 2005-09-21 00:00:00
Reviewed by: Václav Vopálenský Last change: 2007-05-15 00:00:00
IRESite record type:
  natural_transcript
The shape of the nucleic acid molecule translated:
  linear
The quality of the mRNA/+RNA sequence:
  our_best_guess
The abbreviated name of the virus/gene coding for this mRNA/+RNA molecule:
  GBV-B
The genetic origin of this natural mRNA/+RNA:
  viral
The GenBankId GI:# number of exactly this mRNA/+RNA sequence:
13162187
The mRNA/+RNA description: 
Hepatitis GB virus B genomic RNA.
The mRNA/+RNA sequence represented in the +DNA notation:


Credibility of mRNA sequence:
  reverse_engineered_fragment_and_the_rest_is_a_guess
The organism containing this mRNA with IRES segment in its genome:
Hepatitis GB virus B ACY/GBV-B/FL-3
A promoter reported in cDNA corresponding to IRES sequence:
  not tested
The total number of notable open-reading frames (ORFs):
  1
Notable Open-Reading Frames (ORFs; protein coding regions) in the mRNA/+RNA sequence:
ORF
ORF position:   1
Version: 2 Last change: 2007-05-15 00:00:00
Originaly submitted by: Václav Vopálenský Reviewed by: Václav Vopálenský
The abbreviated name of this ORF/gene:
GBV-B
The description of the protein encoded in this ORF:
Hepatitis GB virus B polyprotein.
The translational frameshift (ribosome slippage) involved:
  0
The ribosome read-through involved:
  no
The alternative forms of this protein occur by the alternative initiation of translation:
  not tested
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  446-9040
Remarks:
T7 transcripts were tested in rabbit reticulocyte lysates (RRL) and also in recombinant vaccinia infected
cells. Increasing KCl concentration in RRL decreased translation from first cistron but did not affect
translation from the second. [Grace et al., 1999]

Bicistronic T7 transcripts were tested for their integrity by electrophoresis in agarose gel and translated in
RRL. Also transiently transfected cells were used for the test of bicistronic constructs. Labourious site
directed mutagenesis was performed to disrupt some basepairs in putative stem loop structures or delete
certain loops and the resulting IRES activity was quantified. [Rijnbrand et al., 2000]
Citations:
Grace K., Gartland M., Karayiannis P., McGarvey M. J., Clarke B. (1999) The 5' untranslated region of GB virus B shows functional similarity to the internal ribosome entry site of hepatitis C virus. J. Gen. Virol. 80(Pt 9):2337-2341
Rijnbrand R., Abell G., Lemon S. M. (2000) Mutational analysis of the GB virus B internal ribosome entry site. J. Virol. 74(2):773-783
IRESs:
IRES:
Version: 10 Last change: 2007-05-15 00:00:00
Originaly submitted by: Václav Vopálenský Reviewed by: Václav Vopálenský
The IRES name:
  GBV-B
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  30-445
Conclusion:
  putative_IRES
How IRES boundaries were determined:
experimentally_determined
The sequence of IRES region aligned to its secondary structure (if available):


Remarks:
Deletion of the 5' end to nt 21 had a positive effect on the IRES activity (caused increase to 140 % activity
compared with the full length UTR sequence). A further 5' truncation to nt 61 or to nt 88 caused substantial
decrease in the IRES activity (decrease to 63 % or 8 % respectively; compared with the full lenght UTR
sequence). An influence of downstream coding sequence on IRES activity has been described. [Grace et al.,
1999]
In results shown in De Tomassi et al. (2003) in Fig. 4 in vitro (RRL) translation of non-capped
non-poly(A)-tailed transcriptof NPT gene is GBV-B IRES driven (1st cistron) whereas NS3 is EMCV IRES
driven (2nd cistron). It was found that bases 4-29 of the 5'-UTR do not contribute significantly
to the IRES activity and are not necessary for replication of the virus either (a predicted stem-loop
is located between bases 5-20, shown in Fig. 3). It might be that bases 1-3 are necessary for
replication, though.

In Pizzuti et al. (2003) in Fig. 4 cB76.1/Huh7 cells were transiently transfected by non-capped and
non-poly(A) tailed RNA. It turned out if there is a longer sequence of GBV-B core protein left in
the construct right after the GBV-B IRES the immediately downstream reporter gene (1st cistron) is
translated more effectively. The RNA transcript contained mutation in the RGDD motif or RNA-dependent
RNA polymerase and therefore the presence of beta-lactamase protein could be solely attributed to the
translation (and is not affected by altered replication ability of the subgenomic replicon contained
in the RNA). From in vitro translation experiment in RRL shown in Fig. 5 it can be confirmed that
with longer sequence of the GBV-B core protein left in the efficiency of translation increases as well.
Citations:
Grace K., Gartland M., Karayiannis P., McGarvey M. J., Clarke B. (1999) The 5' untranslated region of GB virus B shows functional similarity to the internal ribosome entry site of hepatitis C virus. J. Gen. Virol. 80(Pt 9):2337-2341
Rijnbrand R., Abell G., Lemon S. M. (2000) Mutational analysis of the GB virus B internal ribosome entry site. J. Virol. 74(2):773-783
Rijnbrand R., Bredenbeek P. J., Haasnoot P. C., Kieft J. S., Spaan W. J., Lemon S. M. (2001) The influence of downstream protein-coding sequence on internal ribosome entry on hepatitis C virus and other flavivirus RNAs. RNA. 7(4):585-597
De Tomassi A., Pizzuti M., Traboni C. (2003) Hep3B human hepatoma cells support replication of the wild-type and a 5'-end deletion mutant GB virus B replicon. J. Virol. 77(22):11875-11881
Pizzuti M., De Tomassi A., Traboni C. (2003) Replication and IRES-dependent translation are both affected by core coding sequences in subgenomic GB virus B replicons. J. Virol. 77(13):7502-7509
De Tomassi A., Pizzuti M., Graziani R., Sbardellati A., Altamura S., Paonessa G., Traboni C. (2002) Cell clones selected from the Huh7 human hepatoma cell line support efficient replication of a subgenomic GB virus B replicon. J. Virol. 76(15):7736-7746
Last change to the database: 2015-04-16 16:45:23 GMT+1