The nucleic acid data:
IRESite Id: 42 Version: 6
Originaly submitted by: Barbora Škaloudová Submission date: 2005-11-08 00:00:00
Reviewed by: Martin Pospíšek Last change: 2009-08-18 16:32:07
IRESite record type:
  natural_transcript
The shape of the nucleic acid molecule translated:
  linear
The quality of the mRNA/+RNA sequence:
  hopefully_full-length_mRNA
The abbreviated name of the virus/gene coding for this mRNA/+RNA molecule:
  HAV
The genetic origin of this natural mRNA/+RNA:
  viral
The GenBankId GI:# number of exactly this mRNA/+RNA sequence:
329582
The mRNA/+RNA description: 
Complete nucleotide sequence of cDNA from Human hepatitis A virus (strain HM175) including non-coding regions.
The mRNA/+RNA sequence represented in the +DNA notation:


Credibility of mRNA sequence:
  guessed_as_the_sequence_was_never_published_by_authors_nor_described_in_sufficient_detail
The organism containing this mRNA with IRES segment in its genome:
Human hepatitis A virus HM175
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: 2007-01-11 00:00:00
Originaly submitted by: Barbora Škaloudová Reviewed by: Martin Pospíšek
The abbreviated name of this ORF/gene:
polyprotein
The description of the protein encoded in this ORF:
Human hepatitis A virus, polyprotein b (alt.)
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:
  no
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  741-7418
Remarks:
The HM175 strain of HAV has two in-frame AUG codons within six nucleotides of each other:
- AUG-11 is located at positions 735-737
- AUG-12 is located at positions 741-743

Although AUG-12 is the preferred initiation codon when full-length 5' UTR is present, either of these AUG
codons can initiate translation in vitro or in transfected BS-C-1 cells if the other AUG codon is absent.
Citations:
Cohen J. I., Ticehurst J. R., Purcell R. H., Buckler-White A., Baroudy B. M. (1987) Complete nucleotide sequence of wild-type hepatitis A virus: comparison with different strains of hepatitis A virus and other picornaviruses. J. Virol. 61(1):50-59
IRESs:
IRES:
Version: 16 Last change: 2009-08-18 23:53:49
Originaly submitted by: Barbora Škaloudová Reviewed by: Martin Pospíšek
The IRES name:
  HAV_HM175
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  151-734
Conclusion:
  strongly_supported_IRES
How IRES boundaries were determined:
experimentally_determined
The sequence of IRES region aligned to its secondary structure (if available):



Remarks:
5'border of the IRES is located between bases 151 and 257. 3'border of the IRES is located between bases 695
and 735. Bases in range 638-666 are not essential for IRES activity. The IRES is about 25x weaker then EMCV
IRES. [Brown et al., 1994]


Activity of the IRES was tested in rabbit reticulocyte lysates using uncapped monocistronic SP6 transcribed
mRNAs. The results merely demonstrate influence of the 5'-UTR secondary structures on translation. The
constructs contained first 2025b of the virus (735ATG is the start of the viral protein). [Brown et al., 1991]

The leftmost base T of the HAV HM175 cDNA used to create the transcripts was not encoded by the genome. The
publication thus refers to bases 2-2026 when 1-2025 is written in the article. Capped, monocistronic
transcripts were partially degraded by RNases within 15 minutes in rabbit reticulocyte lysates. When capped
transcripts were used, the translation was always much higher from the first cistron (and from the second
cistron could be result of degradation?). In case of uncapped transcripts, more product was obtained from the
second cistron, but could that be because of uncapped mRNA being degraded more quickly? [Brown et al., 1994]

Borman et al. (1997) found HAV IRES very inefficient in all human and non-human cell lines tested: HeLa,
FRhK4, HepG2, SKNBE, BHK21, Neuro-2A (cells were infected with recombinant vaccinia virus expressing T7
polymerase to allow for transcription of T7 promoter containing plasmids directly in the cytoplasm).
Citations:
Brown E. A., Zajac A. J., Lemon S. M. (1994) In vitro characterization of an internal ribosomal entry site (IRES) present within the 5' nontranslated region of hepatitis A virus RNA: comparison with the IRES of encephalomyocarditis virus. J. Virol. 68(2):1066-1074
Borman A. M., Le Mercier P., Girard M., Kean K. M. (1997) Comparison of picornaviral IRES-driven internal initiation of translation in cultured cells of different origins. Nucleic Acids Res. 25(5):925-932
IRES trans-acting factor (ITAFS):
IRES trans-acting factor (ITAF):
Version: 2 Last change: 2006-04-14 00:00:00
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
Type of the interaction between ITAF and the RNA subject to translation:
unknown
ITAF protein characteristics:
Version: 1 Last change: 2009-08-18 16:32:07
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
ITAF abbreviated name:
eIF4G
ITAF fullname:
eukaryotic translation initiation factor 4G
ITAF description (long):
eIF4G is the scaffolding protein bridging together the cap-binding protein eIF4E with other proteins of the initiating ribosome
3.1.2. Organisms or in vitro systems where this ITAF was functionally studied:
Organism or in vitro system where ITAF was shown:
Necessity of ITAF for translation in this particular organism or system:
required_but_available_internally
Method used to demonstrate ITAF effect:
in_vitro
In vitro system used to demonstrate ITAF effect:
rabbit reticulocytes lysate
Remarks:
Rabbit reticulocyte lysates were supplemented with human HeLa cell extracts. It was found that cleavage of
eIF4G by FMDV Lb proteinase and by human rhinovirus 2A proteinase inhibits IRES activity. The HAV IRES was
found to require intact eIF4G.
Citations:
Borman A. M., Kean K. M. (1997) Intact eukaryotic initiation factor 4G is required for hepatitis A virus internal initiation of translation. Virology. 237(1):129-136
IRES trans-acting factor (ITAF):
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
Type of the interaction between ITAF and the RNA subject to translation:
direct_interaction_with_rna
ITAF protein characteristics:
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
ITAF abbreviated name:
PTB
ITAF fullname:
polypyrimidine-tract binding protein (unspecified isoform)
ITAF description (long):
polypyrimidine-tract binding protein
3.2.2. Organisms or in vitro systems where this ITAF was functionally studied:
Organism or in vitro system where ITAF was shown:
Necessity of ITAF for translation in this particular organism or system:
required_but_available_internally
Method used to demonstrate ITAF effect:
in_vivo
The organism where action of this ITAF was studied:
Homo sapiens
Remarks:
Cell BS-C-1 and Huh7 expressing additional PTB protein from a plasmid displayed 37- to 44-fold increase of
IRES activity (Gosert et al., 2000).
Citations:
Gosert R, Chang KH, Rijnbrand R, Yi M, Sangar DV, Lemon SM (2000) Transient expression of cellular polypyrimidine-tract binding protein stimulates cap-independent translation directed by both picornaviral and flaviviral internal ribosome entry sites In vivo. Mol. Cell. Biol. 20(5):1583-1595
IRES trans-acting factor (ITAF):
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
Type of the interaction between ITAF and the RNA subject to translation:
direct_interaction_with_rna
OPTIONAL: The interacting RNA base range (if any):
1-157
ITAF protein characteristics:
Version: 3 Last change: 2009-08-30 14:16:52
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
ITAF abbreviated name:
PCBP-2
ITAF fullname:
poly(rC)-binding protein 2 (39 kDa)
ITAF description (long):
Required for poliovirus IRES activity (Blyn et al. 1996 and 1997) and replication (Toyoda et al., 2007). Its amount is not limiting in rabbit reticulocyte lysates (RRL) (Hunt and Jackson 1999).
3.3.2. Organisms or in vitro systems where this ITAF was functionally studied:
Organism or in vitro system where ITAF was shown:
Necessity of ITAF for translation in this particular organism or system:
required_but_available_internally
Method used to demonstrate ITAF effect:
in_vitro
In vitro system used to demonstrate ITAF effect:
HeLa cell lysate
Organism or in vitro system where ITAF was shown:
Necessity of ITAF for translation in this particular organism or system:
required_but_available_internally
Method used to demonstrate ITAF effect:
in_vivo
The organism where action of this ITAF was studied:
Homo sapiens FRhK-4
Remarks:
FRhK-4 are permissive for HAV replication whereas HeLa cells are not (Graff et al., 1998).
Citations:
Graff J, Cha J, Blyn LB, Ehrenfeld E (1998) Interaction of poly(rC) binding protein 2 with the 5' noncoding region of hepatitis A virus RNA and its effects on translation. J. Virol. 72(12):9668-9675
Regions with experimentally determined secondary structures:
A region with the experimentally determined secondary structure:
IRESite 2D Struct Id: 3
Version: 7 Last change: 2009-08-30 03:16:21
Originaly submitted by: Barbora Škaloudová Reviewed by: Martin Pospíšek
The function of the 2D structure:
IRES
The 2D structure causes frameshift:
no
The absolute position of the experimentally mapped region (the range includes START and STOP codons or their equivalents):
151-734
The underlying nucleic acid sequence and structure of the mapped region:



Remarks:
SP6 transcripts containing 2025b of the HAV_HM175 isolates were mapped enzymatically. The structure is a
result of enzyme mapping/primer extension and MFOLD&STAR based modeling and was heavily manually adjusted.
Putative double-stranded regions were originally predicted by the presence of covariant base mutations and
used as prediction constraints.

The possibly unstructured sequence inferred from GenBank immediately upstream of domain III contains
CTCTCCCCTTGC instead of CTCCCCTTGC as shown in Figure 3 (note the extra CT). Thus, the structure is extended
by two dots in that place.
4.1.1. Enzymes used to characterize at least partially the 2D structure.
Enzyme or a combination of enzymes used in a single experiment with respective buffer:
ss_experiment_with_enzyme_id: 3
The temperature (in degrees of Celsia):
25
The enzymatic method used to determine the 2D structure:
S1 nuclease
Enzyme or a combination of enzymes used in a single experiment with respective buffer:
Version: 0
pH
7.50
Li+ [mM]
0
Na+ [mM]
0
K+ [mM]
50.00
Mg2+ [mM]
10.00
Ca2+ [mM]
0
Cl- [mM]
70.00
Tris [mM]
10.00
BSA [mM]
0
HEPES [mM]
0
EGTA [mM]
0
EDTA [mM]
0
cacodylate [mM]
0
Other buffer components and their relative concentrations:
The buffer B contained also 1 mM ZnSO4, 50 U S1 nuclease.
Enzyme or a combination of enzymes used in a single experiment with respective buffer:
ss_experiment_with_enzyme_id: 4
The temperature (in degrees of Celsia):
25
The enzymatic method used to determine the 2D structure:
ribonuclease T1
Enzyme or a combination of enzymes used in a single experiment with respective buffer:
Version: 0
pH
7.50
Li+ [mM]
0
Na+ [mM]
0
K+ [mM]
50.00
Mg2+ [mM]
10.00
Ca2+ [mM]
0
Cl- [mM]
70.00
Tris [mM]
10.00
BSA [mM]
0
HEPES [mM]
0
EGTA [mM]
0
EDTA [mM]
0
cacodylate [mM]
0
Other buffer components and their relative concentrations:
0.2-0.5U RNase T1
Enzyme or a combination of enzymes used in a single experiment with respective buffer:
ss_experiment_with_enzyme_id: 5
The temperature (in degrees of Celsia):
25
The enzymatic method used to determine the 2D structure:
ribonuclease V1
Enzyme or a combination of enzymes used in a single experiment with respective buffer:
Version: 0
pH
7.50
Li+ [mM]
0
Na+ [mM]
0
K+ [mM]
50.00
Mg2+ [mM]
10.00
Ca2+ [mM]
0
Cl- [mM]
70.00
Tris [mM]
10.00
BSA [mM]
0
HEPES [mM]
0
EGTA [mM]
0
EDTA [mM]
0
cacodylate [mM]
0
Other buffer components and their relative concentrations:
0.2-0.5U RNase V1
Enzyme or a combination of enzymes used in a single experiment with respective buffer:
ss_experiment_with_enzyme_id: 6
The temperature (in degrees of Celsia):
25
The enzymatic method used to determine the 2D structure:
ribonuclease T2
Enzyme or a combination of enzymes used in a single experiment with respective buffer:
Version: 0
pH
7.50
Li+ [mM]
0
Na+ [mM]
0
K+ [mM]
50.00
Mg2+ [mM]
10.00
Ca2+ [mM]
0
Cl- [mM]
70.00
Tris [mM]
10.00
BSA [mM]
0
HEPES [mM]
0
EGTA [mM]
0
EDTA [mM]
0
cacodylate [mM]
0
Other buffer components and their relative concentrations:
0.2-0.5U RNase T2
Citations:
Brown E. A., Day S. P., Jansen R. W., Lemon S. M. (1991) The 5' nontranslated region of hepatitis A virus RNA: secondary structure and elements required for translation in vitro. J. Virol. 65(11):5828-5838
A region with the experimentally determined secondary structure:
IRESite 2D Struct Id: 41
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The function of the 2D structure:
IRES
The 2D structure causes frameshift:
unknown
The absolute position of the experimentally mapped region (the range includes START and STOP codons or their equivalents):
1-323
The underlying nucleic acid sequence and structure of the mapped region:



Remarks:
The pseudoknots are not recorded.
4.2.1. Enzymes used to characterize at least partially the 2D structure.
Enzyme or a combination of enzymes used in a single experiment with respective buffer:
ss_experiment_with_enzyme_id: 69
The temperature (in degrees of Celsia):
20
The enzymatic method used to determine the 2D structure:
ribonuclease T1/T2 mix
Enzyme or a combination of enzymes used in a single experiment with respective buffer:
Version: 0
pH
7.60
Li+ [mM]
0
Na+ [mM]
0
K+ [mM]
0
Mg2+ [mM]
10.00
Ca2+ [mM]
0
Cl- [mM]
20.00
Tris [mM]
0
BSA [mM]
0
HEPES [mM]
0
EGTA [mM]
0
EDTA [mM]
0
cacodylate [mM]
0
Enzyme or a combination of enzymes used in a single experiment with respective buffer:
ss_experiment_with_enzyme_id: 70
The temperature (in degrees of Celsia):
20
The enzymatic method used to determine the 2D structure:
ribonuclease V1
Enzyme or a combination of enzymes used in a single experiment with respective buffer:
Version: 0
pH
7.60
Li+ [mM]
0
Na+ [mM]
0
K+ [mM]
0
Mg2+ [mM]
10.00
Ca2+ [mM]
0
Cl- [mM]
20.00
Tris [mM]
0
BSA [mM]
0
HEPES [mM]
0
EGTA [mM]
0
EDTA [mM]
0
cacodylate [mM]
0
Enzyme or a combination of enzymes used in a single experiment with respective buffer:
ss_experiment_with_enzyme_id: 71
The temperature (in degrees of Celsia):
20
The enzymatic method used to determine the 2D structure:
S1 nuclease
Enzyme or a combination of enzymes used in a single experiment with respective buffer:
Version: 0
pH
7.60
Li+ [mM]
0
Na+ [mM]
0
K+ [mM]
0
Mg2+ [mM]
10.00
Ca2+ [mM]
0
Cl- [mM]
20.00
Tris [mM]
0
BSA [mM]
0
HEPES [mM]
0
EGTA [mM]
0
EDTA [mM]
0
cacodylate [mM]
0
Other buffer components and their relative concentrations:
1 mM ZnSO4
Citations:
Shaffer DR, Brown EA, Lemon SM (1994) Large deletion mutations involving the first pyrimidine-rich tract of the 5' nontranslated RNA of human hepatitis A virus define two adjacent domains associated with distinct replication phenotypes. J. Virol. 9(68):5568-5578
Last change to the database: 2015-04-16 16:45:23 GMT+1