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.
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):
Rendering structure of HAV mRNA 584 nt long with energy of -113.00 kcal/mol as calculated by RNAeval using VARNA Java applet with some IRESite improvements (see VARNA modified by IRESite). Hold left mouse button to move structure parts, hold right mouse button to move whole structure, use mouse wheel to zoom. Right mouse-click opens a menu to export into JPG/SVG and many other options.
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).
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.
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).
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:
Rendering structure of HAV mRNA 584 nt long with energy of -113.00 kcal/mol as calculated by RNAeval using VARNA Java applet with some IRESite improvements (see VARNA modified by IRESite). Hold left mouse button to move structure parts, hold right mouse button to move whole structure, use mouse wheel to zoom. Right mouse-click opens a menu to export into JPG/SVG and many other options.
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:
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:
Rendering structure of HAV mRNA 323 nt long with energy of -78.10 kcal/mol as calculated by RNAeval using VARNA Java applet with some IRESite improvements (see VARNA modified by IRESite). Hold left mouse button to move structure parts, hold right mouse button to move whole structure, use mouse wheel to zoom. Right mouse-click opens a menu to export into JPG/SVG and many other options.
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: