The nucleic acid data:
IRESite Id: 596 Version: 6
Originaly submitted by: Martin Mokrejš Submission date: 2009-08-18 14:34:26
Reviewed by: Martin Mokrejš Last change: 2010-03-03 23:22:41
IRESite record type:
  natural_transcript
The shape of the nucleic acid molecule translated:
  linear
The quality of the mRNA/+RNA sequence:
  end-to-end_full-length_mRNA
The abbreviated name of the virus/gene coding for this mRNA/+RNA molecule:
  SV40
The genetic origin of this natural mRNA/+RNA:
  viral
The GenBankId GI:# number of the most similar mRNA/+RNA sequence to this one.
9628421 
The mRNA/+RNA description: 
The SV40 late 19S long-spliced mRNA derived from transcription start site in genomic position 325. This is the
most abundant variant (11% relative of all early and late mRNAs) of the 19S late transcripts.
The mRNA/+RNA sequence represented in the +DNA notation:


Credibility of mRNA sequence:
  end-to-end_sequence_reverse_engineered_and_should_match_experiment
The organism containing this mRNA with IRES segment in its genome:
Simian virus 40 776
A promoter reported in cDNA corresponding to IRES sequence:
  no
The total number of notable open-reading frames (ORFs):
  4
Summary of possible issues when IRES cDNA is experimentally transcribed in vivo:
Summary of experiments studying integrity of the in vivo transcripts in a particular host:
Integrity (uniformity) of mRNA tested using Northern-blot:
not_tested
Integrity (uniformity) of mRNA tested using RNase protection:
not_tested
Integrity (uniformity) of mRNA tested using 5'-RACE:
not_tested
Integrity (uniformity) of mRNA tested using primer extension :
homogeneous_population_of_molecules_confirmed
Integrity (uniformity) of mRNA tested using RT-PCR:
not_tested
Integrity (uniformity) of mRNA tested using real-time quantitative polymerase chain reaction (rtqPCR):
not_tested
Integrity (uniformity) of mRNA tested using RNAi:
not_tested
Integrity (uniformity) of mRNA tested using S1 nuclease mapping:
not_tested
Cryptic promoter presence was confirmed by expression from a promoter-less plasmid:
not_tested
Cryptic promoter presence was confirmed in an experimental setup involving inducible promoter:
not_tested
Integrity (uniformity) of mRNA molecules or possible promoter presence expressed in vivo was tested using another method, please specify in Remarks:
not_tested
The organism used:
Cercopithecus aethiops CV-1 (ATCC CCL-70)
Notable Open-Reading Frames (ORFs; protein coding regions) in the mRNA/+RNA sequence:
ORF
ORF position:   1
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
agnoprotein-truncated
The description of the protein encoded in this ORF:
N-terminal half of agnoprotein fused out-of-frame to VP2 ORF. The resulting protein was not yet directly shown to exist, unlike the non-truncated agnoprotein.
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):
  11-103
ORF
ORF position:   2
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
VP2
The description of the protein encoded in this ORF:
SV40 late structural protein VP2
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:
  yes
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  54-1112
ORF
ORF position:   3
Version: 1 Last change: 2009-08-27 23:02:15
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
VP3
The description of the protein encoded in this ORF:
SV40 late structural protein VP3 (27kDa)
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:
  yes
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  408-1112
ORF
ORF position:   4
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
VP4
The description of the protein encoded in this ORF:
SV40 late structural protein VP4 (14kDa)
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:
  yes
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  735-1112
Remarks:
Yu and Alwine (2006) aimed to proof the IRES is used to enhance translation of the VP3 protein from the 19S
messages. In Figure 1 they only show one type of the 19S messages, the short spliced form. However, there
exist two additional splice forms and even an unspliced form. All of these have the poly(A) tail, contain
the challenged region and encode VP2 and VP3 as well.


Here is a brief overview of abundancies of SV40 late messages (from Good et al., 1988):
11% late 19S long-spliced mRNA (TSS at genomic position 325)
7% late 19S superlong-spliced mRNA (TSS at genomic position 325)
1% late 19S short-spliced mRNA (TSS at genomic position 325)
0.2% late 19S doubly-spliced mRNA (TSS at genomic position 325)
0.1% late 19S unspliced mRNA (TSS at genomic position 325)
<<1% late 19S unspliced mRNA (TSS at genomic position 264)

64% late 16S singly-spliced mRNA (TSS at genomic position 325)
16% late 16S doubly-spliced mRNA (TSS at genomic position 325)
1% late 16S leader-to-leader followed by leader-to-body spliced mRNA (TSS at genomic position 325)


Components of the early and late promoters and transcription start sites of the simian virus 40 are well
studied. The region studied for IRES activity is downstream of the late promoter. Therefore, one is tempted to
presume there is no cryptic promoter in this region. However, it cannot be excluded in the context of the
experimental plasmid used in the work of Yu and Alwine (2006).

From the RNase protection assay shown in Figure 2 (Yu and Alwine, 2006) it can be concluded that the
bicistronic messages were intact in the region spanned by their probe: 102bp of the Rluc ORF, followed by
fragment from SV40 genomic region 565-915 of SV40 further followed by 166bp part of Gal4VP16 ORF. From the
primer extension shown of 4xUAS/565-915 transcripts in CV-1 cells in Figure 3 of their article it can be
concluded there is no promoter in the SV40 565-915 region nor there is a splicing issue with their plasmid
constructs resulting in monocistronic messages containing functional Gal4VP16 or its functional
fusion/shortened product detectable by the primer. In summary, the transcription from their plasmids results
in uniform population of messages.
Citations:
Good P. J., Welch R. C., Ryu W. S., Mertz J. E. (1988) The late spliced 19S and 16S RNAs of simian virus 40 can be synthesized from a common pool of transcripts. J. Virol. 62(2):563-571
Yu Y., Alwine J. C. (2006) 19S late mRNAs of simian virus 40 have an internal ribosome entry site upstream of the virion structural protein 3 coding sequence. J. Virol. 80(13):6553-6558
Daniels R, Sadowicz D, Hebert DN (2007) A very late viral protein triggers the lytic release of SV40. PLoS Pathog. 7(3):e98
IRESs:
IRES:
Version: 3 Last change: 2010-03-03 23:22:41
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The IRES name:
  SV40_661-830
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  153-322
Conclusion:
  unproved_IRES
How IRES boundaries were determined:
experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  50
3'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  219
5'-end of IRES relative to first base of the START codon of the downstream ORF:
  -255
3'-end of IRES relative to first base of the START codon of the downstream ORF:
  -86
The sequence of IRES region aligned to its secondary structure (if available):


Remarks:
Unfortunately, no positive control IRES was employed in assays of Yu and Alwine (2006) to put their results
into scale with other IRESs and therefore it can only be concluded that the putative SV40 IRES activity is
about 18x above the empty bicistronic vector (Figure 2 in their article with p4xUAS vector) whereas in another
vector backbone (pRF with a hairpin structure) only 3.8x (Figure 4 in their article).

It is necessary to mention experiments of Sedman and Mertz (1988). They created mutants with in-frame and
out-of-frame insertions by introducing two slightly different 18bp long oligonucleotides between nucleotides
770 and 771 of the wild-type virus. The former extended N-terminally the VP3 ORF while the other created uORF
within the VP2 region while both introduced initiation AUG codons were in same context. If there would be any
SV40 IRES the first insertion would not be expected to result in a fusion protein if IRES directed
translation from VP3 AUG codon. The second insertion should not result in decreased yields of the VP3
protein either. Similarly, the two 19 and 28bp long deletions within the VP2 region (map positions 759-778 and
759-787) could have truncated the putative IRES, if there was any. Finally, detection of the VP4 protein as
yet another alternatively initiated protein product fits well the hypothesis that both VP3 and VP4 are
synthesized by leaky scanning ribosomes (Daniels et al., 2007). Same authors showed that a cleavage product of
VP4 (7kDa) exists in cells harvested by trypsin.

In this context it is interesting to note, that split of the putative IRES in region 661-830 into two parts
(611-770 and 771-830) destroyed the "IRES activity" in CV-1 cells (pRF backbone). Another region postulated
to have "IRES activity" was 771-915 (Yu and Alwine, 2006).
Citations:
Yu Y., Alwine J. C. (2006) 19S late mRNAs of simian virus 40 have an internal ribosome entry site upstream of the virion structural protein 3 coding sequence. J. Virol. 80(13):6553-6558
Sedman S. A., Mertz J. E. (1988) Mechanisms of synthesis of virion proteins from the functionally bigenic late mRNAs of simian virus 40. J. Virol. 62(3):954-961
Last change to the database: 2015-04-16 16:45:23 GMT+1