Comparing PreXMRV-2 gag sequence diversity in laboratory and wild mice

[Bob]

I do not see it there. It only shows Gag, Pro and Pol.

At viralzone http://viralzone.expasy.org/all_by_species/67.html I see a gammaretro gnome is about
8.3 kb.

The 22Rv1/CWR-R1 is too short

The VP62 is at http://www.ncbi.nlm.nih.gov/nuccore/121104176?report=graph

That is longer and shows the envelope

I do not see it. Can you show it to me please?

The VP62 genbank entry labels the env gene from nt 5754 to 7691.

​

The 22RV1/CWR-R1 genbank entry is labelled as a "complete proviral genome", and the 'putative' env gene is labelled as nt 5754 to 7691, the same as VP62. (Search the webpage for: "putative envelope polyprotein" to find the env gene label.)​

But as Mula says, the genbank entry only has limited value, because the evidence for the virus is limited to a gDNA PCR result. (They neither sequenced the viruses, nor isolated the viruses, nor detected any viral RNA.)​

​

I'm not sure if further research has been carried out on the 22Rv1/CWR-R1 (assumed) virus since the Paprotka study.​

 

[lansbergen]

Thanks Bob

 

[Mula]

The VP62 genbank entry labels the env gene from nt 5754 to 7691.
The 22RV1/CWR-R1 genbank entry is labelled as a "complete proviral genome", and the 'putative' env gene is labelled as nt 5754 to 7691, the same as VP62. (Search the webpage for: "putative envelope polyprotein" to find the env gene label.)

But as Mula says, the genbank entry only has limited value, because the evidence for the virus is limited to a gDNA PCR result. (They neither sequenced the viruses, nor isolated the viruses, nor detected any viral RNA.)
I'm not sure if further research has been carried out on the 22Rv1/CWR-R1 (assumed) virus since the Paprotka study.

A "putative gag polyprotein" is not a gene.

Please go to the database.
http://www.ncbi.nlm.nih.gov/nuccore/334717372

Under customize view, which can be found on the top right, drop down the menu and select Gene, RNA, and CDS features only. Once selected press the Update view button.

Select where it is written gene, by the gag-pro-pol.
gene 613..5814
/gene="gag-pro-pol"

A brown overlay will now appear. At the bottom of the screen is a box with gene. To the right of this you can see that this is 1 of 1. If other genes were available you could select them using this tool.

A new clean lab needs to revisit the 22Rv1 cells and sequence the envelop gene of the unidentified virus.

 

[Bob]

A "putative gag polyprotein" is not a gene.

Please go to the database.
http://www.ncbi.nlm.nih.gov/nuccore/334717372

Under customize view, which can be found on the top right, drop down the menu and select Gene, RNA, and CDS features only. Once selected press the Update view button.

Select where it is written gene, by the gag-pro-pol.
gene 613..5814
/gene="gag-pro-pol"

A brown overlay will now appear. At the bottom of the screen is a box with gene. To the right of this you can see that this is 1 of 1. If other genes were available you could select them using this tool.

A new clean lab needs to revisit the 22Rv1 cells and sequence the envelop gene of the unidentified virus.

The "putative envelope polyprotein" is labelled as a "mat peptide", which I believe is the main portion of the gene which translates to the env protein.
As I've said before, the env gene, in Paptrotka, was handled in exactly the same was as the other sequences, as far as I understand.

 

[Mula]

A mat_peptide is not an gene either. If you would follow the instructions I provided, or if others would like to do so, you can see that there is only one gene listed for this sequence.

Have you read the Paprotka study from which this sequence originates?

 

[Bob]

A mat_peptide is not an gene either. If you would follow the instructions I provided, or if others would like to do so, you can see that there is only one gene listed for this sequence.

Have you read the Paprotka study from which this sequence originates?

Mula, have you actually read any of my posts?

If you had then you would know that: the env gene is clearly labelled; a 'mat peptide' is the main part of the gene that translates for a protein; and, yes, obviously I've read Paprotka (in detail).

 

[Undisclosed]

The "putative envelope polyprotein" is labelled as a "mat peptide", which I believe is the main portion of the gene which translates to the env protein.
As I've said before, the env gene, in Paptrotka, was handled in exactly the same was as the other sequences, as far as I understand.

From what I have been reading, this sounds correct re: the mat peptide.

 

[Mula]

Mula, have you read any of my posts?

If you had then you would know that: the env gene is clearly labelled; a 'mat peptide' is the main part of the gene; and, yes, obviously I've read Paprotka.

I have read your posts. A mature peptide is not a gene.
You may wish to look at the entry for the ENA. Drop down Annotated Nucleotide Sequences (Release) - 1 results.
http://www.ebi.ac.uk/ena/data/view/FN692043

The overview displays genes in orange, which ends with the gag-pro-pol and no envelop gene. Instead the envelope is proposed.

 

[Bob]

I have read your posts. A mature peptide is not a gene.
You may wish to look at the entry for the ENA. Drop down Annotated Nucleotide Sequences (Release) - 1 results.
http://www.ebi.ac.uk/ena/data/view/FN692043

The overview displays genes in orange, which ends with the gag-pro-pol and no envelop gene. Instead the envelope is proposed.

Thanks for that, but it shows exactly the same information as the NCBI genbank database.
The genbank database shows the entire genome for the virus, as detected by PCR in the Paprotka study.
The genbank entry shows the DNA genome, and so it does not show the actual composition of the peptide/polyprotein.
A peptide, or protein, needs to be coded for by a gene, so the env 'mat peptide' shows the portion of the env gene that codes for the env polyprotein.

The Paprotka paper shows that the env gene was treated in the same way as the rest of the genome.
And it has been explained to you why the term 'putative' has been used in relation to the env polyprotein.

By focusing on the env gene, you seem to be missing the weakness of the study as a whole.
All the other genes, listed for the virus, are just as weak in evidence as you have been describing for the env gene. But the term 'putative' doesn't come into it. You are misinterpreting the use of the word. It's irrelevant for the argument that you are presenting.

In the Paprotka paper, as far as I understand, none of the genes for 22RV1/CWR-R1 have been: sequenced; cloned; isolated; nor had any viral RNA detected. So the paper is weak in evidence, as a whole, and it isn't helpful to single out the env gene, as it was processed in exactly the same way as the rest of the genome.

 

[Sam Carter]

...

I'm not sure if further research has been carried out on the 22Rv1/CWR-R1 (assumed) virus since the Paprotka study.​

There's this paper from Silverman, Bob. He says:

"The 22Rv1 cells are known to produce high titers of xenotropic murine leukemia virus-related virus (XMRV). Recent studies suggested that XMRV was inadvertently created in the 1990's when two murine leukemia virus (MLV) genomes (pre-XMRV1 and pre-XMRV-2) recombined during passaging of the CWR22 tumor in mice. The conclusion that XMRV originated from mice and not the patient was based partly on the failure to detect XMRV in early CWR22 xenografts. While that deduction is certainly justified, we examined the possibility that a closely related virus could have been present in primary tumor tissue. Here we report that we have located the original prostate tumor tissue excised from patient CWR22 and have assayed the corresponding DNA by PCR and the tissue sections by fluorescence in situ hybridization for the presence of XMRV or a similar virus...We show that neither XMRV nor a closely related virus was present in primary prostate tissue of patient CWR22. Our findings confirm and reinforce the conclusion that XMRV is a recombinant laboratory-generated mouse virus that is highly adapted for human prostate cancer cells."

 

[currer]

Given the recent findings of a novel virus, XMRV, found in some prostate tumors, we analyzed the BPH affected tissue for viral infection. We found that the majority of tissue from symptomatic BPH patients contained low levels of a virus not previously found in humans. Sequencing confirmed that the virus consists of 2 variants, is not XMRV, and likely produces a protein that has been related to inflammation in other species.

Interestingly, the exact virus sequence differs among patients, suggesting that upon infection of the tissue, the virus undergoes replication. Furthermore, sequencing revealed that the virus is likely transcriptionally regulated by androgens, which is consistent with the fact that cell growth in BPH is androgen-dependent and the classic non surgical treatment for BPH is inhibition of DHT (an androgen) production.
http://www.upmc.com/Services/urology/experts/research-faculty/Pages/denise-s-okeefe.aspx

I have a feeling that the continued argument about XMRV, its supposed ancestry, the elements that make it up etc etc. is becoming increasingly irrelevant. More research is needed and more data. The weakness of the Paprotka hypothesis to me is that it is built on such a small foundation. We know so little to justify all the claims being made. Silverman may be unable to find a retrovirus in the original CWR22 tumor tissue in the cell line but O'Keefe can find one in her patient samples.
Perhaps they should talk to each other.

 

[Bob]

There's this paper from Silverman, Bob. He says:

"The 22Rv1 cells are known to produce high titers of xenotropic murine leukemia virus-related virus (XMRV). Recent studies suggested that XMRV was inadvertently created in the 1990's when two murine leukemia virus (MLV) genomes (pre-XMRV1 and pre-XMRV-2) recombined during passaging of the CWR22 tumor in mice. The conclusion that XMRV originated from mice and not the patient was based partly on the failure to detect XMRV in early CWR22 xenografts. While that deduction is certainly justified, we examined the possibility that a closely related virus could have been present in primary tumor tissue. Here we report that we have located the original prostate tumor tissue excised from patient CWR22 and have assayed the corresponding DNA by PCR and the tissue sections by fluorescence in situ hybridization for the presence of XMRV or a similar virus...We show that neither XMRV nor a closely related virus was present in primary prostate tissue of patient CWR22. Our findings confirm and reinforce the conclusion that XMRV is a recombinant laboratory-generated mouse virus that is highly adapted for human prostate cancer cells."

Thanks for that Sam. It seems to be impossible to keep on top of all the research.
I've read the abstract of that paper, and it seems that they only investigated the original tumour tissue that was used to form the 22Rv1 cell line.
They confirm that XMRV was not present in the original tumour tissue.
So they confirm the conclusion that XMRV entered the cell line from an external source.

 

[currer]

The important question is whether there are MRV sequences in the human population and whether these are causing disease.
XMRV and its origins is a diversion, particularly as it appears that the sequences in patients are not XMRV. Arguments about the origin of the XMRV sequence should not be used to close research into other MRVs and their disease potential in humans.

It looks to me as if the Silverman paper says nothing about the generation of XMRV, merely that it appeared in the cell line at a later stage, We know that XMLVs are efficient contaminants of lab cell lines and have been found contaminating other cell lines than this one.This is not reassuring.

 

[Bob]

It looks to me as if the Silverman paper says nothing about the generation of XMRV, merely that it appeared in the cell line at a later stage...

Yes, that's what I took from the paper.
Except that they say that their findings confirm the Paprotka paper.
I've only read the abstract so far though.

 

[Mula]

Thanks for that, but it shows exactly the same information as the NCBI genbank database.
The genbank database shows the entire genome for the virus, as detected by PCR in the Paprotka study.
The genbank entry shows the DNA genome, and so it does not show the actual composition of the peptide/polyprotein.
A peptide, or protein, needs to be coded for by a gene, so the env 'mat peptide' shows the portion of the env gene that codes for the env polyprotein.

The Paprotka paper shows that the env gene was treated in the same way as the rest of the genome.
And it has been explained to you why the term 'putative' has been used in relation to the env polyprotein.

By focusing on the env gene, you seem to be missing the weakness of the study as a whole.
All the other genes, listed for the virus, are just as weak in evidence as you have been describing for the env gene. But the term 'putative' doesn't come into it. You are misinterpreting the use of the word. It's irrelevant for the argument that you are presenting.

In the Paprotka paper, as far as I understand, none of the genes for 22RV1/CWR-R1 have been: sequenced; cloned; isolated; nor had any viral RNA detected. So the paper is weak in evidence, as a whole, and it isn't helpful to single out the env gene, as it was processed in exactly the same way as the rest of the genome.

That does agree with what I have told you. The sequence in the NCBI and ENA do not contain an env gene. Not that you have disagreed with this. The mature peptide could be assumed to have been treated as the gag-pro-pol mature peptide, but it is the gag-pro-pol gene which is equivalent to the env gene. Sequences cloned from patients have an env gene, whereas 22rv1/CWR-R1 does not, but has a proposed "envelope polyprotein". In all likelihood this virus is an ERV. Silverman's data is limited to the original tumor. If this is an ERV it would not be in that tumor and could not be cloned into the various VPs identified in Urisman.

 

[Bob]

That does agree with what I have told you. The sequence in the NCBI and ENA do not contain an env gene. Not that you have disagreed with this. The mature peptide could be assumed to have been treated as the gag-pro-pol mature peptide, but it is the gag-pro-pol gene which is equivalent to the env gene. Sequences cloned from patients have an env gene, whereas 22rv1/CWR-R1 does not, but has a proposed "envelope polyprotein". In all likelihood this virus is an ERV. Silverman's data is limited to the original tumor. If this is an ERV it would not be in that tumor and could not be cloned into the various VPs identified in Urisman.

I don't think I agree with any of that, at all, except the single sentence about Silverman only investigating the original tumour. But seeing as you are not receptive to any of the evidence that I have presented, I think I will leave it now.

Except just to say that, just because the env gene is not clearly labelled, does not mean it's not there.
If you compare the genbank entry for VP62 with 22RV1/CWR-R1, then you'll see that the env gene of VP-62 is included in 22RV1/CWR-R1 exactly, except for just 2 nucleotide base pairs (out of 1938) which are different.

 

[Mula]

I don't think I agree with any of that, at all, except the single sentence about Silverman only investigating the original tumour. But seeing as you are not receptive to any of the evidence that I have presented, I think I will leave it now. Except just to say that, just because the env gene is not clearly labelled, does not mean it's not there. If you compare the genbank entry for VP62 with 22RV1/CWR-R1, then you'll see that the env gene of VP-62 is included in 22RV1/CWR-R1 exactly, except for just 2 nucleotide base pairs (out of 1938) which are different.

It would be helpful to know what you do think you agree with? The 22rv1/cwr-r1 sequence could be summed up this way. It is from a combined cell line created from 22rv1 and cwr-r1 cells, which has no envelope gene, and would best fit the description of an ERV. The sequence is present in the the NCBI and the ENA databases and has been updated on two occasions without any alteration to either database to include an envelope gene.

 

[Firestormm]

The important question is whether there are MRV sequences in the human population and whether these are causing disease. XMRV and its origins is a diversion, particularly as it appears that the sequences in patients are not XMRV.

No. It only appears thus because Mikovits said so and some people desperately want to believe this. It has never been proved either by her or by others including Lo and Alter. More might come from the 'Lipkin Study' but this is another example of wishful thinking and a belief in the person making the statement in my opinion.

I repeat the ONLY paper allegedly studying CFS patients was looking for and allegedly found XMRV. The publication of Lo et al. then added additional confusion to the mix - hence why (largely) participants in the Lipkin Study are looking for XMRV and MRV's in CFS patients.

Just because XMRV could not be found or Lombardi et al corroborated, in the blinded studies that we have seen, does not mean that Mikovits' later speculations have any more validity i.e. 'Well if it wasn't XMRV it must be MRV's'.

When Lipkin reports (whenever that might be) there will still be those who hone in on minutae claiming that it makes a difference and the whole thing should be done again. It will remain a never-ending argument and source of misplaced belief as well as distress.

When Mikovits made that comment in the NYT or WSJ (I forget) about Lipkin being definitive and 'if we were wrong we were wrong, that's science' (or something similar), I really thought for a moment that she was coming clean and being far more realistic that I have heard her be before.

I only hope that she sticks to this theme when the results are known but a large part of me thinks she won't and that she will continue as she has spreading/adding to the speculation and discord. Even if she doesn't, then more silence will only be used to continue the disinformation.

Hopefully I am wrong. I think a great many people have moved on some time ago.

 

[currer]

Quote "I think a great many people have moved on some time ago."

Ah, Firestormm.
Still keen on science by consensus, I see.

 

[Firestormm]

Quote "I think a great many people have moved on some time ago."

Ah, Firestormm.
Still keen on science by consensus, I see.

I meant the patients including those 'tested'. But whatever.