Generation of replication-competent retroviruses--combining PreXMRV-1 and PreXMRV-2

Sushi

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Delviks-Frankenberry K, Paprotka T, Cingöz O, Wildt S, Hu WS, Coffin JM, Pathak VK.

http://www.ncbi.nlm.nih.gov/pubmed/23966380

Abstract
We previously identified two novel endogenous murine leukemia virus proviruses, PreXMRV-1 and PreXMRV-2, and showed that they most likely recombined during xenograft passaging of a human prostate tumor in mice to generate xenotropic murine leukemia virus-related virus (XMRV).

To determine the recombination potential of PreXMRV-1 and PreXMRV-2, we examined the generation of replication-competent retroviruses (RCRs) over time in a cell culture system. We observed that either virus alone was noninfectious and the RNA transcripts of the viruses were undetectable in the blood and spleen of nude mice that carry them.

To determine their potential to generate RCRs through recombination, we transfected PreXMRV-1 and PreXMRV-2 into 293T cells and used the virus produced to infect fresh cells; the presence of reverse transcriptase activity at 10 days postinfection indicated the presence of RCRs. Population sequencing of proviral DNA indicated that all RCRs contained the gag and 5' half of pol from PreXMRV-2 and the long terminal repeat, 3' half of pol (including integrase), and env from PreXMRV-1.

All crossovers were within sequences of at least 9 identical nucleotides, and crossovers within each of two selected recombination zones of 415 nucleotides (nt) in the 5' untranslated region and 982 nt in pol were required to generate RCRs. A recombinant with the same genotype as XMRV was not detected, and our analysis indicates that the probability of generating an identical RCR is vanishingly small.

In addition, the studies indicate that the process of RCR formation is primarily driven by selection for viable cis and trans elements from the parental proviruses.
 
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Bob

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Delviks-Frankenberry et al. said:
A recombinant with the same genotype as XMRV was not detected, and our analysis indicates that the probability of generating an identical RCR is vanishingly small.
Interesting.

So I think that means that their earlier published paper is flawed.

Is it OK to say "we told you so"?
 
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barbc56

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So I think that means that their earlier published paper is flawed.

Is it OK to say "we told you so"?
Are you talking about the Lipkin et. al. or Mikovitz's study?

So you are saying this study says xmrv is a play in our illness.

I'm so foggy, I can't follow the logic of the study.

Thanks.
 

Bob

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@barbc56

Hi barb, I'm only referring specifically to the Paprotka recombination paper that concluded that XMRV was formed only in a single cell line after recombination of preXMRV1 and preXMRV2.

This latest paper (I've only got access to the abstract) seems to suggest that this was highly unlikely.

If this is the case, then this means that XMRV was not created as Paprotka et al. originally suggested... i.e. a recombination of preXMRV1 and preXMRV2 in the 22Rv1 cell line.

So it seems like quite a major climb-down, because the whole issue of lab contamination was more or less sealed on the basis of the Paprotka study, which was very firm in its conclusion.

My memory is a bit fuzzy on it all now. I'd need a refresher to get up to speed with it all. But there were quite a number of reasons why I found the Paprotka study hard to accept.

XMRV could still be a lab contaminant, but this paper means that we don't know where it originates. It could have originated in a lab as a contaminant via a different mechanism, or it could have originated in the wild, and subsequently contaminated lab mice. Perhaps XMRV is not purely a lab contaminant.

But I've only read the abstract of this paper, so I may have misinterpreted it, or the significance of it.

Remember that PreXMRV2-like gag sequences have been detected in some wild mice.
http://forums.phoenixrising.me/inde...-diversity-in-laboratory-and-wild-mice.18402/

Then there was the paper that demonstrated that there were a large number of possibilities for a recombination event that could create XMRV. But I can't remember the details of that paper. Perhaps it's this paper:
http://www.retrovirology.com/content/8/S1/A235
 
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alex3619

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I think this is the second thread on this paper, but the first was for a pre-publication announcement? I could be wrong about that, but may have read it somewhere else. It is an August paper.

This does not prove that the pre-XMRV viruses can't combine to make it, but it does show that the probability is low. However much I disagreed with the Paprotka paper they do cite the probability as tiny. The argument, if I recall correctly, was that the proof was the virus existed, ergo it must have happened. Improbable things do happen.

My counter argument was that in all of nature, in all of time, if such an improbable event could happen in a lab, then its more likely to have happened in nature, given a wide host range.

So far no source of XMRV in nature has been found.

The Lipkin et. al. study makes it clear that the probability that any ME or CFS patient has XMRV is miniscule.
 
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What this paper is showing is that when PreXMRV 1 and 2 are mixed with another cell line 293T that the virus it generated produced multiple replication competent retroviruses. They went on to say that they did not produce XMRV RCR's. So what were these replication competent retroviruses. It is also a well know fact now that they found Mlv's in cell lines used in cancer research, even in cell lines stored in the same lab where xenografting has taken place. But they found no mlv's in cancer cell lines where no xenografting was done.

And this is from a paper just published in July 2013: "Many additional cell lines that underwent xenotransplantation in mice have been shown to harbor murine gammaretroviruses. Such retroviral infection poses the threat of not only confounding experiments performed in these cell lines via virus-induced changes in cellular behavior but also the potential infection of other cell lines cultured in the same laboratory. Thus, the possibility of xenotropic retroviral infection of cell lines may warrant additional precautions, such as periodic testing for retroviral sequences in cell lines cultured in the laboratory."
 

barbc56

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What does that mean?
Sorry, it's an american saying. I can't even remember it's origin but may be a movie? Or not. Maybe I dreamed it?

All the above are possible as the last few days have been the pain/ fog from hell.

Take care.:)
 
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Does anyone remember the Jacque Monkeys they injected with XMRV? The monkeys later showed XMRV was rampant throughout their bodies and organs during the autopsy. I tested positive for XMRV and I thought there was a lot of conflicting information out there after it was announced XMRV was not it.
Maybe it was just the tip of the iceberg and we can't see it yet. Only history will tell. If the above post is accurate (this paper is showing is that when Pre XMRV 1 and 2 are mixed with another cell line 293T that the virus it generated produced multiple replication competent retroviruses) it may shed some light on it. Onward through the Fog!
 

barbc56

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Does anyone remember the Jacque Monkeys they injected with XMRV? The monkeys later showed XMRV was rampant throughout their bodies and organs during the autopsy. I tested positive for XMRV and I thought there was a lot of conflicting information out there after it was announced XMRV was not it.
Maybe it was just the tip of the iceberg and we can't see it yet. Only history will tell. If the above post is accurate (this paper is showing is that when Pre XMRV 1 and 2 are mixed with another cell line 293T that the virus it generated produced multiple replication competent retroviruses) it may shed some light on it. Onward through the Fog!
Contamination can show up all over the place and that's the problem with xmrv. The tissue samples could have very easily been contaminated.

The Lipton study showed that xmrv is really a moot issue. Other viruses may not be which is to be determined.
 

Bob

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I've seen the full paper now, and it confirms that the likelihood of XMRV being formed from a recombination of the pre-XMRV1 and pre-XMRV2 viruses, is vanishingly small. They've ruled it out, on the basis of the nature of genetic recombinations that they've seen in the study.

This is interesting, as the whole XMRV debate was closed down on the back of the original Paprotka et al. study that firmly concluded that XMRV was formed in the cell line from pre-XMRV1 and pre-XMRV2. My complaint at the time was that the conclusions were inappropriate and far too premature.

There is no speculation in this paper as to the possible origins of XMRV or how it entered the cell line in the first place.

There's an interesting extract in the paper, that I don't understand (my emphasis):
"...the possibility that PreXMRV-1 and PreXMRV-2 are transcriptionally active in some mouse tissue or at some stage in the life of the mouse cannot be excluded. It is also possible that treatment of Hsd nude mice with androgens during the xenograft passaging led to the activation of these proviruses, thereby contributing to the formation of XMRV."

It's news to me that retroviruses can be activated via the action of hormones. This is one of the discussions that we used to have in relation to XMRV. People said that a retrovirus couldn't infect men and women at different rates, so XMRV couldn't be a factor in ME. Well, this piece of text seems to suggest that hormones can affect the behaviour of retroviruses. But I don't understand why yet.
 
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Dan_USAAZ

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It's news to me that retroviruses can be activated via the action of hormones. This is one of the discussions that we used to have in relation to XMRV. People said that a retrovirus couldn't infect men and women at different rates, so XMRV couldn't be a factor in ME. Well, this piece of text seems to suggest that hormones can affect the behaviour of retroviruses. But I don't understand why yet.
Androgen Stimulates Transcription and Replication of Xenotropic Murine Leukemia Virus-Related Virus

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2812331/
 
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This makes sense to me because at the time that I had my blood drawn for the WPI XMRV test I was receiving shots of testosterone trying to raise my low testosterone levels! For what it is worth, the added testosterone did not raise my testosterone levels until I switched to a bio-identical cream. After six months on the shots, it actually lowered my testosterone levels.

Still it probably had something to do with my positive XMRV test result.

XMRV is still confusing, it's a lot like the magic bullet.