Author Topic: Biochemical, inhibition, inhibitor resistance studies XMRV reverse transcriptase  (Read 1003 times)

jemal

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Biochemical, inhibition and inhibitor resistance studies of xenotropic murine leukemia virus-related virus reverse transcriptase

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Tanyaradzwa P. Ndongwe1, Adeyemi O. Adedeji1, Eleftherios Michailidis1, Yee Tsuey Ong1, Atsuko Hachiya1, Bruno Marchand1, Emily M. Ryan1, Devendra K. Rai1, Karen A. Kirby1, Angela S. Whatley1, Donald H. Burke1,2, Marc Johnson1, Shilei Ding3, Yi-Min Zheng1, Shan-Lu Liu1,3, Ei-Ichi Kodama4, Krista A. Delviks-Frankenberry5, Vinay K. Pathak5, Hiroaki Mitsuya6, Michael A. Parniak7, Kamalendra Singh1 and Stefan G. Sarafianos1,2,*

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We report key mechanistic differences between the reverse transcriptases (RT) of human immunodeficiency virus type-1 (HIV-1) and of xenotropic murine leukemia virus-related virus (XMRV), a gammaretrovirus that can infect human cells. Steady and pre-steady state kinetics demonstrated that XMRV RT is significantly less efficient in DNA synthesis and in unblocking chain-terminated primers. Surface plasmon resonance experiments showed that the gammaretroviral enzyme has a remarkably higher dissociation rate (koff) from DNA, which also results in lower processivity than HIV-1 RT. Transient kinetics of mismatch incorporation revealed that XMRV RT has higher fidelity than HIV-1 RT. We identified RNA aptamers that potently inhibit XMRV, but not HIV-1 RT. XMRV RT is highly susceptible to some nucleoside RT inhibitors, including Translocation Deficient RT inhibitors, but not to non-nucleoside RT inhibitors. We demonstrated that XMRV RT mutants K103R and Q190M, which are equivalent to HIV-1 mutants that are resistant to tenofovir (K65R) and AZT (Q151M), are also resistant to the respective drugs, suggesting that XMRV can acquire resistance to these compounds through the decreased incorporation mechanism reported in HIV-1.

http://nar.oxfordjournals.org/content/early/2011/09/08/nar.gkr694.full

jemal

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They are talking about mutants of XMRV and said mutants have acquired resistancy to certain antiretrovirals. This is significant I think?

omerbasket

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It seems to me that these are artificial mutants, meaning, not mutatns that they have found in nature, but mutants that they intenitionally created.

jemal

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It seems to me that these are artificial mutants, meaning, not mutatns that they have found in nature, but mutants that they intenitionally created.

Thanks. Then it is less significant, I guess :(

I haven't read the full paper yet, but it does seem they were artificially created:

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Drug resistant XMRV RT mutants Q190M and K103R (equivalent to HIV-1 Q151M RT and K65R) were generated by site-directed mutagenesis using forward and reverse primers 2 and 3.

(Isn't it a bit scary these people are making mutant retroviruses by the way?)
« Last Edit: September 09, 2011, 08:08:55 AM by jemal »

Tango

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Gammas don't need reverse transcriptase.  They propagate in blood using colonal expansion.

You don't get replication or recombinants until they get to certain organs.

This being the case, APOBEC is irrelevant, because it is inactive in replicating cells.  Glycogag also protects against APOBEC.

They are not saying it won't make you sick.

Mutants have already been found in Lo et al.
« Last Edit: September 09, 2011, 10:15:41 AM by V99 »
"I suspect there have been a number of conspiracies that never were described or leaked out. But I suspect none of the magnitude and sweep of Watergate." Woodward

"I would favor any name that does not impose (or give the appearance of imposing) taxonomic preconceptions on the nomenclature." Coffin

joy

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Cowa bunga teenage mutant retrovirus  :P

seriously
What does this means in plain English - the for Dummies version that is.
The trick is to keep breathing.

Tango

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It is a study looking at reverse transcriptase in XMRV and HIV.  But HGRVs use mitosis.

So what does it tell us - not much in some respects.

We know a gammas don't use this to propagate.  Gammas are not like HIV.

They don't mutate much because they don't use reverse transcriptase.

Total load of bollocks.
« Last Edit: September 09, 2011, 11:05:34 AM by V99 »
"I suspect there have been a number of conspiracies that never were described or leaked out. But I suspect none of the magnitude and sweep of Watergate." Woodward

"I would favor any name that does not impose (or give the appearance of imposing) taxonomic preconceptions on the nomenclature." Coffin

Gerwyn

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http://nar.oxfordjournals.org/content/early/2011/09/08/nar.gkr694.full

 
Do these people never read they should compare the reverse transcriptase activity of HIV and moloney murine leukemia virus another gammaretroviruses.This study has in fact been done and the results are almost identical. Moloney murine leukemia virus is a deadly pathogen
 
It does however draw attention to how absurd Gregory Tower,s model of gammaretroviral evolution was in his latest spinscience study

They are assuming that all retroviruses replicate primarily by RT----this lack of knowledge is quite incredible
« Last Edit: September 09, 2011, 06:44:57 PM by Gerwyn »

Tango

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"I suspect there have been a number of conspiracies that never were described or leaked out. But I suspect none of the magnitude and sweep of Watergate." Woodward

"I would favor any name that does not impose (or give the appearance of imposing) taxonomic preconceptions on the nomenclature." Coffin

Dr. Yes

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This study is mainly basic science on the properties of XMRV's reverse transcriptase enzyme (RT) without drawing conclusions about the life cycle of XMRV itself.  They did comparative analyses between XMRV RT, HIV-1 RT and MoMLV RT, in order to add to the knowledge base about RTs in general; this study does not attempt to say anything about XMRV as a pathogen.

They found that, in vitro, XMRV RT showed relatively better ability to avoid 'mismatch' errors while creating DNA strands (as compared to HIV-1 or MoMLV), but not in vivo (as compared to HIV-1 and related amphotropic MLV); therefore this study is inconclusive about how relatively mutation-prone it really is.

They looked at the known mutations for HIV-1 that make it resistant to AZT and tenofovir and engineered XMRV RT clones with mutations at the equivalent sites.  Their engineered RTs did turn out to be less susceptible to AZT and tenofovir.  That suggests two things - that XMRV has the potential to develop such resistances if the appropriate mutations evolve, and that the same mechanisms by which HIV-1 develops resistance to these drugs can be used by a relatively distantly related retrovirus like XMRV.  Again, the latter point is more about understanding RTs via comparison than anything else.

It is useful to know as much as we can about XMRV RT, particularly what drugs might inhibit it, as it does indeed have to replicate in certain tissues and at some points in the infectious cycle.  But as for their conclusions about likely mutation rates or recombination rates, I think the differences in this study's in vivo results suggest that they can't extrapolate yet from their in vitro ones.  And of course, as G pointed out, anyone trying to predict mutation rates of MLVs in the wild via in vitro-observed mutation rates has to be aware of the likelihood that there will be little if any variation in the blood compartment, where replication takes place primarily via clonal expansion (where there will be no mutation) rather than RT activity.  In blood at least, any relative differences in RT fidelity are likely to be irrelevant; the bulk of wild variation of the virus will not show up in blood isolates unless the actively replicating virus in tissue reservoirs actually makes it to the blood.
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joy

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Love you Dr YES Understood that  :-* :-* :-*
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currer

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Thanks for the explanation, Dr Yes.

This helps explain why the Switzer paper found the expected variation in xmrv because it looked in prostate tissue.


I do wish people would get on with tissue studies.
« Last Edit: September 10, 2011, 07:17:34 AM by currer »

jemal

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Thanks for the explanation Dr. Yes, this study was way out of my league. Too bad it's not as significant as I first thought (at first glance).