More on Nicholas Bennett and Montagnier's RT activity 28 September 2004
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Eleni Papdopulos-Eleopulos,
Biophysicist
Department of Medical Physics, Royal Perth Hospital, Western Australia, 6001,
Valendar F Turner, John Papadimitriou, Barry Page, David Causer, Helman Alfonso, Sam Mhlongo, Todd Miller, Christian Fiala

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Re: More on Nicholas Bennett and Montagnier's RT activity

More on Nicholas Bennett and Montagnier's RT activity

In his rapid response: "Various responses to the Perth Group", 9 September, Nicholas Bennett wrote: "And I was boggled, absolutely stunned, to read at the end of their reiteration of misunderstandings regarding RT that: "This is the first time that we have read (or heard) that the specificity of the retroviral reverse transcriptase can be determined on the basis of the "RELATIVE amount"." This clearly highlights their inexperience in the field of practical scientific research, or more likely, common sense. It is mind-numbingly obvious: endogenous "RT" activity can be detected in certain cells at certain times, the Perth Group have repeatedly given us references to support this which have not been denied. This "RT" might indeed be the reverse transcriptase component of telomerase, the mitochondrial DNA polymerase, or perhaps other less well-known enzymatic activities. But how does one distinguish it from viral RT? By simply comparing the activity to a baseline in normal, uninfected cells. This is common sense, in the same way that one can demonstrate an increase in lung cancers in smokers above the level seen in non-smokers (so-called "attributable risk"). The trick is called "subtraction" or sometimes "division" to get a relative increase. I have provided example values from a real experiment here before, but shall do so again. Blank buffer-only control: 4-6 counts. Uninfected cells: 20-30 counts. Infected cultures: 100-2000+ counts (depending on efficiency of infection/transfection and days post-infection) Positive control: 0.5ul MMLV-RT 10,000+ counts. Sometimes the uninfected cells give higher background levels, say 50-100 counts, in which case unless the virus counts are also much higher they have to be discarded. The actual counts are based on variables such as stringency of the SSC washes, age of radioisotope, as well as the amount of RT present: this is why positive and negative samples are run every time. I typically expressed virus RT as a value relative to uninfected cells, since in a 200-day long experiment it best accounted for changes in radioisotopes, buffers etc. In a typical 14-28 day replication experiment, if all samples are run at once there's no need to do that. If the Perth Group's original rebuttal of Montagnier's work was based on a mere lack of understanding of relative RT expression (exactly the same as seen in any other enzyme assay) then it's almost laughable, if it weren't so sad. So much angst created from ignorance."

In "HIV" research (including in Montagnier's and Gallo's 1985 and 1984 papers respectively) reverse transcriptase activity is determined by the use of the template primer An.dT15. This template-primer may be transcribed by the "HIV" reverse transcriptase, by the reverse transcriptase of endogenous retroviruses, by the reverse transcriptases of other infectious agents which may be present in the cultures, by the cellular reverse transcriptase, as well as by the cellular DNA polymerases, beta and gamma. Since the early 1970s retrovirologists have spared no effort trying to develop methods to discriminate at least between the activity due to the enzyme reverse transcriptase, and the activity due to the cellular DNA polymerases. To date all efforts have been unsuccessful. Nobody has ever suggested Nicholas Bennett's "method".

Not surprising. There is no scientific basis for the claim that the level of reverse transcription by the cellular reverse transcriptases, including the reverse transcriptase of endogenous retroviruses as well as the DNA polymerases beta and gamma is "20-30 counts" and of the "HIV" reverse transcriptase "100-2000+ counts". In Montagnier's 1984 paper [1] the level of RT activity by the DNA polymerases beta and gamma is at least as high as that of the "HIV" polymerase. It is equally unscientific to discard data which do not meet with one's expectation.

Enzymes are released into the bloodstream following myocardial infarction. One of these, creatine phosphokinase (CK), is used diagnostically. However, large amounts are also present in skeletal muscle and brain. Because of this many other conditions such as, muscular dystrophies, myopathies, polymyositis, stroke, surgery, convulsions, trauma involving muscles cause an elevation of CK. Even intramuscular injections may cause diagnostic confusion. It is not possible to claim that a particular level of enzyme is the result of a particular cause.

Repeat, scientifically it is not possible to determine what is the cause of reverse transcriptase activity on the basis of "RELATIVE amount".

Reference

1. Rey MA, Spire B, Dormont D, Barre-Sinoussi F, Montagnier L, Chermann JC. Characterization of the RNA dependent DNA polymerase of a new human T-lymphotropic retrovirus (lymphadenopathy associated virus). Biochem Biophys Res Commun 1984;121(1):126-33.

Competing interests: None declared