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« Richard Strohman: Maneuvering in the Complex Path from Genotype to Phenotype | Main | Celia Farber: Moving AIDS to the Third World »

October 30, 2006

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Otis

It is with a modicum of trepidation that I leave the comments to this exchange open but given the higher level to which comments in general have risen lately (see here for example), and the incredible surge in the number, and change in distribution, of referring addresses (more on that later in the week), I am going to risk the intrustions of the peanut gallery comment crew whose briefest of bios can be found here.

But fair warning, any comment that is in the least bit disrespectful will be deleted with no further remark and the poster's IP banned forever with no appeal. We are seriously funny when we wish to be, and deadly serious all the time.

ErnestD

On the one hand, the 2000 paper that Dr. Depressed refers to (along with several later pubs on the same topic) contains, in my view, some of the rigor issues that plague the HIV research field and which Professor Duesberg and others here have called out. But on the other hand, this nuclear import paper contains some interesting data on HIV replication, and I would be appreciative of any explanations that Professor Duesberg or another expert could offer.

Dr. Depressed quotes a Stanford website's paraphrase of the abstract of a 2000 Cell paper from Pierre Charneau's group, and you, Professor Duesberg, reassure the Dr. by correctly pointing out the abstract's lack of reference to viral replication.
However, I would respectfully submit that the authors' goal in their abstract is to convey what they consider to be their main point: that a feature of the viral pre-integration complex is necessary for nuclear import. In the paper itself, the authors do present evidence of viral replication by two viruses in both primary cells (PBLs) and a cell line (MT4), but no such replication of an HIV lacking a functional "central DNA flap." With this defective virus in hand, the authors use in vitro and culture experiments to eliminate, in their view, several possible stages of the viral life cycle at which the virus could be defective: reverse transcription (they find DNA in the cells); integration (a mutant viral construct integrates in vitro); and "viral production" (viral DNA is inserted into cells to bypass the need for nuclear import...and viral protein is generated).

I mentioned weaknesses in the 2000 import paper. Specifically, I refer to the lack of controls. Take Figure 3A, for instance, representing the production of viral protein after transfection of cells with a viral plasmid. We can of course take potshots at this artificial, highly contrived situation and speculate as to its relationship with what happens in a cell in an organism, but, then again, scientists have to start somewhere. My criticism of the experiment is that it lacks controls. If p24 was made by all cells transfected with the various viral constructs, there should be a transfected control that shows no protein production. This is similar to the situation seen in most cell culture experiments (and even animal "model" work) when viral particles are involved: the "mock" infections or "mock" inoculations are not sufficiently related to the real infection or inoculation procedure to rule out artifacts.

At the same time, though, I have a difficult time examining Figure 2 (where three viruses, including the flap mutant, are used to infect cells, and only the two non-mutants result in measurable RT activity) in Charneau's paper without concluding that virus replication may indeed be occurring in culture. I am well aware of the criticisms of RT assays (including that all cells are capable of producing RT enzymes). But this one experiment is refreshingly well-controlled; equal amounts of viral particles (as determined by protein levels) are placed on the cells, but one type of virus, with really minimal differences from the others at a biochemical level, does not result in RT production. If substances in the virus prep are killing cells and releasing endogenous RT activity (assuming sufficient endogenous to measure), then why would the third virus preparation not kill cells and release RT? If it is the virus, same question. There are only two explanations I can think of, other than outright falsification and manipulation, which I reject: 1)the authors are correct in proposing that a biological functionality of the virus (here, the DNA flap) is needed for viral entry into the nucleus and eventual replication of the virus (including RT release into the culture supernatant with newly-formed virions); or 2)the absence of the central DNA flap somehow keeps the mutant virus from killing the cells or otherwise mediating a release of RT activity into the culture supernatant.

My questions for you, Professor Duesberg, are the following:
1) Do you maintain that HIV cannot infect non-dividing cells, even in culture?
2) Although both RT assays and p24 assays may have serious problems, how is it possible that well-controlled experiments, like that shown in Figure 2A of the above paper, result in several viruses producing markers of viral replication, while a third (or other) virus does not?

I look forward to your response.
-ED

Otis

Dr. ED,

Very interesting and good question set and observations in my humble estimation.

However, I cannot say how quickly you might receive a reply from Prof. Duesberg, although I am certain you will, and quite a fullsome one, in due course.

But in the interim, and to avoid this becoming a series of posts that might to an unsophisticated reader seem very similar to your own, but which in fact would not be any more than pretentious regurgitation of material cobbled from internet search engines, I am going to now close these comments until I read something from the very busy, but very conscientious iconclast prof.

If anyone really, really thinks that they have material that is so important that it cannot wait one or two days to be posted here, they can send me an email containing it, and perhaps I will open the post briefly again.

I am, or try to be, even-handed in how people who write to YBYL are treated. Courtesy begets courtesy, always, and the DTs get treated in kind as well.

Otis

Dr. ED,

Prof. Duesberg informs us that he has been extremely busy the past weeks and has not had the time to properly read the Cell paper you are so interested in.

However, "an undergraduate intern in the lab, who is doing an independent study of retrotransposition, wondered if she might have a go at your question."

Since you are clearly interested in a technical, scientific discussion of these biochemical details and not simply in arguing with prof. Duesberg, I trust the following will be satisfactory.

In the event you wish to pursue these erudite, even esoteric, matters further, you may write to Simone (retrosimone@gmail.com), and if your ensuing correspondence appears to hold interest to other than a very small circle of "molecular retrovirologists", I will publish it here. Until such time, these comments are resealed. [Otis]

"A discussion of the“lentivirus” mutant created by the Pasteur and its wild type equivalent to be used by Stanford researchers interested in engineering plasmids for safe gene therapy.

By Simone

A genetic property of HIV: “…plus strand overlap, the central DNA flap, is created following a strand displacement in the center of the genome.” (14)

Response:

This looks like Step 6 of “reverse transcription of retroviral genomes”, “plus strand DNA synthesis” shown in Fig 2, Temin and Hu, (15) not unique to “lentiviruses”.

The eight steps (15) leading to a “pre-integration complex” could apply to LTR retrotransposons with at least two complete RNA templates “handy”.

It is important to note that nothing in the reactions described by Dr. ED requires the presence of env.

Cell paper: “… a feature of the viral pre-integration complex is necessary for nuclear import.” (14)

Response: You (Mr DNA) have no business being in the nucleus without the secret handshake (ability to promote transcription).

Dr ED: “At the same time, though, I have a difficult time examining Figure 2 (where three viruses, including the flap mutant, are used to infect cells, and only the two non-mutants result in measurable RT activity) in Charneau's paper without concluding that virus replication may indeed be occurring in culture.”

Response:

This is the central question, applying to retrotransposons as well, the “flap mutant” seems to have a disabled transcriptional apparatus, thus it will not produce the standard markers.

“Replication” in this experiment, testing the necessity of the “central DNA flap (cDF)”* (16) means nothing more than demonstration of transcription, perhaps from plasmid as an extra-chromosomal element, resulting in the RT and p24.

In the case without the cDF – no transcription, no RT and no p24! This would also happen with a retrotransposon, since the DNA with LTR promoter “flap”* also makes it back to the nucleus from the cytoplasm.

Dr ED: “… but one type of virus, with really minimal differences from the others at a biochemical level, does not result in RT production…”

Response: More than a “minimal difference”, if the transcriptional apparatus is disabled in the mutant.

Dr ED says, “the absence of the central DNA flap somehow keeps the mutant virus from killing the cells or otherwise mediating a release of RT activity into the culture supernatant.”

Response: This is the “safety” issue, somehow the virus needs to be disabled so that vectors can be injected into cells without killing them. “(T)he genomic viral DNA is inserted into the target genome as a promoter-less sequence. The lack of active viral promoter avoids both the possible transcription of the viral sequence and detrimental effects on eukaryotic gene expression.” (16)

Here it is better to be safe whenever injecting humans, of course, but does not prove that wild type HIV transfers are hazardous in vivo.

As far as the question re dividing cells; this is a true “replication” via mitosis of any provirus within the genome.
Can a retrovirus enter a non-dividing cell? Why not, it may have an important message for necessary “signal transduction” to the nucleus.

* “plus strand strong stop DNA, the cis-promoter region (ppt-U3-R-U5-PBS)” (15)

14. Zennou V, Petit C, Guetard D, Nerhbass U, Montagnier L, Charneau P. HIV-1 genome nuclear import is mediated by a central DNA flap. Cell. 2000. 101:173-85.

15. Temin and Hu, Science, 250, 1228 (1990)

16. Helix-HIV lentivirus vectors

Otis

The discussion that is begun above was indeed continued, and with profit to both of the discussants as can be seen here.

We hope that this exchange might serve as an example of how even totally anonymous correspondence between two persons of good faith, ie. who are interested only in the written communication and not the "identity bona fides" of the other*, can be stimulating, cross-fertilizing and possibly of some real interest.

Quite a far cry from the usual such internet "debates", as they are called by some.

*On Jan 17, Simone received the following email from Dr. ED, which casts into some shadow the good faith assumption above:

Simone,

I apologize in advance but I will be unable to continue our correspondence. This morning I received a message from an interested party who had read our correspondence on Mr. Barnes' page. This person made the case that you are not Prof. Duesberg's undergraduate student and presented evidence that I sadly must admit was very compelling.

Otis introduced you as Prof. Duesberg's undergraduate student and you confirmed your student status with me later. I wrote you since Prof. Duesberg would not write me. My correspondent's info places you on the other side of the continent from Prof. Duesberg's lab. Anonymity is fine but correspondence that starts with a lie is ill-fated. I do not appreciate that you and Otis lied to me.

I enjoyed our correspondence and I'm sorry it has to end.

-ED


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