Re: New and repeated questions and requests to Brian Foley 8 June 2004
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Brian T Foley,
HIV Researcher
Los Alamos National Lab, Los Alamos, NM 87545

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Re: Re: New and repeated questions and requests to Brian Foley

The Perth group wrote:
“…
Q1. If the virus preparation is not 100% pure but contains extraneous elements such as cellular vesicles which contain proteins and poly(A)-RNA by what means can anyone prove what is cellular and what is viral? For example, how can anyone determine whether a protein with molecular weight of 41000 or a poly(A)-RNA present in the impure virus preparation is cellular or viral?

Note that Brian Foley agreed that the poly(A)-RNA is not specific to retroviruses and that the antibody-antigen reaction is also non-specific.
…”

The poly-Adenylate tail of an RNA is indeed not specific, but the rest of the RNA is specific. Likewise although antibodies, even monoclonal antibodies, are not 100% specific, the specificity of polyclonal antibodies is closer to 98% specific than it is to 90% specific. The specificity of monoclonal antibodies can be greater than 99.9% specific, i.e. a monoclonal antibody can often be shown to bind to only one protein in a mix of more than 10,000 proteins.

There are textbooks on molecular biology which describe in great detail the hundreds of different ways that proteins and genes can be analyzed. It is really not my responsibility to be a one-on-one tutor for a group of unwilling students who have no interest in the subject matter, but only want to claim that they have all the answers before doing any studying themselves.

I might be inclined to be more patient with answering the Perth group’s repetitious and purposefully ambiguous questions, if they would answer mine, such as “Where did you ever get the idea that there was one set of rules for the purification/characterization of retroviruses?” or “Can you show me ONE example of a virus that has ever met the rules that you think exist?”

Because people who understand molecular biology already know that the Perth group has no clue what they are talking about, and most people who do not understand molecular biology will never take the time to read a few textbooks on the subject, I will attempt to explain this in other terms. DNA is just an information transfer system. It is very much analogous to the binary code used in computers. DNA without a cell and proteins to interpret it, is similar to ones and zeroes on a disk or tape, with no computer to read it. Prior to the mid 1970s geneticists had figured out that DNA and not proteins, was the genetic information, but they had little ability to read that information. The basic method of studying genetics was to induce mutations with X-rays or other DNA-damaging agents and then attempt to find which defective processes matched up with damage to which region of a genome. It was sort of like making scratches on a computer disk and then trying to find out whether the scratch inactivated Microsoft WORD or Microsoft EXCEL. Damage to key genes such as those involved in DNA replication were much more difficult to study, than damage to visible but non-important traits such as fruit fly eye color. This is analogous to changing the data in a single cell of a spreadsheet, rather than changing some part of the operating system or the EXCEL.exe program itself.

Viruses are very small and very simple organisms. There are viruses that infect bacteria, called “phages” and viruses that infect almost all other life forms. They are so simple that it is very debatable whether or not they can be considered to be “alive”. They require cells and cellular proteins for much of their ability to replicate. Computer viruses are likewise able to replicate only with the aid of both computers and other software (usually the operating system, but sometimes a program such as Microsoft WORD which can replicate viruses known as macro viruses which use the WORD macros to copy themselves, or a program such as a mail program to spread a type of virus known as a worm to addresses found in the mail program’s address bool).

There is nothing really special about either biological viruses or computer viruses, in terms of the methods used to study them as compared to the methods used to study the more complex biological organisms or the more complex computer programs. Biological viruses move from cell to cell by packaging up their genome in some proteins into what is known as a virion or viral particle. The ability to separate the viral particle from the host cell, made them ideal for studying their genetics, in the early days of genetics before monoclonal antibodies, DNA sequencing, cloning and other tools became available. The genetics of the Lambda phage Lac operon was one of the very first successes in molecular biology. While it took decades of work by dozens of people working in several different labs to gain a crude understanding of the Lac operon using those pre-cloning techniques. Today, a new operon in a newly discovered phage can be studied and understood in much greater detail by a single graduate student in a single lab working for 4 years. It’s similar to the difference between writing a program in BASIC to manage your household finances on a Comodore 64 computer, vs using QUICKEN or some other off-the-shelf accounting package on a modern computer.

This ability to obtain a virus genome when it is outside the host genome was quite critical in the days before cloning made it possible to obtain any gene or genetic fragment separated from the rest of the genome. It was similar to being able to obtain a program on a 5 and ¼ inch floppy disk, such that it could be moved from one computer to another, before anyone knew how to use e-mail or wires to move data from one machine to another. Maybe the use of punch cards would be a better analogy, as far as historical timing goes. At any rate, just because floppy disks were “state of the art” at one time, does not mean that they are the only acceptable method of moving data from computer to computer today. Likewise, just because plaque purification of phages, or sucrose gradient centrifugation of retroviruses was “state of the art” at one time, does not mean that they are required methods in the study of phages or retroviruses today.

Asking how it is possible to know that a retrovirus genome is a retrovirus genome today, is similar to asking how we know that a string of ones and zeroes on a computer hard disk or attached to an e-mail message is a computer virus. The short answer is that we have now seen enough of both of these things that they are easily recognizable to the people who study them on a daily basis, but they are still difficult to precisely describe to people who are not in our fields of study. I am a biological scientist and for me it is simple to use a few tools such as BLAST to tell me that this:
ccgaagcagg agcagaaaga cagggaacag ggaccgcctt tagtttccct caaatcactc
tttggcaacg acccctggtc acagtaaaaa tagcaggaca gctaaaagaa gctctgttag
atacaggagc agatgataca gtattagaag atataaattt gccaggaaaa tggaaaccaa
aaatgatagg gggaattggg ggttttatca aggtaaaaca gtatgatcaa atacttatag
aaatttgtgg aaaaagggct ataggtacag tattagtagg acctacgcct gtcaacataa
ttggcagaaa tatgttgacc cagattggtt gtactttaaa ttttccaatt agtcctattg
agactgtacc agtagcatta aaaccaggaa tggatggccc aaaggtgaaa caatggccat
tgacagaaga gaaaataaaa gcattaacag aaatttgtac agagatggaa aaggaaggaa
aaatctcaag aattgggcct gaaaatccat acaatactcc aatatttgct ataaagaaaa

Is HIV-1 M group pol gene from a virus that is not easily classified into one of the M group subtypes.

And that this:
aaggaactag agaggtatta gcaaaaatac caagtaagtt ttgtcctcgt cttcttggta
atccaacttg atttaataaa gccatcgctt cattagcttg cccttcttcc acactgatat
tccataaaac tccttttgca ccaccaccac cttcggaaac tgcttgaact ttattggcat
tgattccttt attggataga aaaacgagta tttcgttagc ttctttttct tctaatccat
ttacaatgac gcgtcttgat tcacagcttg tgaccaaccc catgagagtg attaataaga
aaaattgacg aagaaaataa tacaaatttg tttgtgaaaa agtatttttg gtagtcatga
accgctctgg attaagctta tttttgagag ataattttaa ccagatccta gcagaataat
gaatttagct atagatcttc taagcaaaac aatgaaaatt tctaaagatt cgagggttaa
atagattttg gagttccaga aatacacttt tttcaaatca ttttgcaata aaatttgata
attttaggca aaattcagat gaataatgaa tagccaagat aaaaaagagg ccggattatc
cggcctctga agctgaaaac tcaattactt caaacgatta agctttaatt ttaatgtcta

Is probably part of an elongation factor G gene from a bacteria in the Chlamydia family.

I am not enough of a computer scientist to know if a string of ones and zeroes is part of a computer virus or part of a GIF image, but I have little doubt that there are tools available that can distinguish between the two. If computer scientists all over the world agreed that such tools existed and were accurate, I would either just take their word for it, or begin reading some textbooks and using some tools to make the determination for myself. I would not try to convince a government of some African nation that they should stop scanning internet traffic within their country for a worm or virus string until there were endless “debates” about whether or not the global community of computer scientists can convince me that they know what they are talking about. Just because I can make clever arguments about computer viruses needing to be found in the boot sector of 5 and ¼ inch floppy disks, does not mean that all over the world computer scientists, systems administrators, and people who have had there computers infected by the Netsky virus are lying about the Nestsky computer virus.

Computer viruses can’t always be found in boot sectors of floppy disks, and mammalian retroviruses can never be purified to 100% purity using sucrose gradients. Serology alone or DNA sequencing alone or protein sequencing alone is not enough to fully classify a new biological virus, but any two of those three can in most cases provide all the evidence we need to determine whether or not a virus is present, and what broad class of virus it belongs to.

Nobody has ever produced an electron micrograph of a computer virus. Nobody has ever produced and electron micrograph of carbon dioxide or the human insulin protein. There are many things that human logic can demonstrate to exist that are not possible to see, even with a good microscope. Viruses happen to be on the lower edge of the scale of the sizes of things that are worth looking at. If we could see carbon dioxide it would probably look just like many other molecules. It is the chemical properties, molecular mass and other characteristics of carbon dioxide that are most interesting, rather than what it looks like. The same holds true for viruses.

The interesting properties of both biological viruses, and computer viruses, are what they are observed to do to the systems they interact with. Once we have a computer virus on a disk, or a biological virus cloned, we can begin to study how it reproduces, and how it affects the cells or computers that it infects. The fact that there is some technology involved, that can be difficult to understand if one becomes confused by arguments about “specificity”, “similarity” and “identity”, does not necessarily mean that the people who use those technologies are making up lies about them.

The Perth group wrote:

“… Q2. Isn’t it true that to do serological and molecular characterisation of the virus first one has to obtain its proteins and genome.
…”

No. Cloning a viral genome is so simple with modern technologies that cloning and sequencing the genome is now the fastest and most economical route to gain the most information about a viral pathogen, but it is not “required”. There are still dozens of other methods that could be used to characterize a virus. The techniques used to clone and sequence the first genomes of the SARS coronavirus last year were not identical to the techniques used with HIV-1 in 1983. Molecular biology makes advances in technology that are similar to those made in the field of electronic communications.

The Perth group wrote:
“…
We would be grateful if Brian Foley would please give us Levy’s reference(s) where the complete genome and its genes were determined.
…”

Already given above, but here they are again:

Levy JA, Hoffman AD, Kramer SM, Landis JA, Shimabukuro JM, Oshiro LS. Isolation of lymphocytopathic retroviruses from San Francisco patients with AIDS. Science. 1984 Aug 24;225(4664):840-2. PMID: 6206563

Luciw PA, Potter SJ, Steimer K, Dina D, Levy JA. Molecular cloning of AIDS-associated retrovirus. Nature. 1984 Dec 20-1985 Jan 2;312(5996):760-3. PMID: 6096718

Sanchez-Pescador R, Power MD, Barr PJ, Steimer KS, Stempien MM, Brown-Shimer SL, Gee WW, Renard A, Randolph A, Levy JA, et al. Nucleotide sequence and expression of an AIDS-associated retrovirus (ARV-2). Science. 1985 Feb 1;227(4686):484-92. PMID: 2578227

The Perth group wrote:
“…
As we stated in our rapid response “A paraphrased request and a question to Brian Foley” 14th May 2004, “We did not ask for the titles of 90 studies. Especially studies conducted in HIV-2, SIVs, SHIVs, BIV and FIV. Neither for “HIV-1” studies which have no evidence for the existence of “infectious molecular clones”. Let us paraphrase our request: Would Brian Foley please give us a summary of the evidence (not just the title) of a study as well as the evidence from a few confirmatory studies where the existence of an “infectious molecular clone” (as defined by Brian Foley) of “HIV-1” has been proven.”
…”

Yes. I will give a summary of that, right after the Perth group gives me a summary of any one virus that they feel has been characterized to their satisfaction. This is not a one-way street, I have the right to ask a few questions too.

The Perth group wrote:
“…
In the Fisher, Gallo et al study, the authors “used a transfection technique to investigate the biological properties of molecular cloned HTLV-III DNA.” The clone used was lambda-HXB2 whose ultimate origin is a poly(A)-RNA originating from the 1.16g/ml band.
…”

This is a blatant lie. The Perth group has been repeatedly informed that the Lambda-HXB-2 infectious molecular clone of HIV-1 M group subtype B was NEVER passaged through a sucrose gradient.

The Perth group wrote:
“… (b) “Expression of the HTLV-III gag-related proteins p15 and p24 by transfected celled was demonstrated using special monoclonal antibodies.” That is, by a totally non-specific reaction.
…”

That is another blatant lie. Binding between monoclonal antibodies and proteins is not “totally nonspecific”. Monoclonal antibodies are VERY HIGHLY specific, closer to 99.99% specific than to 99.0% specific.

The Perth group wrote:
“…
Nowhere in references 14,15 and 16 is there any evidence that the ultimate origin of the molecular clone, that is, the stretch of DNA called 93IN101, is an RNA which originated from “HIV” particles. In other words, there is no evidence that ultimately 93IN101 is the cDNA of an RNA found in “HIV” particles, the “HIV” genome.
…”

The evidence is in there, the Perth group is just not going to admit that they do not understand the evidence.

The Perth group wrote:
“…
Q4. Is It true that the "proviral DNA" is a transcript (reverse) of a poly(A)-RNA identical to the poly(A)-RNA which, in sucrose gradients, bands at the density of 1.16g/ml and thus that its ultimate origin is this RNA.
…”

No. Proviral DNA is nearly identical to viral RNA, but because of the error rate of the reverse transcription process, nearly all integrated proviral genomes contain one or more mutations relative to the RNA genome from which they were reverse transcribed. In addition to this, the viral particles contain two RNA genomes, sometimes derived from two different proviruses, and the reverse transcriptase can and does switch templates between these two genomes during the reverse transcription process. Thus any proviral genome can be a recombinant between two different viral RNAs.

Proviral genomes are similar, most often very highly similar, such as 99.98% identical, to RNA genomes from the same isolate of virus, but they are almost never “identical”.

The Perth group wrote:
“…
Q9. Is it true that the molecular clones, ëHXB-2 and ëHXB-3 were obtained from the “HIV” provirus and thus that the ultimate origin of ëHXB-2 is a poly(A)-RNA identical to the poly(A)-RNA from the 1.16g/ml band.
…”

We can never know the answer to that question. Robert Gallo’s lab and Luc Montagnier’s lab both ended up creating molecular clones of HIV-1 M group subtype B virus that were so nearly identical to each other as to be indicative of being derived from the very same patient. During the investigation as to how that could have possibly occurred, many other clones were made from the patients known as “LAI” and “BRU”. None of them were 100% identical to Lambda-HXB-2, Lamda-HXB-3, Lambda-BH8 (all from Gallo’s lab) or the LAV-1(BRU) clone (from Montagnier’s lab), but all of them were far more similar to all of those clones than to the clones of HIV-1 M group subtype B obtained from any other patient. See for example:

Guo HG, Chermann JC, Waters D, Hall L, Louie A, Gallo RC,
Streicher H, Reitz MS, Popovic M, Blattner W
Sequence analysis of original HIV-1.
Nature. 1991 Feb 28;349(6312):745-6. No
PMID: 2000145

Both the Montagnier lab group, and the Gallo lab group made many different preparations of viral RNA. Not all of them came from the same patient from which the Lambda-HXB-2 clone was derived from. RNA preparations from viruses derived from that same patient would be on average 98% or 99% identical to the Lamda-HXB-2 clone, whereas RNA preparations derived from other people infected with other HIV-1 M group subtype B viruses in the 1982 to 1984 time period would be on average closer to 92% identical to the Lambda-HXB-2 clone in the env gene, and 95% to 97% identical to the Lambda-HXB-2 clone in the pol gene. For comparison purposes, the pol gene of any lentivirus is roughly 60% identical to any HTLV-I or HTLV-II virus, which is toward the lower limit of sequence similarity needed for meaningful hybridization.

The Perth group wrote:
“…
Furthermore, we would be grateful if Brian Foley would please answer all our previous questions and well as those asked here.
…”

For a group that claims to know all about the isolation and characterization of retroviruses (or lack therof), you have far too many questions that would be better answered by reading some textbooks. But if you can give me one example of a virus that you think has met your criteria for isolation and characterization, I will illustrate for you how the characterization of various isolates and clones of HIV-1 have also been done.

Is it just monoclonal antibodies to HIV-1 M group subtype B virus that you believe are “totally nonspecific” or do you believe that all monoclonal antibodies are “totally nonspecific” despite tens of thousands of publications to the contrary?

Competing interests: None declared