Brian T Foley,
Los Alamos National Lab, Los Alamos, NM 87545
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The Perth group wrote:
Unfortunately he gave a lengthy response and a long list of inappropriate references but did not answer our questions.
It is unfortunate that the Perth group denies that the references I provided were appropriate. If they read the content of those papers, they would not need to keep repeating their questions, which are answered in those papers.
The Perth group wrote:
Yes, there were HIV-1 M group subtype B poly-adenylated RNA genomes in many of the 1.16g/ml bands that were analyzed by the Gallo and Montagnier labs (those that came from infected cell lines, but not from uninfected cell lines). There were also some polyadenylated subgenomic RNAs in the 1.16 g/ml bands, which were produced by cellular splicing machinery which removed introns from the viral genome to make mRNAs that can be translated to produce Tat, Rev and other viral proteins which cannot be produced from unspliced full-length genomic RNAs. These three papers cited by the Perth group [1,2,3] are only a small sampling of the many papers published by the Gallo and Montagnier labs in the 1984-1985 time period. I would advise the Perth group to do more reading on this subject, if they wish to understand why the Gallo and Montagnier groups were given credit, over other labs such as the one that Jay Levy worked in, for being the first two labs to “isolate” the virus which became known as HIV-1 M group subtype B.
I assume that when the Perth group refers to “the” HIV genome, they are speaking of one or more of the genomes that were cloned and sequenced by the Gallo and Montagnier labs. In that case, not all of these clones were derived DIRECTLY from any 1.16 g/ml band. As I clearly stated before, most molecular clones of HIV-1, including those that were sequenced in 1985, did not originate directly from any 1.16 g/ml band. For example, if the Perth group could read and comprehend the papers they cited, they would know that the Lambda-HXB-2 and Lambda-HXB-3 clones in  originated from two of the several HIV-1 M group subtype B genomes which were integrated into the genomic DNA of one particular HIV-infected infected H9 cell culture. These two molecular clones of HIV-1 M group subtype B virus are not identical, as is readily apparent from the restriction maps in figure 2 of , but they are nearly identical, as would be expected from two clones which originated from pooled sera from several HIV-infected people, and were passed into clone 9 of HT cells (H9) as stated in . Likewise the HIV genomes found in the Lambda-HXB-2 and Lambda-HXB-3 clones were nearly identical to the Lambda-BH-5, Lambda-BH8 and Lambda-BH-10 clones, each of which originated from circular pre-integration complex DNAs isolated by the procedure of Hirt et al. [4,5]. This extrachromosomal DNA was probed with radiolabeled cDNA made from the HIV-1 RNA isolated by oligo-dT column from HIV-infected cells, after “double banding” meaning that the first 1.16 g/ml band was reloaded and recentrifuged .
The Alizon paper  reports the cloning and preliminary analysis of 3 subgenomic fragments of HIV-1 M group subtype B cDNA, called plasmid-LAV13, plasmid-LAV75 and plasmid-LAV82. The Alizon paper states that an HIV-infected cell line called FR8 was used, and cites a 1984 Levy paper  . It is not 100% clear whether Alizon is citing Levy as the source of the LAV-infected cell line that was used, or only the source of the procedure used to purify HIV-1 RNA in order to make the cDNA. At any rate, yes, these 3 clones (although not infectious, as they are not full-length genomes) were in fact derived directly from virion particles isolated from one or more 1.16 g/ml sucrose density bands.
If the Perth group had read and comprehended the Arya paper , they would know that in this case, yes the authors obtained their HIV-1 genomic RNAs, from which they made cDNAs, from the 1.16 g/ml band of sucrose-density gradient-purified virions. The Arya paper does not report cloning any of their cDNA, they only labeled it and used it to probe HTLV-1 and HTLV-II cloned genomes in an effort to determine how closely or distantly related HIV-1 is to these other two complex retroviruses. However,  reports the cloning the integrated proviral genomic DNA from these very same cells, as discussed above.
The Perth group wrote:
Yes, as I have said twice or more times before already. The clone must produce virus particles that are identical by serology, morphology, protein sequences, RFLP, Southern blotting, etc. to the parental virus, and the particles must also be infectious. If a cloned viral genome does not meet these criteria, it is not an INFECTIOUS molecular clone of the virus, be it HIV-1 or any other virus. Morphology by electron microscopy is the least important of those criteria, because as I have stated many times before, all lentiviruses look alike by electron microscopy. Also, it is not always possible to detect the difference between infectious and non-infectious virus particles by electron microscopy.
1. Alizon M, Sonigo P, Barre-Sinoussi P, Chermann JC, Tiollais P, Montagnier L,
2. Ayra SK, Gallo RC, Hahn BH, Shaw GM, Popovic M, Salahuddin SZ,
3. Shaw GM, Hahn BH, Arya SK, Groopman JE, Gallo RC,
4: Hahn BH, Shaw GM, Arya SK, Popovic M, Gallo RC, Wong-Staal F.
5: Hirt B.
6 : Levy JA, Hoffman AD, Kramer SM, Landis JA, Shimabukuro JM,
Competing interests: None declared