1 U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES PUBLIC HEALTH SERVICE FOOD AND DRUG ADMINISTRATION CENTER FOR BIOLOGICS EVALUATION AND RESEARCH INTERNATIONAL ASSOCIATION FOR BIOLOGICALS NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES NATIONAL VACCINE PROGRAM OFFICE WORLD HEALTH ORGANIZATION + + + + + EVOLVING SCIENTIFIC AND REGULATORY PERSPECTIVES ON CELL SUBSTRATES FOR VACCINE DEVELOPMENT + + + + + WORKSHOP + + + + + Thursday, September 9, 1999 + + + + + The workshop took place in the Plaza Ballroom, Doubletree Hotel, 1750 Rockville Pike, Rockville, Maryland, at 8:00 a.m., Naomi Rosenberg, Ph.D., Kathryn Zoon, Ph.D., and Keith Peden, Ph.D., Session Chairs, presiding. 2 PRESENT: KATHRYN ZOON, Ph.D., Session Chair NAOMI ROSENBERG, Ph.D., Session Chair KEITH PEDEN, Ph.D., Session Chair JOHANNES LOEWER, M.D., Panel Chair STEPHEN HUGHES, M.D., Panel Chair RON DESROSIERS, Ph.D., Speaker BEN BERKHOUT, Ph.D., Speaker HSING-JIEN KUNG, Ph.D., Speaker MAXINE LINIAL, Ph.D., Speaker JOHN KAPPES, Ph.D., Speaker JOHN PETRICCIANI, M.D., Speaker DONALD BLAIR, Ph.D., Speaker RUTH RUPRECHT, M.D., Ph.D., Speaker EUGENE MAJOR, Ph.D., Speaker HAIG KAZAZIAN, M.D., Speaker BERNARD MEIGNIER, Speaker GIRISH VYAS, Ph.D., Speaker ALEX VAN DER EB, Ph.D., Speaker THOMAS BROKER, Ph.D., Speaker PAUL, SANDSTROM, Ph.D., Speaker JAMES McDOUGALL, Ph.D., Speaker BRIAN VAN TINE, M.D., Ph.D., Student Speaker SANDRA RUSCETTI, Ph.D., Panelist 3 PRESENT (Continued): CLIVE PATIENCE, Ph.D., Panelist LEONARD EVANS, Ph.D., Panelist DAMIAN PURCELL, Ph.D., Panelist ARIFA KHAN, Ph.D., Late Breaker 4 C-O-N-T-E-N-T-S Retroviral Recombination as an Adjunct to Viral Propagation and Pathogenesis (HIV Model), Ron Desrosiers, Ph.D. . . . . . . . . . . . . . . . . .6 Reactivation of Replication-Defective (HIV) Retroviruses, Ben Berkhout, Ph.D. . . . . . . . . 18 Retrovirus Insertion into Herpes Viruses, Hsing-Jien Kung, Ph.D. . . . . . . . . . . . . . 33 Foamy Virus Replication: Implications for Transfer of Cellular Sequences, Maxine Linial, Ph.D. . . . 57 Recombination in Lentivirus Vectors, John Kappes, Ph.D. . . . . . . . . . . . . . . . . . . 76 Panel Discussion of Viral-Viral and Viral- Cellular Interactions . . . . . . . . . . . . . . 91 Designer Cell Substrate, Telomerase Activity and Cell Immortalization, James McDougall, M.D. . . .137 Introduction to Cell DNA Issues, John Petricciani, M.D. . . . . . . . . . . . . . . . .151 Evaluation of the Transforming Activity and Tumor Inducing Capacity of Tumor Cell DNA, Donald Blair, Ph.D. . . . . . . . . . . . . . . . . . .166 Infectivity of Retroviral Provirus DNA, Ruth Ruprecht, M.D., Ph.D. . . . . . . . . . . .185 Cells and Their DNA Containing Integrated DNA Viral Genomes: Differentiated Phenotypes of the Human Central Nervous System, Eugene Major, Ph.D. . . .197 Mobile Elements in Mammalian Genomes and Their Implications for Cell Substrate Safety, Haig Kazazian, M.D. . . . . . . . . . . . . . . . . .217 Panel Discussion of Cellular DNA as a Potential Source of Oncogenic Activity or Infectious Agents . . . . . . . . . . . . . . . . . . . . .234 Introduction to Evening Session, Keith Peden, Ph.D. . . . . . . . . . . . . . . . . . .289 5 C-O-N-T-E-N-T-S (Continued) Industrial Experience with Life Polio Vaccine Prepared on the VERO Continuous Cell Line, Bernard Meignier . . . . . . . . . . . . . . . .294 Proteins of Replication-Incompetent Virions for HIV Vaccination, Girish Vyas, Ph.D. . . . . .313 Unusual Response to Apoptin of Diploid Fibroblasts From Cancer Prone Syndromes, Alex van der Eb, Ph.D. . . . . . . . . . . . . . . . . . . . . . .327 Human Papillomaviruses: a Window into Eucaryotic Cellular DNA Replication Mechanisms and Regulation, Thomas Broker, Ph.D. . . . . . . . . . . . . . .341 Facilitated Detection of Adventitious Agents Using Genetically Engineered Cell Lines, Paul Sandstrom, Ph.D. . . . . . . . . . . . . . . . .352 In Situ Transcriptional Analysis of Integrated Viral DNA, Brian Van Tine, M.D. Ph.D./Student . .368 Late Breakers: Arifa Khan, Ph.D. . . . . . . . . . . . . .376 Damian Purcell, Ph.D. . . . . . . . . . . .387 Johannes Loewer, J.D. . . . . . . . . . . .395 6 1 P-R-O-C-E-E-D-I-N-G-S 2 (8:03 a.m.) 3 CHAIRPERSON ROSENBERG: Good morning. 4 Please take your seats. We would like to start the 5 session so that we try to stay on time. 6 This morning's session is going to 7 continue. I think you'll hear many similar issues to 8 those that we heard addressed yesterday afternoon. So 9 we'll continue discussing retroviruses. 10 However, this morning we'll move more 11 toward the complex retroviruses with presentations 12 addressing issues relating to lentiviruses. 13 We'll start with our first speaker, Ron 14 Desrosiers. 15 DR. DESROSIERS: Thank you very much. 16 My laboratory -- excuse me -- my 17 laboratory uses simian immuno-deficiency virus, SIV, 18 infection of rhesus monkeys as an animal model for 19 AIDS, and I'm not exactly sure why I'm here, but I 20 think I'm here to -- 21 (Laughter.) 22 DR. DESROSIERS: -- I think I'm here to 23 give some examples of retroviral recombination from my 24 experiments or where there may have been opportunities 25 for retroviral recombination in our SIV system and 7 1 where we have not seen it. 2 So I only have three slides in the 3 carousel, so I'll try to give some explanation of each 4 of these three, and I should finish well ahead of 5 time. 6 One of the things we've been doing in this 7 SIV system is knocking out the so-called auxiliary 8 genes. These are genes that are not absolutely 9 required for the viruses' ability to replicate, but do 10 contribute to the virus' ability to replicate in 11 monkeys and to cause disease. 12 So we've done such experiments with the 13 nef gene, the vpr gene, the vpx gene, and the vif 14 gene. 15 When we introduced a premature stop codon 16 into the nef reading frame to eliminate the ability of 17 nef to be expressed and used that nef stop virus to 18 infect rhesus monkeys, there was very quickly -- by 19 two weeks after infection of rhesus monkeys, all we 20 could detect was revertant virus, that is the 21 premature taa stop codon had universally reverted by 22 two weeks of monkey infection to a stop codon, 23 indicating that there was strong selective pressure 24 for open functional forms of nef in vivo, and that 25 such revertant viruses, although they might be rare, 8 1 appear rarely as a rare event. The strong selective 2 advantage of such a virus can very quickly result in 3 that virus being the vastly predominant virus. 4 We've also made forms of virus with a 5 large, gaping deletion in the nef gene, and infection 6 of monkeys with such nef deletion virus shows that 7 this nef deleted virus is clearly an attenuated virus. 8 It replicates much less than the parental virus, and 9 is much less prone to inducing disease. 10 So one of the things that we've done is to 11 look very hard to see whether or in which ways a 12 virus, such nef minus virus, could regain pathogenic 13 potential. 14 We've looked both in cultured cells 15 producing this nef deletion virus and in rhesus 16 monkeys infected with nef deletion virus for possible 17 recombinant viruses. 18 In discussing use of such strains as 19 experimental vaccines, the question has been raised 20 whether such viruses could possibly acquire cellular 21 sequences in an analogous way, the way the Type C 22 retroviruses can acquire oncogenes or other such genes 23 to bring back nef function or to provide some 24 additional oomph to the virus to replace the loss of 25 nef. 9 1 And despite extensive efforts looking for 2 such recombinant viruses, where there would be strong 3 -- so in the absence of nef, there would be selective 4 pressure for capture of any gene that created a 5 selective advantage, and we should be able to see it 6 in vivo because of the strong selective advantage that 7 could impart. 8 And despite extensive efforts looking for 9 such viruses, we've never found such recombinant 10 viruses that have picked up extraneous sequences. 11 We've taken animals infected with nef 12 deletion virus, and we've serially passaged virus in 13 rhesus monkeys, and we have been able to increase the 14 pathogenic potential of such nef deleted SIV by serial 15 passage in monkeys, and the increased pathogenic 16 potential is not due, at least in the two lineages 17 we've looked at, is not due in either case to capture 18 of cellular sequences or extraneous sequences, but is 19 actually due to compensatory changes in SIV elsewhere 20 in the genome that allow the virus to make up for the 21 loss of the nef gene. 22 So similarly using nef minus virus, vpr 23 minus virus, vpx minus virus, and vif minus virus, 24 we've not seen any examples, despite extensive efforts 25 looking for it; we've not seen a single example where 10 1 the virus has captured an extraneous sequence and made 2 it increase selective advantage. 3 Now, a few years ago we published a paper 4 where we attempted to directly look for -- we designed 5 an experiment to see if we could directly demonstrate 6 retroviral recombination in an infected monkey. So 7 this is now -- we're not talking about recombination 8 with cellular sequences, but recombination between two 9 different SIV strains. 10 So what we did -- how do I go backwards 11 here? -- so we used two different SIV strains for this 12 experiment. We used one strain with the deletion in 13 nef, and the second strain was deleted in both vpx and 14 vpr. That is about four kilobase pairs away. 15 So we inoculated the nef deleted virus in 16 one leg, and we inoculated the vpr/vpx virus in the 17 other leg. Both of these are attenuated viruses. 18 By two weeks after infection, as reported 19 in that Journal of Virology paper, by two weeks after 20 infection all we could detect is wild type virus 21 resulting from a recombination between these two 22 different strains. 23 For recombination to occur, one needs 24 infection of the two different retroviral strains, 25 infection of the same cell, and one can easily achieve 11 1 that in cell culture, but there had been some question 2 how likely an event that would be in a whole infected 3 organism with the massive pool of cells, and obviously 4 at least initially a very, very low multiplicity of 5 infection. 6 For such recombination to occur, as I just 7 said, you need infection of a single cell with both 8 strains, and although at least theoretically there are 9 a number of or several different mechanisms by which 10 retroviral recombination can occur, the major way such 11 recombination occurs relates to the fact that virions 12 contain two RNA molecules, and when you have two RNA 13 molecules in a single virion of the two different 14 genotypes, when a cell is newly infected through the 15 reverse transcription process and the copy choice 16 mechanism in the reverse transcription process, one 17 can then get recombinant viruses, in this case 18 representing a wild type sequence and fully 19 pathogenic. 20 So these experiments illustrated two 21 things. They illustrated the ease with which 22 recombination can occur even in vivo in the large pool 23 of infected cells in the rhesus monkey. 24 And the second thing that they illustrated 25 was the enormous power of selective advantage and how 12 1 selective forces can very quickly selected for strains 2 of virus with a growth advantage. 3 And the final thing I want to talk about 4 is a virus deleted in the vif gene. So although vif 5 is generally considered an auxiliary, nonessential 6 gene, which it certainly is with HIV for growth in 7 some cell types, it turns out that vif in most cell 8 types for both SIV and HIV is very important for the 9 virus' ability to replicate. 10 In fact, for the strain of SIV that we 11 work with, the only way we've been able to grow a vif 12 minus virus is in a vif complementing cell line. 13 So this shows the growth curves of vif 14 deleted SIV, which has been produced in a vif 15 complementing cell line. Growth of this vif minus SIV 16 in the vif complementing cell line, viv CEMX compared 17 to growth of the same virus stock, same amount in the 18 parental line CEMX 174. 19 Now, the vif complementing cell line 20 contains the full vif open reading frame, and the vif 21 minus the deletion in the vif gene in the virus that 22 we used is within the vif reading frame. So it is at 23 least theoretically possible that there could be 24 recombination in this complementing cell line between 25 the vif minus viral sequences and the vif gene 13 1 present, integrated in the host cell genome used for 2 growing the virus. 3 And obviously if there was a recombination 4 for vif-plus virus, that virus would have an enormous 5 selective advantage both in cell culture and in 6 monkeys, and we could quickly, quickly see it. 7 So we've made a number of vif-minus virus 8 stocks and vif complementing cell lines. We've 9 infected at least six different monkeys with vif 10 deleted SIV grown in vif complementing cell lines, and 11 we've not seen a single example of virus regaining the 12 vif sequences from the cell line that it was grown in. 13 That's basically the sum total of my 14 experiences involving recombination either with viral 15 sequences or cellular sequences in the SIV system. 16 So I'll stop there and take questions. 17 (Applause.) 18 DR. KUNG: Hsing-Jien Kung, UC-Davis. 19 Hi, Ron. 20 DR. DESROSIERS: Hi, Hsing-Jien. 21 DR. KUNG: So much of the -- in terms of 22 detection of the recombinants, usually you need a 23 selection. So I was asking you in the nif experiment, 24 did you grow the virus on just wild type non- 25 complementing virus in an effort to pick up -- to 14 1 actually pinpoint the selection of that virus? 2 DR. DESROSIERS: Well, the -- 3 DR. KUNG: The vif, the last experiment 4 you were talking about. You said you didn't see the 5 pick-up of the host gene. 6 DR. DESROSIERS: That's correct. So vif 7 minus virus does not grow in CEMX 174 cells or other 8 cell lines, but the vif plus virus does. So we've 9 transfected vif minus SIV DNA into the vif 10 complementing cell line to make virus stocks. We've 11 expanded those virus stocks in the vif complementing 12 cell line. 13 If the vif minus virus had picked up a vif 14 gene, it would now grow in a non-complementing cell 15 line, and we've repeatedly tested our virus stocks, 16 undiluted, large amounts. We've taken undiluted virus 17 stocks and put it into six different monkeys, two 18 different stocks prepared independently. We've never 19 seen -- there there was clearly the enormous selective 20 advantage of minute amount of vif plus virus would 21 quickly grow out, and we've not seen it in the 22 examples we've done. 23 MR. BROKER: Tom Broker, UAB. 24 In that last experiment, how much flanking 25 homologous sequence might you have had, and can you 15 1 consider expanding that outward to ask whether you'd 2 favor recombination with enough flanking homologous 3 things allowing crossover? 4 DR. DESROSIERS: Yeah, I don't remember 5 off the top of my head exactly how much, but it was 6 not a lot. It was maybe 100 base pairs or so flanking 7 on each side, but you're right. I think if one wanted 8 to investigate it, I think that would be a pretty good 9 system to do it. 10 MR. BROKER: It would be great. 11 DR. DESROSIERS: could expand out further 12 and further and see under what conditions one might 13 see it. 14 MR. BROKER: Okay. 15 DR. DESROSIERS: I think in the experiment 16 when there was recombination between two retroviruses, 17 I've always kind of hoped that people would have used 18 that system a little bit more, too. One could 19 actually -- we never went to the trouble to try to 20 look. One could actually use that system to look at 21 where the recombination events were occurring by 22 putting in third base changes. You could actually map 23 where the crossover, quote, crossover events occurred, 24 and we never took the trouble to do that. 25 But I think anyone who's interested in 16 1 studying recombination in retroviruses, that would be 2 a pretty nice way to do it, but, no, we've not done 3 that experiment. 4 It was a short stretch of sequence. We 5 were actually -- for the experiments we wanted to do, 6 we wanted to avoid getting recombinant virus, and so 7 we kept the length of overlap short, but if one wanted 8 to study it, exactly right. You could just expand the 9 length and see if you had a much larger length of 10 flanking sequences, whether you had increased chances 11 of seeing recombination 12 PARTICIPANT: It's really an extension of 13 the same experiment. We know that retroviruses will 14 promiscuously package all sorts of cellular messages. 15 Do you know within the virion population that's coming 16 out of the complementing line whether you actually do 17 get vif message actually packaged in the virions that 18 might be a substrate eventually for a strand jump 19 recombination? 20 DR. DESROSIERS: Yeah, that's a -- we 21 haven't actually looked at it. My mind is turning in 22 theoretical terms, what that means. It's generally 23 believed that vif minus virus in a noncomplementing 24 cell line is capable of a single round of infection 25 events. 17 1 PARTICIPANT: Sure. 2 DR. DESROSIERS: It can get into one 3 round, but then not pass. I think there's really 4 only room in the virion -- some of our retroviral 5 experts here can correct me if I'm wrong -- but 6 there's really only room in the virions for two RNAs, 7 and I think if a virus did package cellular RNA and 8 that would leave room at most for a single viral RNA. 9 I think that would result in a defective particle, 10 would not be infectious, but still could be propagated 11 as a defective virus conceivably anyway in the 12 presence of a helper. 13 CHAIRPERSON ROSENBERG: I need to remind 14 the questioner to give their names. 15 DR. LINIAL: Maxine Linial, Hutchinson 16 Center. 17 About your second point, we have shown 18 with ALV that you can package a full length genome and 19 a cellular RNA and get recombination. So it can 20 occur. 21 But my main question was: how much 22 replication did you get with the delta vif virus, and 23 if you did see any, in what kind of cells was it 24 replicating in the monkeys? 25 DR. DESROSIERS: The vif minus virus? 18 1 DR. LINIAL: Un-huh. 2 DR. DESROSIERS: The only way we were able 3 to replicate the vif minus SIV is in the vif 4 complementing cell line. So the vif minus virus, 5 we've tested it. We can clearly generate high titred 6 stocks in the complementing cell line, and we've used 7 that to infect a variety of cell lines, primary rhesus 8 PBMC, primary macrophage cultures, and inoculated into 9 monkeys. 10 And in cell culture, the only way -- the 11 only system we detect any replication is in the vif 12 complementing cell line. 13 In monkeys, the vif minus virus is at the 14 very most -- the very least the most attenuated strain 15 we've seen. The only thing we've been able to measure 16 in the delta vif inoculated monkeys is very, very weak 17 antibody responses. 18 CHAIRPERSON ROSENBERG: Thank you. 19 The next speaker is Ben Berkhout. 20 DR. BERKHOUT: Okay. I would like to 21 continue on some of the themes that have been 22 introduced by the previous speaker, and I guess we can 23 quickly go over the introduction. 24 Of course, most of you should be familiar 25 with the idea of live attenuated viruses. These 19 1 entities should replicate and elicit supposed immune 2 response that confers lifelong protection against 3 challenge with a pathogenic wild type virus. 4 Of course, the concept has proven to work 5 in several systems, for instance, vaccinia, polio, and 6 measles, and for HIV-1 good results have been 7 described in SIV infection models of macaques by 8 several labs, including that of the previous speaker, 9 and it has been proposed recently by some groups that 10 one should start clinical trials on humans with the 11 HIV-1 versions of live attenuated viruses with 12 multiple gene deletions. 13 But there are still several safety 14 concerns. One of them is the induction of a fulminant 15 infection in immunocompromised hosts, and second, and 16 that's the one I would like to focus on today, is the 17 potential reversion of an attenuated vaccine strain to 18 a virus variant that can replicate fast and that 19 potentially can cause AIDS. 20 Now, all of the studies I will present are 21 done in tissue culture settings. So we will only look 22 at replication potential of viruses, and therefore, of 23 course, we don't have any measurements of 24 pathogenicity. 25 Now, let me start out with a purely 20 1 hypothetical graph. The idea here is to delete genes. 2 As you know, HIV-1 has nine genes; simple 3 retroviruses, only has three. So the idea is that we 4 can probably removed a couple of the accessory genes 5 and thereby we will credibly decrease the replication 6 potential of such a virus. 7 Probably that also has a consequence for 8 the pathogenicity of this virus, and I did draw 9 parallel curves. There's no evidence for that, but I 10 think it's reasonable for this graph at least. 11 Probably also the immunogenicity of these 12 viruses will slowly drop off, and the idea is to reach 13 a level that is below a threshold for the 14 pathogenicity such that this virus will not cause 15 AIDS. 16 However, of course, replication should 17 still suffice to induce a good immunogenicity, and of 18 course, these thresholds are, again, hypothetical, and 19 we don't know how big this window is. 20 Even if you managed to identify such a 21 virus that has these properties, one of the key 22 questions is how stable is this virus and will it 23 perhaps not be able to regain replication capacity and 24 thereby going over this threshold. 25 Now, why should we release genes? We 21 1 already heard in the previous talk that when you put 2 stop codons in, for instance, the nef gene, it's 3 repaired in two weeks. Even small deletions or 4 substitutions will be removed by this virus very 5 rapidly, and that was shown early on in a paper 6 published in '95 using the SIV system and virus 7 evolution in macaques. 8 A four amino acid deletion was introduced 9 in nef, and that's shown here. It was still present 10 two weeks after inoculation. However, after 17 weeks, 11 the virus managed to repair this sequence, at least to 12 fill in this gap, by duplicating the sequences that 13 are located over here. 14 Officially at this point in time, you do 15 not have the wild type sequence back, but credibly 16 after 25 weeks, we do see a wild type tyrosine appear 17 at this position. It's like the wild type, and after 18 45 weeks, we also have an aspartic acid here, and we 19 are almost back to the wild type sequence 20 demonstrating the enormous repair capacity of this 21 virus. 22 So small deletions will not suffice and 23 we, therefore, have to make bigger deletions. 24 Now, the system we are going to us is a 25 tissue culture evolution system, and I will use one 22 1 slide to introduce the system. The method we have 2 called forced evolution. Basically we start out with 3 a molecular clone of HIV-1 in which the deletions in 4 accessory gene products have been introduced. 5 We start with a massive transfection. Ten 6 to 40 microgram of molecular clone is electroparated 7 (phonetic) into cells, and then initially the cells 8 are cultured, but when there is a sign of virus 9 replication, it's seen in either syncytia or GAG E24 10 production. We start to passage the culture 11 supernatant onto fresh and infected cells. 12 Initially these large inocula up to 1 mL, 13 later on credibly reducing the amount of virus that is 14 transferred. 15 So in Section A we will be able to pick up 16 revertants, and I guess it's important to realize that 17 this reversion or evolution is a two-step process. 18 One needs mutations, and those will be introduced 19 primarily by the first transcriptase enzyme, which is 20 error prone. Of course, these mutations will be 21 introduced in a random manner, and then will get 22 selection of those virions that are better than the 23 input virus. 24 So its evolution, as I guess it must be 25 proposed by Darwin, although he used different terms. 23 1 It's variation and survival of the fittest. 2 Now, because I think a presentation may 3 not be complete without seeing an RNA stem loop 4 structure, I actually show you one example of a study 5 that we performed on a stem loop structure. That's 6 the so-called TAR hairpin that is found at the extreme 7 five prime and three prime end of HIV-1 genomes. This 8 is the wild type situation. 9 This is a mutant that is completely 10 replication defective. Here we have open to the lower 11 left-hand side of the stem, and after about six months 12 of culturing initially the cells, later on the virus 13 passage, we ended up with this revertant. It has one, 14 two, three, four substitutions. It doesn't go back to 15 the wild type sequence, but what this revertant is 16 telling us is that this hairpin structure is critical. 17 Sequences are less critical, but one needs 18 a base pair lower region here, and of course, we also 19 have some idea of what this hairpin is doing. It's 20 critical in transcription regulation of this virus, 21 but also because it's present in both ends, it plays 22 a role in the mechanism of reverse transcription. 23 So we obtained a rather complete set of 24 deleted HIV-1 genomes from Ron Desrosiers, and I will 25 today only focus on one of these deletion virions, and 24 1 that's the so-called Delta 3 virions with three 2 deletions. 3 One deletion is in the accessory vpr 4 protein, and this five prime half of the genome was 5 combined with this one, which has a deletion in the 6 nef gene. Then some of the sequences here at the 7 border of the three prime LTR are still present 8 because they are important for replication. 9 And then there is a second deletion in the 10 three prime LTR. 11 Now, it's important to realize that this 12 deletion in the LTR will be inherited in the progeny, 13 also in the five prime LTR, and the five prime LTR, as 14 you probably know, is the transcription promoter at an 15 untelldish (phonetic) virus. 16 Over here I've indicated some primers that 17 will be used in subsequent PCR analysis to actually 18 check whether these introduced solutions are still 19 present. 20 One more thing. We tried to do efflution 21 (phonetic) in tissue culture in primary cells. That's 22 indicated over here, but in particular, for this Delta 23 3 variant we found that the replication, while that 24 much delayed, that it probably will take years to 25 really obtain the fertent (phonetic) viruses. 25 1 So to solve that problem, we are going to 2 use a transformed T cell line, in this case Sup. T1 T 3 cell line, because there the replication defects of 4 this kind of mutants are less feared (phonetic). 5 There certainly is a replication, delay in 6 replication, but it's much less feared than in primary 7 cells. 8 So the virus with three deletion was 9 passaged for up to four month in the Sup. T1 T cell 10 line. Samples were taken at day 14, 53, 83, 125, and 11 we analyzed the samples with similar input virus 12 amounts in a parallel infection, and as you can see 13 over here, the initial virus or the virus present 14 after two weeks has a hard time replicating, and then 15 you see a gradual increase in replication capacity and 16 a big jump actually is seen already at day 53. 17 So we were interested to find out what has 18 happened with this virus. So we performed PCR essays, 19 PCR analysis. PCR analysis across vpr gene didn't 20 show any insertion or deletion of sequences, and in 21 fact, the sequenced region, nothing has happened. 22 This is a PCR across the nef and LTR 23 deletion, and as you can see over here, something 24 happens around day 55. We see the appearance of a 25 variant that is about 14 base pairs longer, and 26 1 apparently this variant is much more fit than the 2 input virus because it out competes the input virus 3 within a couple of weeks. 4 So at this point we got excited. We 5 thought, well, perhaps there is an insertion of 6 perhaps cellular sequences in these deleted virus, and 7 of course, we sequenced this region. 8 It turned out that the two introduced 9 deletions were maintained. The nef deletion was there 10 and also the deletion in the U3 region was perfectly 11 maintained. 12 There also was no insert of cellular 13 sequences, but what has happened is that in the core 14 promoter region, and as indicated here this is a 15 transcription start (phonetic) side, three binding 16 sites for the constitutive transcription factor, SP-1, 17 and a tandem repeat for nf kappa B binding. The 18 deletion that was introduced is indicated over here. 19 Nothing happened over there. 20 What we did see is a gross duplication of 21 all the three SP-1 sites. In addition, there are 22 seven nucleotides of unknown origin, and we have seen 23 an onser (phonetic) duplications. 24 Yesterday in one of the talks, and I must 25 say that this apparently is a very popular theme in 27 1 mutant retroviruses, they easily change the number of 2 repeat elements in their genome, and in fact, we 3 probably know why that is so, because this duplication 4 is probably introduced during the first inscription by 5 a mechanism called slippage realignment. 6 So RT enzyme has probably copied the 7 sequences over here. Then RT and the cDNA is 8 partially removed and it reanneals back to the 9 original sequences, and in one step one gets a 10 duplication of three SP-1 sites. 11 So we end up with this promoter. Does 12 this make a better transcriptional promoter? 13 (Unintelligible) in transient LTR reporter assays, and 14 what we compare is the wild type LTR, the one with the 15 U-3 deletion, and then the one that's the duplication 16 of SP-1 site. So as you compare the last two bars, we 17 tested that in the absence of the transcriptional 18 activate of protein TAT and also in the presence and 19 then in cells that were not more activated by PMA and 20 PHA. 21 In the absence of TAT protein, we, in 22 fact, see that the LTR promoter does not gain function 23 by duplicating of the SP-1 site. In fact, it gets a 24 little worse. 25 However, in the presence of TAT protein 28 1 and, of course, in virus replication in this system, 2 the TAT protein will be around. We do see a partial 3 restoration of the LTR function, and in this case 4 actually it's becoming a little better than the wild 5 type. 6 So the transcription promoter is, indeed, 7 improved in the presence of TAT protein. 8 Does this single mutation also improve the 9 replication of the delta 3 virus? Well, indeed, it 10 does, and that is shown here. So we reconstructed a 11 molecular clone with three deletions, and then in 12 addition the six SP-1 sites. So here is the wild type 13 virus. 14 If you only delete vpr there is a small 15 replication. So this is the input virus with three 16 deletions, and if you in this virus then introduce the 17 six SP-1 sites, there is a rather dramatic increase in 18 replication capacity. In fact, this revertant virus 19 replicates much faster than the virus with a single 20 vpr evolution. 21 Now, we thought it was also of interest to 22 test the replication of this virus not only in the 23 Sup. T1 T cell line, in which it's evolved, but also 24 in primary cells, and that's shown here, and there we 25 do see a different picture. 29 1 This is the wild type virus replication in 2 primary cells, and over here we see that there hardly 3 is a difference in the replication of the original 4 delta 3 virus and the one with six SP-1 sites. 5 So it seems that the SP-1 site duplication 6 is beneficial only in the Sup. T1 T cell line that was 7 used for the evolution experiments. That may have to 8 do with the pool of transcription factors that are 9 present in that particular T cell line. 10 And, for instance, it has been reported 11 that Sup. T1 cells have an extremely low amount of nf 12 kappa B, and one can imagine that perhaps, therefore, 13 SP-1 is more important for this virus. 14 So just to sum up, we show that this delta 15 3 strain is genetically unstable. It retains 16 replication capacity in tissue culture, and we have 17 seen duplication of SP-1 sites. 18 Now, this is all tissue culture, but I 19 think there are some additional evidence to suggest 20 that these viruses are unstable. The Ruprecht 21 laboratory has recently demonstrated that some of the 22 viruses, the delta 3 viruses, do eventually cause AIDS 23 in infected monkeys, and there's also some evidence 24 from cohort studies in humans where people infected 25 with a nef deleted and, therefore, attenuated virus 30 1 strain do over time show a decline in CD-4 T cell 2 numbers and, therefore, perhaps they are at risk for 3 going on to AIDS. 4 So the conclusion will be that these 5 deleted viruses are safe. 6 Now, in the context of this meeting, I 7 should say that the one example I showed you today we 8 did not find any incorporation of cellular sequences 9 in these -- in these deletion virions, and like the 10 previous speaker, in all of the evolution experiments 11 that we have done so far, we have not come across any 12 insertion of cellular sequences in these virus 13 strains. 14 Now, finally, I will discuss a putative 15 route to perhaps a more safe vaccine. What I showed 16 is that this deleted and attenuated virus is able to 17 regain replication capacity, and that's indicated by 18 this arrow. 19 Now, perhaps this virus may be a nice 20 starting point to remove additional functions, thereby 21 again going down on the replication letter. Perhaps 22 then again we can try to evolve a faster replicating 23 variant, and by repeated cycles of gene deletion and 24 subsequent evolution, we perhaps may end up with a 25 virus with only three to five genes that is able to 31 1 replicate efficiently, and that perhaps will be more 2 stable in genetic terms. 3 And with that I would like to stop. 4 (Applause.) 5 CHAIRPERSON ROSENBERG: Is there a 6 question? 7 MR. FALLEAUX: Hi. Frits Falleaux. 8 This may be a silly question for 9 someone -- 10 CHAIRPERSON ROSENBERG: Be sure to 11 identify yourself clearly. 12 MR. FALLEAUX: Yeah, Frits Falleaux, 13 right. 14 This may be a silly question for someone 15 from working with adenoviruses and also HIV, but did 16 someone think of trying to make an attenuated to HIV 17 which is, in part, replication efficient and in 18 combination with, for example, a useful promoter that 19 dries (phonetic) nef so that you have partial 20 replication in the presence of the inducer, which you 21 take away after the introduction of protection? 22 DR. BERKHOUT: People have worked along 23 these lines. For instance, one has introduced the 24 constitutive CMP promoter in the context of a 25 replicating HIV-1 virus. 32 1 One of the problems with introducing 2 exogenous sequences in this final genome is that in 3 most cases the virus doesn't accept them, and they are 4 over time kicked out of the genome. 5 So the only way to safely introduce 6 exogenous sequences in this final genome will be to 7 introduce elements that are absolutely required for 8 replication, and then I think, indeed, it would be of 9 interest to study better and inducible promoters, but 10 that hasn't been tested so far. 11 DR. EVANS: Leonard Evans, Rocky Mountain 12 labs. 13 Did you try to put that promoter, the 14 duplicate promoter back into the wild type? 15 DR. BERKHOUT: We did, and in 16 straightforward replication curves, there is no 17 difference in replication. If you do fairly sensitive 18 competition assays so the wild type virus, and the 19 wild type has six SP-1 sites, you do see that in the 20 wild type context, in fact, the fitness goes down a 21 little bit. So -- 22 DR. EVANS: It goes down? 23 DR. BERKHOUT: Yes, it does down a little 24 bit. 25 DR. EVANS: Okay. 33 1 DR. BERKHOUT: But that difference is 2 marginal. So you have to do very sensitive 3 competition assays with the two viruses to find out. 4 So six SP-1 sites are clearly beneficial in the 5 context of this deletion variant, but they don't make 6 wild type virus much better. 7 DR. EVANS: Okay. Thanks. 8 CHAIRPERSON ROSENBERG: Thank you. 9 The next speaker is Hsing-Jien Kung. 10 DR. KUNG: Thank you. 11 So in today's talks and yesterday's, you 12 have heard that retrovirus have significant potential 13 to recombine with another retrovirus or recombine with 14 hos genes under selective pressure. Today I'd like to 15 discuss with you about experiments that I would 16 summarize as experiments regarding recombination 17 between retroviruses and the herpes viruses. 18 If I can have the first slide. 19 Let's consider the following scenario. In 20 co-infected cells with the retrovirus and the herpes 21 virus, retrovirus copying to RNA, then into DNA, RNA 22 copying to DNA, and has a choice of going into the 23 cell or the genome or has the choice to go into the 24 herpes viral genome if in the co-infecting cells. 25 We can argue what is the probability. 34 1 What is the chance? Well, based on the most 2 simplistic view, the simple calculation, simply based 3 on the mass ratio, you would argue that if the herpes 4 virus -- and let's take the worst case scenario. It's 5 only present as one to ten copies -- one copy, let's 6 say, in the latent state. Based on mass ratio, you 7 would calculate that in every 10,000 integration into 8 the host genome, you would have one into this size of 9 herpes virus. 10 Now herpes virus sometimes will also 11 replicate. So it can reach -- so okay. Let's take 12 that calculation and say you'll have massive infected 13 cells, about a million cells, in fact, with 14 retrovirus. Then there should be 100 integrants if 15 herpes virus is in latent state. 16 Now, herpes virus, of course, also can 17 replicate, let's say, 2,000 copies of 10,000 copies. 18 If it were 10,000 copies, then every integration into 19 the host chromosome, there's a chance into the herpes 20 virus genome. 21 So if you calculate, you say this is not 22 too rare, but, on the other hand, you can make the 23 argument that herpes viral replication usually are 24 combined in certain subnuclear structure, and they may 25 not be as successful to the retrovirus integrative 35 1 complex, and they may be encapsulated very quickly, 2 and so all of these theoretical considerations may 3 also argue against genotypic mixing or recombination 4 between these two. 5 Okay. So I think only experiments will 6 tell. 7 Next slide, please. 8 So today I'm going to talk about 9 retrovirus integration into herpes virus. The system 10 under study is avian retrovirus using reticular 11 endothelial virus as a model system, REV. It's a 12 typical nonacute Type C retrovirus that cause T cell 13 lymphoma and the B lymphoma in chickens, and we show 14 before that the mechanism of oncogenesis is through 15 insertion or activation of C mixing. 16 Avian herpes virus, Marek's disease virus, 17 is very, very prevalent. Until 1970 that was the most 18 important economic loss in the poultry industry, until 19 the live attenuated vaccine was developed. 20 Now, both viruses infect T cells, infect 21 the same T cell types. The tumors are derived from 22 very similar T cell types. So co-infection did exist 23 and has been observed and reported in many papers, and 24 in fact, many of the retroviruses were isolated from 25 Marek's diseased chickens, including the chicken 36 1 syncytia virus strain of the REV and avian 2 myeloblastosis virus, some of you probably know, that 3 carries the mip gene. 4 So there is a preponderance of evidence 5 that these two viruses coexist in chickens. 6 So the implication of the study -- I will 7 show you some experimental evidence, but the 8 implication of the study is, of course, that you can 9 generate a hybrid virus with altered gene expression 10 patterns, which I will show you, and the phenotypes, 11 emergings of new pathogens, if you will, and can 12 certainly sometimes broaden the host range because 13 some of the herpes virus, indeed, can carry retroviral 14 information. 15 So with that in mind, let's first tell you 16 a little bit more about the herpes virus, this Marek's 17 disease virus. This virus, although it's 18 lymphotropic, really the structure is very much 19 similar to alpha herpes virus with the repeating 20 sequence flanking the unique, long reaching, and 21 repeating sequence flanking unique short. 22 And just in a typical alpha herpes virus, 23 you are reaching and encode mostly structural and 24 replication enzymes. In fact, this virus, now Lucy Li 25 has entire sequence and, indeed, show a strong 37 1 correlation with herpes simplex virus. 2 Unique small region also encodes some of 3 the genes, however. They are not important in the in 4 vitro replication. 5 Now, the interesting part are usually 6 confined in the more, but divergent region, in the 7 repeat region, and we now know that in the Marek's 8 disease virus that we have worked in the past few 9 years, that it encodes a protein that we can call it 10 oncogene, called the mac (phonetic), which is in the 11 Joan Foss family of loosing zipper protein, and if you 12 remove this, the virus becomes nononcogenic. 13 Okay. So in this sense the virus is 14 similar to herpes simplex virus, but it's not, due to 15 some of the coding sequences in the repeat region. 16 Okay. But for our purpose, I'd like to 17 point out this virus is extremely oncogenic, probably 18 most potent oncogenic herpes virus. It causes tumors 19 within four to six weeks in experimental animals, and 20 these tumors are of polyclonal origin. Okay. So it 21 smells like this oncogene does do something in a 22 direct way. 23 But today we'd like to talk about how this 24 virus can be used as a template for retroviral 25 insertion and what happens after that. 38 1 Just to give you a sense, this virus is 2 probably, again, the most successful live attenuated 3 virus vaccine against oncogenic virus. I'll tell you 4 a little bit about the vaccine. 5 The serotype 1 is oncogenic strand, the JM 6 MD11 and GA stand, but serotype 2 and 3 are vaccine 7 strands. They share about 70 percent homology with 8 serotype 1. However, they are not oncogenic in 9 chickens. 10 Now, there's another way of making this 11 virus attenuated virus, by taking serotype 1, okay, 12 and simply passage. It's a mysterious way, but it 13 worked every time. You passage for a long time. It 14 could be five to ten years experiments, but you would 15 get attenuated virus. 16 People still do not know exactly what 17 happened, but there is a correlation of expansion with 18 certain repeat sequence in the region. We now know 19 these attenuated viruses actually still maintain the 20 mac oncogene. So what happens most likely is that 21 these viruses do not replicate very well. Therefore, 22 they cannot induce T cell lymphoma in chickens. 23 In vitro, however, after long passage 24 viruses tend to replicate quite well. 25 Okay. So we said that moving to the real 39 1 experiment. Now, our story began with this particular 2 experiment. They were intended to study our REV -- at 3 the time it was still retrovirologist -- REV insertion 4 of the T cell lymphoma. 5 So Bob Isford took some of the T cell 6 lymphoma generated by Marek's disease virus and looked 7 for whether REV virus is present or not. 8 It turned out REV viruses are not present, 9 but there was some surprise in that in the low passage 10 of serotype 1 virus, not serotype 2 or 3, that he 11 actually could detect hybridization. This 12 hybridization was under 30 percent mismatched, but he 13 could detect hybridization against REV or TR. 14 And this suggests to us there are some 15 sequences related to REV, and we call it ALTR remnant 16 present in the present day serotype 1 viruses. So 17 this virus has never seen REV recently, but you can 18 see that they do have some sequence. 19 The fact that they show specific bands and 20 they can map in the band D and band F region, which is 21 close to the repeat sequence, convinced Bob Isford at 22 that time that these are not a fluke. These are 23 probably real homologous sequence even though it has 24 diverged significantly. 25 There are the high passage one now, 40 1 attenuated one. You can see that the sequence begin 2 to diverge because of expansion of the repeat 3 sequence. 4 However, in this, this is something else. 5 I will come back to this. This GM high virus has 6 multiple LTR related sequence. I'll come back to 7 this. 8 So then Bob Isford began to sequence of 9 them. So just to give you summary, indeed, there are 10 several patch homologies ranging from 70 to 81 11 percent, with some nucleotides ranging from 22 to 33 12 nucleotides. 13 Now, if there's only one site, you can say 14 this is very skeptical, but with all of these sites 15 clustered together in the right order and many of them 16 diverging at the junction of the retroviral genome, 17 and that suggests to us this probably is real. 18 So we took that as LTR remnants. There 19 are also some sequence related to the retrovirus, but 20 the LTR sequence was most, most prevalent. 21 And this stretch of sequence is most 22 interesting. This stretch of sequence turned out to 23 be that it's in the enhanced region of the REV LTR. 24 This sequence now is present as in the enhancer region 25 of the herpes virus alpha tif or the VP-16 equivalent 41 1 of the Marek's disease virus, and it's a T cell tropic 2 enhancer. 3 So with that we figured that in nature 4 this had happened, and let's see what happens, whether 5 in recent -- this is what we call the ancestral 6 insertion. We like to see whether in recent time 7 whether there was any evidence of recent insertion, 8 and we recall this one, the same picture. 9 This one has a multiple one, and now 10 again, this is a virus derived from low passage, 11 simply by culture them for five years. Okay? This is 12 a passage of 211, and it's completely attenuated, and 13 they can be potentially used vaccine, but this is a 14 vaccine experiment that went exactly as intended. 15 So what happens once we discover there's 16 some retroviral insertion, and these LTR hybrids under 17 stringent condition and still stick to the filter 18 paper. So we know these are more recent insertion. 19 And Dick Witter, our collaborator, 20 collected all these viruses. So he then did the 21 following smear by cloning this separately. So with 22 that he actually was able to separate these clones and 23 these LTR sequences of segregate, indicating indeed 24 they are part of the viral genome, and they are 25 genetically stable because they have been passaged for 42 1 about 200, 200 passages. 2 And so what happened is that during this 3 long term propagation you'll feed DEF, duck embryo 4 fibroblasts, the primary cells. We talked about it 5 two nights ago, that DEF, and it turns out in passage 6 about 87, that DEF they used for fuller infection by 7 Marek's disease virus turned out to contain reticular 8 endotheliosovirus. So during this evident of clone 9 mixing fraction, that retrovirus integrate that, but 10 now they become very stable. 11 Now, you would argue why do they persist 12 for so long. Well, when Dick Witter compared the 13 replication rate with the wild type, they all in vitro 14 replicate much better. We do not know whether that's 15 due to LTR insertion. However, the LTRs seem to be a 16 persister, and the LTR integrants seem to dominate the 17 culture at passage 211. 18 So this was a five year experiment ten 19 years before we began the study. We could not 20 control. We could not add any control. So Dick 21 Witter and I think that if this happened nature, on an 22 evolution or scale; if it had happened in the ten 23 years prior to our experiments, can we do a more 24 controlled experiments by doing a co-infraction 25 (phonetic), and can we make this work in five weeks or 43 1 five days? 2 So the experimental protocol turned out to 3 be very straightforward, that you mix the MDV, 4 infected DEF with REV. MDV is cell associated virus. 5 So it's actually a little bit difficult to do 6 experiment, but if you mix them together, you passage 7 them every week, and then by feeding fresh DEF, okay, 8 and then we took individual passage mass culture, 9 isolate MDV, and in fact just isolate the cell and 10 look for MDV mini chromosome imposed fiogel 11 (phonetic), and the free retroviral DNA should run out 12 of the gel, and then you probe with REV LTR to monitor 13 the kinetics of integration. 14 It turned out it's not difficult at all. 15 Okay. We have repeated this several times now. 16 Basically after, in fact, five weeks you can see a 17 little bit. This is a southern blot. So it's not a 18 most sensitive method, but you can see that retroviral 19 LTR integration increase, okay, after passage 16. 20 It's a huge amount radioactively. 21 This is just a load to show you that MDV 22 DNA were loaded at about the same amount. 23 Now, if you do TRP CPCR, you could 24 actually detect within one to two passage. Okay. So 25 the integration certainly can be very efficient, 44 1 although as I said, this is radioactive. You really 2 do not know what is the population of the integrants 3 versus no integrated one. 4 Now, this increasing intensity, of course, 5 are due to two reasons. One is that REV is still 6 present. It can infect more herpes virus, but, 7 secondly, of course, it may be the herpes virus with 8 LTR, in fact, replicated better in some fashion. 9 There is no selective pressure except in vitro 10 replication. So this may actually contain the LTR 11 integrate certain places that can enhance the 12 replication. 13 So what Dan Jones did and Rhonda Koss did 14 was -- were to actually look at the insertion sites, 15 and then something rather interesting was revealed. 16 So by looking at -- this is 17 kilobase genome, but 17 they looked at the integration sites. Integration 18 sites are tightly clustered with two insertions in the 19 GD region, but others are tightly clustered around the 20 -- close to the boundary between repeat and the unique 21 sequence. 22 Now, at Alsets (phonetic), if you look at 23 the sequence, okay, just very briefly, you found 24 actually these are the individual integration sites. 25 I do not mean to have you look at the sequence, but 45 1 look at the arrow indicating that there's no sequence 2 facility. It's a regional facility. 3 Okay. Now, at the outset we knew the 4 integration should not be totally random because we 5 were selecting viruses, replication virus. So any 6 insertion in the essential genes would disrupt its 7 ability and we may not be able to pick up. So, again, 8 this underscored the importance that if you want to 9 study some recombinant, that the selection pressure 10 turned out to be an important one. 11 So we figured that maybe these are the 12 sequence, the regions. There are no coding sequence. 13 Therefore, it integrates better, but that still cannot 14 fully account for this tight cluster because the U.S. 15 region shown by Robby Morgan and Marc Purcell can be 16 completely deleted, yet in vitro replication, and 17 still very viable. 18 So we then began to think this actually 19 also looked very much like insertion of mutagenicity 20 oncogene that we studied before, especially the RB 21 oncogene in avian erythroleukemia in terms of the 22 tight clustering. 23 So we began to entertain the hypothesis 24 maybe this can activate some of the genes near the 25 boundary. 46 1 But before I say that, before I show you 2 the data that I have, we have to isolate the virus in 3 order to do the experiment. This is a simple whole 4 cell PCR mapping insertion sites. We did not have the 5 virus yet. 6 So in the past few years Dick Witter was 7 able to isolate the virus. This was a heroic effort 8 because the retrovirus that we used had no markers, no 9 selection marker. It's a wild type retrovirus. So it 10 depends on how prevalent these integrations are in the 11 whole mass population. 12 So Dick was able to isolate several, and 13 I'm just going to talk about one, quote, RM-1. For 14 the first retrovirus, the REV MDV hybrid virus number 15 one, and this is very interesting because it contains 16 a solo LTR. No other retrovirus sequence are present, 17 and it integrates at a hot spot, the tight cluster 18 area. 19 And this shows the retrovirus duplication 20 of the MDV genome. So it's authentic integration. 21 It has a solo LTR, our first in this 22 region, and then it homogenized to the other region as 23 well. 24 Now, the most interesting is the phenotype 25 of this virus. This virus, okay, RM-1 -- these other 47 1 two are the clones -- had everything. Everything else 2 is very similar to the wild type in that it can 3 replicate very well inside bursa, inside T cells in 4 the chicken. 5 The only thing different is it has no 6 oncogenicity. Okay? So this is the viral clone that 7 did the test, in vivo test, and this is the wild type, 8 okay, the oncogenicity seven out of eight, five out of 9 eight, eight out of the eight, and this is the 10 attenuated one. Of course, after long passage it also 11 has no oncogenicity. 12 But this virus N and attenuated virus 13 differ significantly. This virus can replicate very 14 well, whereas the attenuated virus does not, do not 15 replicate very well in vivo. 16 Okay, and as a result, when Dick Witter 17 did the challenge protection experiment, so in fact, 18 it was RM-1, then challenged with the virulent, very 19 virulent MDV, it turned out this has much better 20 protectivity than the other attenuated strand. 21 And this is showing here -- maybe we could 22 just locate the protections. So this was done by 23 taking the RM clone, infect the first as a vaccine. 24 Then you challenge it with the very virulent virus, 25 and it turned out the protection is 100 percent, 48 1 whereas the current vaccine virus is 34 percent, which 2 this is the Basta (phonetic) vaccine virus, about 78 3 percent, and so on and so forth. 4 And the reason is that it replicate very 5 well inside the chicken, the only things that cannot 6 cause oncogenesis, and I'd be happy to speculate to 7 the reason why that's the case, but also because it 8 can spread very well. So it serves as very good 9 protection. 10 Okay. Now, I don't mean to say this is 11 the vaccine virus because it causes other associated 12 diseases. Other than oncogenicity, it does cause 13 thymic atrophy, like the wild type. So it cannot be 14 a vaccine yet, but this certainly gives us some clue 15 as how perhaps to manufacture better vaccine for MDV. 16 But in the context of this discussion, 17 this shows that LTR insertion can change the phenotype 18 of the virus. We look at other regions, whether 19 there's any gross change. There's no gross change, 20 but we cannot rule out point mutations. 21 So now, at the molecular level, we'd like 22 to see whether it activates anything. Indeed, this 23 RM-1 has a solo LTR integrate in there, and these are 24 the northern blot to show, indeed, there is LTR 25 insertion in here, and there is a transcript with a 49 1 link to LTR from here to here. That would transcribe 2 polysystronic message carrying soft 2 open reading for 3 US-1 and US-10. 4 Since this is the closest to the five 5 prime end, we think this product may be relevant. We 6 don't have evidence to show that this is relevant. 7 We're just beginning to do that, but I can tell you a 8 little bit about it. This is a novel sequence, unique 9 to the oncogenic herpes MDV strain virus. The 10 sequence is novel. So we do not know the function, 11 but it does share some homology with US-22 gene family 12 of this cytomegalovirus as well as HHV-6. 13 In the case of HHV-6, this particular 14 protein, it's not the same protein, but it shows some 15 similarity. It has been implicated in the 16 transactivation of HIV. 17 So we think it may be a co-factor of the 18 transcriptional factor. Indeed, recent experiments 19 show that by Hous Chan's lab that this is a 20 nucleoprotein. 21 I cannot tell you more about this simply 22 because we don't have much data about this protein 23 yet. So let me give you a conclusion. 24 So what I have shown you is that we have 25 shown retroviral insertion of herpes virus at least in 50 1 this system at several levels. We show there's 2 ancestral insertion; that avian retrovirus REV LTR 3 remnants are present in the serotype 1 MDV, indicating 4 that infection into the herpes virus in nature 5 probably happened after the divergence between the 6 vaccine virus and this virus. 7 I also show you in the acute co-infection 8 experiments REV insertion into MDV detectable as early 9 as second passage. REV insertion sites are non- 10 randomly distributed. REV LTR insertion of mutagen of 11 MDV genes demonstrated MDV with altered pathogenicity 12 in the phenotypes also generated. 13 I'd like to take this in a broader context 14 and tell you about what happened since our original 15 discovery. Now this has been repeated by several 16 laboratories, and that demonstrate that herpes 17 retrovirus insertion to herpes virus. 18 First, that our own actor (phonetic) has 19 extended REV insertion into the oncogenic strand 20 experimentally to the vaccine strand, herpes virus of 21 turkey, and we can see its insertion with no problem, 22 again within one to two passages. 23 But here there's a very interesting clone. 24 One of the clones -- I'm sorry. So this you saw 25 already, but this is REV insertion into HVT. 51 1 There's one clone here that actually 2 carries the full length of REV. In fact, we were able 3 to show this can produce virus after transfection. So 4 this is not anything specific for the oncogenic strand 5 of MDV. 6 We also showed that rav 1, rouse 7 (phonetic) associated virus, can also integrate in the 8 MDV or HVT. Okay. So, again, there is nothing 9 special about REV. 10 This is probably very significant. From 11 Japan, Hiria's group about two years ago showed that 12 this MDV virus isolate from chicken actually carry 13 endogenous REV 0. Now, this experiment did not see in 14 vitro culturing at all. So it was an in vivo isolate, 15 and REV 0 very nicely integrated MDV, again, at a hot 16 spot that we show for REV. 17 Furthermore, there are two more 18 laboratories that I actually did not update this 19 slide, show last year that the avian erythroblastosis 20 virus LTR also integrates into MDV and another REV 0- 21 like sequence integrated into MDV. So this has been 22 repeated in the four laboratories independently. 23 Recently Eric Davidson in Israel did in 24 vivo experiments to see whether in vivo recombinant 25 can be detected or not, and they used PCR knowing that 52 1 the cluster region -- so they designed PCR primary 2 that can easily detect recombinant, and they were able 3 to detect about 20 of them and were helping them 4 analyze it. 5 Now, a year ago this paper is rather 6 profound to us. It's not retrovirus insertion of 7 herpes virus, but this group found that REV can 8 integrate into fowlpox virus, and the pox virus is a 9 vaccinia virus group. 10 This is profoundly significant to us 11 because this shows retroviruses can integrate into a 12 virus that only has a cytoplasmic life cycle. They 13 may not need the DNA pk. You may not other things in 14 the nucleus to do the job, but you can. 15 And again, I emphasize this was an in vivo 16 isolate. The virus never sees in vitro culture. So 17 its recombination in vivo. 18 Then finally, George Miller's lab found in 19 EBV infected cells there is a fusion of cDNA. They 20 did an isolated virus. So we do not know the fate of 21 the virus. They do find a junction that carried both 22 retroviral LTR and EBV sequences. 23 So this may happen in other systems as 24 well, and I do not -- I have no reason to believe it's 25 a special for chicken, but I think, again, selection 53 1 is very important. If you do not have selection, this 2 kind of integration come and go. 3 I think I will stop here. Thank you very 4 much. 5 (Applause.) 6 PARTICIPANT: I have a comment and a 7 question. I think for the sake of the audience it's 8 also worth noting that there is evidence for ancestral 9 capture in a number of herpes viruses, ancestral 10 capture of cellular genes by a process involving 11 retroviruses. 12 So, for example, if you look at the 13 members of the gamma-2 herpes virus group, they -- 14 from new world primates, old world primates, and 15 humans -- those herpes viruses have genes for 16 dihydrofolate reductase, cyclin D, and a few other 17 genes, in all cases lacking introns (phonetic), the 18 lack of introns suggesting it's acquired by a process 19 involving reverse transcription, and that's likely to 20 have been acquired by a process involving co-infection 21 of cells with a retrovirus. 22 My question has to do with why do you 23 think -- it looked to me like that many of your 24 examples of capture were pieces of LTR and not whole 25 LTRs. You showed one example of a single LTR. Why do 54 1 you think there are so many -- I mean, how does that 2 happen, and why does it just have small pieces or a 3 single LTR, and is there any evidence in any cases 4 that these LTRs or LTR pieces are actually driving 5 expression of some viral gene? 6 DR. KUNG: Okay. First, I did snow in RM- 7 1. I just went through so quickly. I'm sorry. The 8 LTR is promoting insertion activate transcript. Okay? 9 Secondly, in terms of why it's LTR, I 10 think that what happens, LTI/LTR direct recombination 11 which especially in herpes virus, that you have the 12 flip-flop of the R region. So the recombination is 13 very, very acute. You have direct recombination 14 between LTI/LTR you would delete the sequence of. In 15 fact, the full length sequencing, the herpes virus 16 genome, it's not very stable. It's about ten 17 kilobase. So the virus has a tendency in my mind to 18 spit out the extra sequence, and LTR seems to be 19 harmless at least. Yeah. 20 MR. MINOR: Philip Minor from NIBSC. 21 I'm completely ignorant about 22 retroviruses. The integrations that you described 23 were all with co-infections, I guess. Is it possible 24 that you could get an integration if you were just 25 looking at an endogenous retroviral sequence that was 55 1 maybe being transcribed? Can you pick it up in that 2 way? 3 DR. KUNG: Yeah. Thank you for asking the 4 question. 5 Again, I went so quickly. There was an 6 endogenous virus REV 0 integration into the herpes 7 virus shown by the Japanese group, but also more 8 importantly is that the retrotransposon, popping -- I 9 guess later the speakers will be talking about that -- 10 it's very frequent. 11 I give you one example, not related to 12 herpes virus, but you probably know the bacula virus, 13 the autografa californa nuclear polyhedral viruses, 14 that the Freezen (phonetic) and Miller and their 15 colleagues did a beautiful study, show that the TET, 16 it's a transposal element, number four, integrating to 17 the bacula virus genome transcribed the gene and 18 contained the entire sequence. 19 So, yes, my feeling is it's -- all we're 20 talking about is selection. If you have a selection, 21 I think you will be able to detect those endogenous 22 transposable -- retrotransposable like, and I think 23 that's also related to Ron's comment. 24 Many of the herpes viruses captured, 25 especially like the KSS-3, Kaposi's sarcoma, capture 56 1 a lot of cellular homologs, and many of them are 2 internalist (phonetic) even though herpes virus are 3 known to be able to splice out intron. 4 So my feeling is that some of them may not 5 be entirely due to retrovirus, but could be due to 6 retrotransposable. 7 DR. PEDEN: Keith Peden, CBER. 8 You may have said, but does the 9 integration site have the hallmarks of genuine 10 retroviral integration, the duplications at the ends? 11 DR. KUNG: Oh, yes, yes. 12 DR. PEDEN: It does? 13 DR. KUNG: Duplication at the end, yeah. 14 MR. COFFIN: John Coffin, Tufts. 15 Just to clarify a little bit your answer 16 to the previous question, I think it was asked whether 17 the endogenous viruses could actually be moving 18 without an infection cycle, and the answer probably 19 is no, for all that we know about endogenous 20 retroviruses. You still need to get -- in order to 21 move them from one place to another, you still need to 22 -- they still need to undergo a complete replication 23 cycle. 24 DR. KUNG: Yes. 25 CHAIRPERSON ROSENBERG: I think we need to 57 1 move on because we are somewhat behind, although I 2 don't want to cut this short. 3 The next speaker is Maxine Linial. 4 DR. LINIAL: Okay. Today I'm going to 5 talk mostly about foamy viruses, but I wanted to start 6 off raising a couple of issues, some work in my lab 7 about packaging of avian retroviruses. 8 Okay. So for many years my lab has been 9 interested in defining the minimal packaging region of 10 the avian retroviruses, and this is a schematic of 11 what from the literature appears to be defined as the 12 minimal packaging regions of NLV HIV and the ALV 13 viruses, and we had previously defined a packaging 14 region of about 160 nucleotides from the five prime 15 end of the genome, which are sufficient for packaging. 16 That is, you can place this region on any heterologous 17 RNA, and that RNA will be packaged with a high 18 efficiency into retroviral particles. 19 And in fact, we find that such a small 20 region on a neo or a hygro RNA is packaged only about 21 2.7 times worse than the intact ALV genome. So we 22 believe this is a sufficient packaging region. 23 More recently we've done a series of 24 experiments on this 160 nucleotide region and have, in 25 fact, found that we can delete off the entire three 58 1 prime end, leaving an 82 nucleotide region with this 2 set computer predicted secondary structure that has 3 several stem loop regions, and by mutagenesis RNAase 4 protection and looking at a variety of viruses, we 5 know that there is one, two, three stems that are 6 important, and that possibly the region that's 7 involved in protein binding and recognition of this 8 RNA may be only a four nucleotide loop. 9 So we're getting very close to 10 understanding what the structure of the packaging 11 region looks like for this virus. 12 And one other point I want to make is that 13 retroviral packaging seems to be a hierarchy of 14 sequences. The retrovirus most avidly packages 15 sequences with its own psi regions, but then, of 16 course, we know that vector RNAs containing psi can be 17 packaged with very high efficiency, and probably after 18 that the retrovirus is packaged cellular RNAs or other 19 elemental RNAs containing psi-like sequences, as we 20 heard for MLV. VL-30 sequences have psi-like 21 sequences and are avidly packaged, but they also 22 package random cellular RNAs. 23 And work from my lab and a variety of 24 other labs has shown that such RNAs can be a player in 25 packaging cell lines, and we did a lot of work on a 59 1 quail packaging cell line which contains a single 2 integrated RSC lacking psi, and the unique thing about 3 this packaging cell line is that the provirus is 4 exceedingly active and produces about 100 times more 5 virus than any of the other packaging cell lines that 6 we looked at, and because it made so many particles, 7 we could so that what was packaged was random cellular 8 RNAs, and that we could also show that these cellular 9 RNAs could be reverse transcribed in new cells and 10 integrated into the genome with detectable frequency. 11 And we could find about 100 using neo as 12 a cellular RNA. We could find about 100 transductions 13 of the neo gene in an infection with this virus, and 14 we propose that this kind of event can occur with any 15 packaging system if it's looked for, but this kind of 16 a looking for integrations in the new cell is not 17 generally assayed in any of these packaging systems. 18 So now I'd like to turn to foamy viruses. 19 This is one of the seven genera of retroviruses, the 20 spuma retroviruses. They're a tightly knit family 21 that's fairly divergent from all the other groups of 22 retroviruses. 23 One aspect of the virus that's very 24 interesting is that unlike, for instance, the gamma 25 retroviruses which are know -- can be pathogenic in 60 1 their native host with long latency, the gamma 2 retroviruses or the alpha retroviruses are unlike the 3 lentiviruses which are known to be pathogenic and 4 accidently infected hosts but generally not in their 5 natural host. 6 The spuma viruses or foamy viruses are not 7 pathogenic in any host but spar. So that there is a 8 whole variety of these viruses, and their life long 9 infections in their host, and there's no 10 pathogenicity, and when there's an accidental 11 infection, for instance, several of these simian 12 viruses are known to infect people. 13 Again, there appears to be no 14 pathogenicity in those hosts, and this is a very 15 interesting question, is why the life style of this 16 virus is so different from those of its other 17 retroviral cousins. 18 The genome of the foamy virus is very 19 similar structurally to other retroviruses, has gag, 20 pol, and env genes. It also has several open reading 21 frames and two known products, the transactivator 22 protein called tas, which is absolutely required for 23 transcription from the LTR promoter, and uniquely to 24 this group of viruses there's a second promoter in the 25 envelope gene, the internal promoter, which seems to 61 1 be responsible for transcription of the accessory 2 genes, tas, self, and a second gene which is a spliced 3 variant from tas and bel 2 called bet (phonetic). Bet 4 is a major product of this virus. It's completely 5 dispensable in tissue culture, but is believed to play 6 some important role in vivo, although we have no idea 7 what the role of this protein is. 8 Some features of this virus that make it 9 very different than other retroviruses is, first, the 10 pol protein is not made as a gag-pol fusion. It's 11 made from its own splice pol message. This makes it 12 more similar to the hepadenoviruses that goes to the 13 retroviruses. Again, it has an internal promoter 14 unlike all the other retroviruses. 15 And a third feature is that you cannot get 16 particle egress from the cell with gag alone. You 17 must have the envelope protein. Again, we're similar 18 to the hepadenoviruses like HBV than to the 19 retroviruses. 20 Foamy viruses have been isolated from a 21 variety of species. It's extremely prevalent in cats, 22 both domestic and wild cats. Recent studies say about 23 70 percent of individuals cats are infected. It's 24 highly prevalent in many bovine flocks. 25 Recently a virus has been isolated from 62 1 horses, although the prevalence of this virus is not 2 clear at the moment, and from a variety of primate 3 species, in fact, every primate species that's been 4 studied does have a foamy virus. 5 And in some groups of primate in primate 6 centers, essentially all of the individuals infected 7 -- Jonathan Allen at Southwestern, I think, has shown 8 that all of the baboons there do have foamy virus. So 9 although there aren't so many studies in the wild, 10 these viruses have been isolated from wild animals, as 11 well. 12 There have been several isolates from 13 human, and I'd like to speak briefly about that. The 14 type species is called HFV, human foamy virus. This 15 was isolated from a nasopharyngeal carcinoma cell 16 culture from a patient from Kenya many years ago. 17 Recent studies, however, from the group at 18 CDC have clearly shown that this human virus is 19 basically a chimpanzee virus, and interestingly, it 20 clusters very closely with isolates from 21 Schweinfurthiae chimpanzees, which is the only 22 chimpanzee that's present in East Africa where the HFV 23 isolate came from, which clearly suggests that HFV was 24 in this culture because the patient might have had 25 contact with the chimp and acquired the virus through 63 1 a zoonotic infection. 2 And all the other isolates from people are 3 clearly linked to having been bitten by a monkey or a 4 chimpanzee. 5 So one interesting thing about HFV is 6 despite the lack of pathology in vivo, as far as we 7 know, in culture you get two types of infection. The 8 first and most dramatic is a cytopathic infection of 9 fibroblast, and this was what gives the foamy virus 10 their name because these cells become highly 11 multinucleic and syncytia, multi-syncytia form. 12 But there's also a second type of 13 infection, a long term, persistent infection, and this 14 is seen and our lab has found it in a variety of human 15 cell lines, T cells, erythroid cells, monocytic cells, 16 et cetera, and in these long term, persistent 17 infections you see absolutely no CPE. The cells 18 become infected. They grow perfectly normally, and 19 you would not know that they were foamy virus infected 20 without doing PCR or assaying the virus. 21 So this is clearly a problem when one 22 deals with material from primates. One needs to 23 assume that the primates are probably infected with 24 foamy virus, and although many isolations of cell 25 lines from primates lead to cytopathicity and clearly 64 1 show their foamy virus, since we know very little 2 about the cells in vivo that are actually infected, 3 it's very possible that you could have cell lines from 4 primate cultures that are infected with foamy virus 5 without CPEs. 6 This is an example. This is an indicator 7 cell line where we have LTR driving betagal in a 8 hamster fibroblast line, and when you infect these 9 cells with foamy virus, you can see that that turns on 10 the betagal expression from the tas transactivator. 11 You get highly cytopathic cultures. 12 And here's an example of a really giant 13 multi-nucleus syncytia that can occur at high 14 multiplicity. On the other hand, when we infect a 15 variety of human cell lines, these cell lines grow for 16 years. This only goes up to 40 weeks, but we've grown 17 these cells. They continually produce virus, but they 18 are never cured of the infection, and there's no CPE. 19 Here we show that in this experiment we 20 could not infect a V cell, but this is not a problem 21 of infectivity. This is a problem of viral 22 replication. In fact, now that we have vectors marked 23 with gfp and work from other laboratories as well 24 shows that there are basically no vertebrate cells 25 that are immune to foamy virus infection. Everything 65 1 from fish on upwards, and in fact, in humans no cell 2 types to be seen to be immune to foamy virus. So 3 whatever the receptor is, it's extremely widespread. 4 So the life cycle of foamy virus is very 5 similar to that of other retroviruses with a couple of 6 striking exceptions. As I mentioned or I will mention 7 again, the virus, most of the viral budding is through 8 the endoplasmic reticulum, and although some virus 9 does bud from the plasma membrane, again, in order for 10 the virus to bud, there must be glycoprotein. 11 If you have an M minus mutant, virus does 12 not bud from the cell, and this tends to be a 13 cytopathic event. If there is no envelope, the cells 14 die very rapidly. 15 The other important thing to mention is 16 that work from our lab, as well as another lab, 17 strongly suggests that reverse transcription in this 18 virus is a late step in infection, and that means that 19 the functional genome in foamy viruses is really DNA 20 rather than RNA. 21 And this is done using AZT as an inhibitor 22 and show that the stuff that is sensitive to AZT is a 23 late step rather than an early step in the life cycle 24 of other retroviruses. 25 Another point to be made is that there are 66 1 huge numbers of intracellular particles. In fact, 2 most foamy virus is intracellular. Only about one to 3 five percent of the virus buds from the cell. The 4 rest of it is cell associated, and this has suggested 5 that perhaps like hepadenoviruses, there could be some 6 type of recycling step where some of these 7 intracellular particles get back into the nucleus, and 8 I'll get to that in a moment. 9 So in terms of the HFV genome, by doing 10 very sensitive PCR and RT PCR, we found that about 25 11 percent of the particles released from cells contain 12 apparently full length, double stranded DNA. The AZT 13 experiment strongly suggests that the functional 14 genome is DNA so that even though there are a large 15 number of RNA particles, we don't believe that these 16 are infectious, and they're probably remnants of 17 abortive reverse transcription events. 18 And we've also been able to show that if 19 you extract DNA from extracellular particles, it is 20 infectious if you put it back into cells with 21 lipofectamine. 22 Despite the fact that the foamy virus 23 functional genome is DNA, what is packaged is RNA, and 24 this is RNA's protection experiment using wild type 25 human foamy virus or a deletion mutant in the pol 67 1 gene, looking at RNA's protection. 2 And I haven't shown you the controls here 3 for particle numbers, but when you look, and this is 4 RNA's protection that really only just looks at RNA in 5 the particles and not DNA. If we RNA, we don't see 6 any nucleic acid in the particle, and the same is true 7 for the pol mutant. 8 And what we found from this study when we 9 compared the amount of nucleic acid packaged in the 10 pol mutant and wild type, it's exactly the same when 11 it's normalized to the number of particles. 12 So you don't need polymerase to get genome 13 into the particle. Of course, this is completely 14 dead. So what's packaged is RNA, and DNA just occurs 15 sometime during assembly or egress from the cell. 16 We also believe there are large numbers of 17 particles in the intracellular particles that contain 18 DNA. So when you look at a copy number of foamy virus 19 persistently infected cells or acutely infected cells, 20 you can find hundreds or thousands of copies of DNA 21 per cell, and this has made it very difficult to look 22 at integrated genomes in these cells. 23 This is a schematic of what we think the 24 foamy virus looks like. Instead of having an RNA 25 genome, it has a DNA genome. 68 1 Another feature of the foamy virus which 2 I didn't mention, but which is also very striking is 3 that the gag polyprotein is not cleaved except for 4 four KD at the C terminus. So you never get 5 maturation to caps at matrix and nuclear caps as in 6 other retroviruses. Basically we believe that this 7 gag protein is multi-functional, that the carboxy end, 8 which has many basic residues, probably behaves like 9 the core protein of hepadenovirus and interacts with 10 the DNA genome in the particle, and that probably the 11 amino terminus behaves somewhat like matrix and part 12 of this protein also behaves like tapsin (phonetic). 13 So we were interested in looking at 14 integration, and to do this we used a chronically or 15 persistently infected H-92 cell line, and we cloned 16 out single cell clones of this virus, and in these 17 experiments, these southern blot experiments, we cut 18 with NAG-1, which cuts ounces in the genome, and use 19 the bet probe. 20 And if you do such an experiment, you can 21 see a large number of -- a large amount of viral DNA. 22 This is the cut DNA here. This is the small amount of 23 DNA that was not cut. 24 And in many experiments this obscures the 25 background of integration. So what we do is we treat 69 1 the cells for several weeks with AZT, and this gets 2 rids of all the intracellular DNA in the particles, 3 and then you can easily see the integrated copy number 4 in these single cell clones, and what's very striking 5 is that there is a large number of integrated DNAs. 6 And we've counted upwards of 20 bands per 7 cell of, as I said, single cell clones of foamy virus 8 infected, which were leukemia cells. We've confirmed 9 that these are single integrations by cloning out the 10 junction fragments and showing that they're not 11 duplications or repeats. 12 These are bona fide new integrations. 13 We've also done FSH analysis. This isn't a very good 14 slide, and the arrows are not pointing to all of the 15 integrated copies. You'll just have to take my word 16 for it. There's a rough correlation between the 17 integration number that we see by a southern blot and 18 by FSH analysis, and we do see up to about 20 19 integrated copies per cell genome of this virus as 20 well. 21 And we have been very interested in the 22 mechanism of these multiply integrated pro viruses. 23 I'm not showing you the data. There is, in fact, a 24 NLS sequence within the gag genome foamy virus which 25 Axel Rethwelm's lab shows behave as an NLS. So after 70 1 infection, much of the newly synthesized gag protein 2 goes back into the nucleus. 3 And by using a mutant in the NLS, so that 4 if you delete the NLS the virus can grow fine in 5 tissue culture, it does have a slightly lower titre, 6 and we've shown that by deleting the NLS we prevent 7 accumulation of all of those multiple integrated 8 copies, and we now have clones of cells that only have 9 one, two, or three copies per cell instead of ten or 10 20 copies. 11 So this implicates that there could be 12 some kind of intracellular recycling mechanism that's 13 responsible for the high copy number. However, what 14 complicates this is that we've recently found that the 15 foamy viruses also have another unexpected feature, 16 and that is we have made a vector in which we have 17 replaced the bet gene with GFP, and in this case we 18 also put in the RSV strong promoter. 19 And when we use a vector to infect either 20 BHK cells or these erythroleukemia cells, we get a 21 high infectivity. This is shown with the GFP 22 fluorescent, but surprisingly if we compare 23 infectivity of either these uninfected H-92 cells or 24 this single cell clone that has about 20 integrated 25 copies, we find there's basically no difference in the 71 1 ability to reinfect the uninfected -- the infected 2 cells versus the uninfected cells. 3 So at least in these persistently infected 4 cells that have huge copy numbers, they're not immune 5 to super infection, and this is very surprising for a 6 retrovirus. 7 So, therefore, we can't say whether we're 8 getting accumulation of all of these integrated copies 9 by an intracellular pathway or an extracellular 10 pathway, and this also suggests that even if a cell is 11 infected by foamy virus, it would not be immune to 12 superinfection by another foamy virus or more foamy 13 virus. 14 The other point about foamy -- foamy 15 viruses are not very well studied. There's very 16 little information about the packaging sequences of 17 these viruses. Several groups have tried to make 18 viral vectors, and so far this isn't an attractive 19 genome for viral vectors. 20 For one thing, the virus, as I said, is 21 probably not pathogenic in either accidental or 22 natural hosts. It has a very large genome, greater 23 than 11 KV. It has at least one gene that we believe 24 we can delete without -- at least in vitro -- without 25 any untoward effects on the virus. It has two 72 1 promoters, so it's a very flexible genome. 2 And another point is work from Germany has 3 shown that in infected monkeys you can find foamy 4 virus DNA in every organ in the body, including the 5 brain, although there's very low viral replication in 6 vivo. 7 So it might be a good gene delivery target 8 to evoke a wide variety of organs. So in vector 9 development several groups have found that, in fact, 10 there are probably at least two packaging or two sys 11 acting regions in the RNA, and one is at the five 12 prime end of the genome where you would expect a psi 13 sequence to be, but surprisingly there's also a region 14 in the pol gene that's required for transfer of vector 15 sequences. 16 Whether this is another packaging sequence 17 or has another sys acting RNA function is not known. 18 For instance, since pol is not made as a gag-pol 19 fusion protein and the method of incorporating pol 20 into the particles does seem to require a gag-pol 21 interaction, but we can't rule out that perhaps it 22 also needs to bind to the RNA, and so this region of 23 pol could be a pol binding sequence. We don't really 24 know. 25 So the packaging regions of this virus are 73 1 not at all well understood at all. 2 So in summary, all retroviruses have 3 packaging signals. Unfortunately those of the foamy 4 viruses have not yet been delineated. Foamy viruses 5 package RNA, although the functional genome appears to 6 be DNA. 7 In some cells, at least in tissue culture, 8 foamy virus infection leads to multiple integration. 9 This is an interesting point because one would think 10 that if such a thing occurred in vivo, and we have 11 absolutely no evidence for it, that foamy viruses 12 would be all set up for promoter insertions and 13 inductions of tumors in infected animals. Yet this 14 has never ever been seen. 15 So it's possible that the replication in 16 vivo is so meager that the virus never really does 17 multiply integrate, but nobody has ever been able to 18 or nobody has ever looked in vivo for foamy virus 19 integration sites. 20 Nothing is known about the recombination 21 between foamy virus and other retroviruses. Clearly, 22 experiments that are done in monkeys, infecting them 23 with other viruses such as SIV or viral vectors, there 24 is probably foamy virus in all of those animals. So 25 it would be very interesting to know something more 74 1 about how foamy virus interacts with other types of 2 retroviruses. 3 And also any packaging cells derived from 4 primates, bovine or feline species need to assess the 5 effect of foamy virus as well. 6 And I think that's all I have to say. 7 (Applause.) 8 CHAIRPERSON ROSENBERG: We need to keep 9 the questions, I'm afraid, brief because we are 10 seriously behind. So I believe we're supposed to be 11 at the break, but we still have one more speaker. 12 PARTICIPANT: Maxine, does anybody know if 13 vaccines have been checked for foamy virus 14 contamination? 15 DR. LINIAL: As far as I know, no. 16 PARTICIPANT: You mean nobody has looked 17 or as far as you know? 18 DR. LINIAL: I don't know. There are very 19 few reagents. I mean, there are reagents for the so- 20 called human or chimp foamy virus, but as far as I 21 know, there are no good antibody reagents. 22 PARTICIPANT: There are. 23 DR. LINIAL: There are? 24 PARTICIPANT: There are? They are 25 checked. Okay. 75 1 DR. LINIAL: Are they checked for all of 2 the simian foamy viruses? 3 PARTICIPANT: No. 4 PARTICIPANT: By PCR? Is that -- 5 PARTICIPANT: (Inaudible.) 6 CHAIRPERSON ROSENBERG: Could someone 7 repeat this so that -- 8 PARTICIPANT: I think it's by a 9 combination of tests, including PCR and infectivity 10 tests; is that right? Okay. 11 DR. LINIAL: One problem is that these 12 monkeys do get cross-infected with other foamy 13 viruses. So, you know, I don't know how many you're 14 looking at. 15 PARTICIPANT: Since foamy viruses seem to 16 be breaking all of the rules, I wonder if it's worth 17 asking or is it known whether integration is required 18 for replication, whether there's significant -- since 19 there are so many copies of DNA, et cetera, whether 20 there can be significant expression and replication in 21 the absence of integration. 22 DR. LINIAL: My lab, as well as a lab in 23 Germany, have made a DD35E integrate mutants, and at 24 least in tissue culture it's completely dead. So we 25 believe integration is required. 76 1 CHAIRPERSON ROSENBERG: I think we need to 2 move on. The last talk in this session is by John 3 Kappes. 4 DR. KAPPES: Just a momentary delay. 5 Technology, MacIntosh, a little slower in booting. 6 Perhaps I'll begin a short introduction. 7 The focus of my work -- there we go -- has 8 been on the possible use of lentiviral vectors for 9 gene therapy. Off to a bad start. That is the second 10 slide. 11 The principal concern for using lentiviral 12 vectors for gene therapy is that they may recombine to 13 produce replication competent retrovirus, and 14 underlying the concerns for replication competent 15 retrovirus is genetic recombination. 16 There have been a number of different 17 types of -- and I'm going to focus really just on HIV- 18 based vectors, although I'll probably refer to them 19 many times as lenti -- but there have been a number of 20 different HIV-based lentiviral vectors produced to 21 minimize the pathogenic properties of any RCR that 22 could emerge, and those include deletions of most of 23 the accessory genes, deletions of even the TAT and REV 24 regulatory genes, the lesions in U3, and while I won't 25 focus on a lot of these details, I will focus on, 77 1 again, what I think is fundamental to understanding 2 the risks associated with generating replication 3 competent retrovirus, and that is genetic 4 recombination. 5 The assays which have been used thus far 6 include, that is, to measure recombination or, if you 7 will, really more because of the way they've been 8 applied measurements of RCR, include gag transfer -- 9 oh, TAT transfer it should be -- gag transfer, and DNA 10 mobilization of marker rescue assays. 11 It's unlikely, and I'm sure they have not 12 been suggested by the authors who published on these 13 assays to be adequate indicators of the risk 14 associated with these viruses or these viral vectors 15 in vivo. Especially that's true in the long term. 16 So today I will present data from an 17 approach that I devised to understand the risk, if you 18 will, of using HIV-based vectors through an analysis 19 that focuses on genetic recombination. 20 First, I'll present one slide, the unique 21 difference that I've used to enable the detection of 22 recombinant viruses and their analysis, and then 23 several slides I'll show the detection and 24 characterization of these recombinants, both 25 biologically and genetically, and finally, in one of 78 1 two slides I'll show how I have further disarmed or 2 dismantled or split the functions of the lentiviral 3 based vector to improve safety. 4 This depicts the three component systems 5 for HIV-based vectors, not necessarily analogous to 6 what might be thought of as third generation, but the 7 important point is that there is a packaging 8 construct, a vector construct, and an envelope 9 construct, and just for this one slide, I wanted to 10 point out in particular the TAT gene because my 11 recombination assay is based on TAT. 12 That is, if recombination occurs between 13 the vector and the packaging construct, and if TAT is 14 included, I will be able to select for the recombinant 15 using this approach. 16 If lentiviral vectors are generated 17 through transfection of 2-9-3 T cells, which is what 18 I will show in every case, genetic recombination can 19 occur during reverse transcription to generate an LTR 20 TAT containing structure. It could contain gag; it 21 could contain the full packaging construction, but 22 minimally if it contains TAT and TAT is expressed, it 23 could confer resistance to puromycin in the cell line 24 that I call Hela-puro. 25 This cell line was transduced with this 79 1 construct, which confers resistance to puromycin 2 selection when the cells infected with a virus or a 3 vector containing and expressing TAT. 4 A couple of other points worth noting for 5 this slide because it's really what I will use in 6 terms of the components that generate the vector and 7 the data I'll show in every slide, except for a couple 8 at the end. 9 The packaging construct is driven by a CMB 10 promoter. It contains gag-pol-vif. Vpr, vpu, mpf 11 (phonetic) are deleted, and also importantly nef is 12 deleted, importantly because there is no overlapping 13 sequence at this end of the genome, which will become 14 important for reasons which hopefully will be obvious 15 later. 16 So to generate the HIV vectors, I 17 transfect these three constructs into 2-9-3 T cells, 18 and that's depicted here. 19 Through transfection bioparticles are 20 generated, and these bioparticles can be used to 21 infect the Hela-puro cell line. 22 If TAT is expressed, the cell should be 23 resistant, and that's exactly what is shown here. 24 These are the colonies stained with crystal violet, I 25 believe, about nine days after puromycin selection, 80 1 and what's shown is that there is approximately 1,000 2 colony forming units per ten to the seventh infectious 3 particles. 4 Also, importantly, if this infection is 5 done in the presence of a niverapine, there are no 6 resistant colonies detected, implying that it's 7 mediated through the HIV-1 reverse transcriptase, and 8 in particular, I chose niverapine because it's 9 specific for HIV-1 RT. 10 As I suggested, recombination could occur 11 through any region of the packaging construct. As 12 long as TAT was picked up, resistant colonies could be 13 produced. So to confirm TAT was actually present, we 14 designed primers to amplify the first exon, and that's 15 what's depicted here in what I call the lenti pool. 16 This is the lenti pool. After the cells 17 were expanded, the high molecular DNA was extracted, 18 and PCR shows detection of a 219 base pair fragment 19 similar to that detected in proviral DNA. 20 Because or at least in part because data 21 had been published that showed there was no TAT 22 transfer, no gag transfer, no marker rescue using 23 lentiviral vectors, I wanted to address each of those 24 points. 25 And the first series of slides I just 81 1 showed would suggest that there is TAT transfer if the 2 system is sensitive enough to detect it. 3 This slide detects a slightly different 4 approach for looking toward gag transfer. This is the 5 path I just described for TAT transfer. Because 6 recombination can occur in many ways where an 7 infectious virus would not be generated, I chose this 8 pathway for gag transfer because I was really looking 9 for an open gag reading frame. 10 So the difference is instead of going at 11 the heel of puro cells, I infected the virus particles 12 that were generated by transfection into 2-9-3 T, and 13 in 2-9-3 T, if a recombinant forms such that you had 14 an open gag-pol rating frame, you would expect that 15 particles could be generated, and if those particles 16 were pseudotyped by transfecting VSVG into the 2-9-3 17 T cells two days after infection, then they could 18 infect the Hela-puro cell line and confer resistance, 19 and that data is shown here. 20 We detected 540 resistant colonies when 21 the virus derived by 2-9-3 T cells was used to infect 22 the Hela-puro cell line, suggesting both DNA 23 mobilization and gag transfer. 24 Another way of looking at DNA -- oh, let 25 me point out that we also confirmed from the 82 1 supernatants of the puromycin resistant cells the 2 presence of gag protein, both in culture supernatants 3 and in viral pellets. 4 Importantly, the amount of gag detected 5 was increased by about tenfold if TAT was transfected 6 into those puro resistant cells, suggesting that the 7 LTR was truly linked, that is, expressed in sys, with 8 the packaging construct. 9 Another experiment, which was just a 10 further extension of what I've already shown related 11 to the gag transfer, to more specifically and more 12 clearly differentiate between artifacts and true DNA 13 mobilization is the depicted here. 14 The recombinant, which I referred to as 15 mobilizing gag or gag transfer is depicted here. We 16 don't know at this point whether this is the exact 17 structure, but it should contain an LTR gag-pol 18 reading frame, and certainly tat and rre. If this 19 structure is respent in the Hela-puro line, it's 20 possible that when it produces particles because the 21 Hela-puro cell line contains puromycin and a packaging 22 signal further puro RNA strained, that it, too, could 23 be packaged into these particles, mobiled to heal a 24 TAT line in this case, and confer resistance to 25 puromycin. And indeed, that's the result shown here. 83 1 So the data indicate by three assays 2 recombination of the lentiviral vector componen