John James Skehel Oral History

June 28, 30 1999
Recorded and edited by
Sondra Schlesinger

John Skehel is currently the Director of the National Institute for Medical Research, Mill Hill London, a position he has held since 1987. He received his Ph.D. in 1966 at the University of Manchester where he worked on ion transport mechanisms in yeast. He then became interested in mechanisms of derepression in eukaryotic systems and decided to do postdoctoral research on the induction of interferon with Derek Burke at the University of Aberdeen. Initially viruses were only a means to induce interferon, but - as virologists know - viruses quickly capture the interest and imagination of scientists and John Skehel was no exception. Semliki Forest virus was used to induce interferon, but then John and Alan Hay began to work on proteins synthesized in cells infected with this virus. John claims that they switched from Semliki Forest virus to the influenza virus, fowl plague virus, because they wanted to look at a more complicated virus that had many more proteins "to mess around with." John left Aberdeen to spend a year with Professor Wolfgang (Bill) Joklik at Duke University where he worked on reoviruses and specifically on in vitro transcription of reovirus mRNAs. He claims that it was "therapeutic" just to see how much RNA could be made and could be seen on stained gels. In 1969 John returned to the UK, to the National Institute for Medical Research at Mill Hill and to influenza research. Initially he worked on replication. As he said: "Replication was what it was about." John continued to have an interest in influenza virus replication and mRNA transcription, but he also began to pay more attention to the surface glycoprotein, the hemagglutinin. One of the initial reasons was that Colin Brand who was a graduate student began to work with him on the viral nucleoprotein and that is where this oral history begins.

John Skehel Dick Compans had just digested viruses with bromelain to make them bald. That looked like a good starting point to get rid of all this (outer lipid and glycoprotein) "crap" to get the nucleoprotein. So he (Colin Brand) did that. We talked about it. This would be a good project for a thesis. You look at every fraction and see where everything had gone. It turned out that after bromelain digestion the protein (the hemagglutinin) had been released into the supernatant and was still pretty large. Vivien Mautner had just crystallized the (adenovirus) fiber. You know Helio Pereira had crystallized the (adenovirus) hexon here, first. Those were the two first animal virus proteins to be crystallized, the hexon and the fiber. So everybody knew in this lab that the thing to do if you got protein was - crystallize it. Not that they ever did anything with them because life was too short. Colin Brand - just in getting rid of the sucrose and concentrating it - got crystals and it probably is the case that he had crystals of neuraminidase.

SS I'm glad you said that because I had been told not to say anything about that unless you did.

JS No that's O.K. It's interesting because I said probably because I don't know. There are obvious things you do for your thesis. One of them is to take those crystals and see what that protein is. And he (Colin Brand) did that and it was HA so whether he ever had crystals of HA was his business. We published it as crystals of HA. That must have been one of the things I wrote up just before going to Australia (in 1972).

One of the other things he had done as a consequence of that was - because we had done electron microscopy on the soluble HA - we wanted to do the same for the neuraminidase. So Hans Noll had made neuraminidase years before, in the early 60's, by treating flu B with either pronase or trypsin and he had neuraminidase purified. There had been quite a lot of work done on neuraminidase so we did the same just for completion on the glycoproteins and Nick Wrigley got some fantastic EMs on the neuraminidase showing it was a square box, looking at it from the side and the top (Wrigley, NG, Skehel, JJ, Charwood, PA, Brand, CM, Virology, 51: 525,1973). So we compared the hemagglutinin with the neuraminidase and we didn't have neuraminidase in the HA that he was crystallizing. There were no two ways about it. While I was in Australia I did some more stuff on neuraminidase and in fact got crystals of neuraminidase while I was there but I started from flu B neuraminidase and never did anything with it. I still have pictures of the crystals.

JS I sort of decided when the bromelain results came - I remember seeing Derek Burke who was then in Warwick and was in his office and he said "what are we going to do?" I more or less just decided "I am going to do the hemagglutinin". That was just before I went to Australia, this was in '72.

JS No, in terms of the size of the protein and how much work it would involve I was completely naïve. It wasn't just the crystals that I wanted to do, it was the sequence and all that. So I started a collaboration with (Michael) Waterfield downtown (at the Imperial Cancer Research Fund) on the protein sequencing and that continued from '72-'73 when I came back from Australia and went right through to '82-'83. And it went very well, I think. It wasn't easy. The glycosylation stuff wasn't easy. The fact that it was membrane associated wasn't easy.

SS By the time you were doing things with Waterfield, did you have the bromelain piece?

JS Yes, largely that, but by comparing that with the intact protein, we knew that the hydrophobic piece was at the C-terminus of HA2. We could define the cleavage site. We knew what the biosynthetic amino terminus was. That was fine, that was by 1974-75. An interesting snippet was the business of the fusion peptide. We did the amino terminal sequence of HA2 for the H3 subtype of HA. The amino terminus of HA1 was blocked anyway and so the only sequence you could get was HA2. So we did that from a lot of different strains and showed that it was conserved and we also knew that that was the site of cleavage of the precursor. (Jack) Strominger put that into PNAS for us. In fact, taking that to Strominger was the first time I met Don (Wiley) although I'll tell you how that started.

Jack put it into PNAS (Proc Natl Acad Sci USA, 72: 93-97, 1975) but we didn't make a big deal about calling it a fusion peptide or fusion sequence because Lazarowitz and Choppin had published that viruses with HA0 were quite infectious. That was in '73 so we didn't have a function for the cleavage like you did with Sendai. So we didn't discuss it from that point of view.

JS Well Klenk and also Lazarowitz and Choppin eventually showed in '75 that you needed to have it cleaved. After I came back from Australia in '73, Don called up one day - I mean I had never heard of him - and just asked if I could send him the procedure for making the crystals - making the HA because he wanted to do it. So I sent him the procedure. And then eventually he came back and said they hadn't really managed to get the protein so I said "OK I'll send you the protein". So I sent him the protein and that's how it started, really.

JS I visited the lab with Waterfield in '74 and I remembered they were quite keen to say they were getting somewhere and we looked forever down this bloody microscope at a chunk of glass - did he tell you that?

JS Then Waterfield and I cleared off to the West Coast as he had worked with (Lee) Hood and California was his stomping ground. I think I certainly met him (Don) in '75 because I went to talk. I turned up at Heathrow and I got one of these messages over the loud speaker to report to somewhere, the sort of thing you always dread. It turned out that the old man had been taken to the hospital so I went just for a day (to Madrid) to give the talk and came back again. I did meet Don but I had met him before in 74, maybe he's forgotten. I don't think he had forgotten it, but he obviously remembers Madrid better. And then maybe he was fixing up in '75 to come on sabbatical. He came on sabbatical in '76 I guess.

JS Well, it was interesting because Dick Bellamy was on sabbatical with me at the same time. So they became very good friends as a consequence of that and that's one of the reasons why Steve (Harrison) is involved with Dick on rota and the reovirus. We were doing with Dick transcription stuff and with Alan (Hay). I think that's an interesting part of flu, at least for me because at that stage we were doing things with RNA probes which a lot of other virologists were only able to do when DNA became available. Well we had large amounts and so we were doing autoradiography of double stranded RNAs after hybridization and showing what messages were made at what time after infection. This didn't actually change when cDNAs became available - it was sort of interesting.

Don initially had come to only work half-time with me and half time in Cambridge with Bretscher I think on glycophorin. Well, it didn't work out that way. He finished that work and was about 90% here. What he did was the cross linking of HA.

JS I worked in the lab all the time. I haven't worked in the lab properly since I've done this job (Director of the NIMR) but before that, up until the early 90's, I worked in the lab all the time.

SS How did you respond to having Don here in terms of structure? Did you feel in a sense now that you had made the decision to do hemagglutinin, here was somebody who could do it?

JS Oh yes, there were no two ways about it. Most of my lab was involved by that stage in doing hemagglutinin, one thing or another about hemagglutinin. Most of the protein at that stage was going into sequencing so I would go down to ICRF (Imperial Cancer Research Fund) half the time and mess around with Waterfield and the rest of the time making the protein here and doing the fragmentation.

JS Well we chose - the crystal thing was easy, that was the only one that crystallized. We tried to crystallize Jap but it didn't go. There seem to be some small crystals of Jap now.

SS Is that the one that Don told me about? He said that he had crystals of something else.

JS No. There are different conditions of cleavage, so you can get them all cleaved by and large. We finished up doing Jap primarily for sequence because we got a good cyanogen bromide fragment profile. That's basically what it boiled down to for HA1 which we didn't get for H3. And that was the limitation in those days - getting fragments of suitable size to do Edmans. So I guess when Don was here, most of the time I was preparing protein and doing amino acid sequencing.

SS Let's talk about flu itself now. The problems were obvious at that time in terms of antigenic variation. Did you sort of know what the answers were going to be?

JS No it was interesting. There was another guy in the lab, a post doc who shared a desk with Don actually.

JS Well, he didn't but in the corner that he used as his desk was this guy John Davies who occupied it most of the time and that's why Don didn't get it. He was trying to do the antigenicity of HA so he was trying to use the fragments that I was making - cyanogen bromide and stuff like that to get reactivity with serum. We had no monoclonals, we had lots of sera and we had HA. He was just trying to do standard immunochemistry. It never really went anywhere. It went as far as defining the flu HA1 as the place that has all the antigenic sites, HA2 doesn't have anything. But not at the level that you get out of sequencing variants which is where it comes from. We knew we wanted to understand what antigenicity was about and variation. We knew we wanted to understand what receptor binding was about in hemagglutination. We didn't think that much about membrane fusion. In terms of my mind set at that time, HA didn't need cleavage in order for flu to be infectious and it obviously took a while to get out of that.

SS If we try to remember what we thought about how viruses got into cells, we didn't think very much about it.

JS That's probably right. It was pretty damn vague. But Waterfield had Mary Jane Gething as a post doc and she started to work on Sendai and they noticed - and this was in parallel with HA - and they noticed the sequence similarity between the amino terminus of F1 and the amino terminus of HA2. So that's when it became crystal clear that this thing which was called the fusion protein had similarities to the HA and that's when it seemed likely that it had something to do with fusion.

SS But now you have to go back because actually one knew that Sendai had the F protein and did fusion, but you didn't know that HA caused fusion.

JS You knew this common sort of sequence and that's all. People didn't think about fusion.

SS So when Don was here, was the major contribution to show that the molecule was a trimer?

JS Clear as day and that was all done by stained gels - clear as day, well (laughter). I mean what was absolutely clear was that we generated two additional bands on cross linking and that you could vary the concentration of the protein and the cross linker but you could never put it all up into trimers. Because identification of the trimers depended on the molecular weight estimates, you could never be absolutely sure. It could have been monomer, dimer, tetramer but that's not what we favored largely because of the molecular weight, but you could never be absolutely sure because of the glycosylation that that was the molecular weight of the thing.

JS No, the sequencing didn't get finished until 1980-81. People in Australia, Colin Ward and Theo Dopheidi were doing it. I had Barry Davidson here on sabbatical for awhile and he did a reasonable amount on the glycopeptides. I had Lynn Dalgarno and him on sabbatical at the same time here, that was slightly after Don had been here.

SS So by that time when you were doing the sequencing, did you have enough sequence from more than one strain to begin to look at the variation?

JS Yes we did, but did we make any sense out of it? Not really. No and there still is no sense out of that because what we were doing was looking at subtypes. But you know and we knew that there was less than this percentage sequence identity but the stuff that changed the antigenicity was the single amino acid substitutions. And that we hadn't done and nobody did before nucleic acid sequencing.

Do you remember Helen Donis-Keller? She was a student in Wally Gilbert's lab and we met one time when I was over there. I mean I used to spend long periods of time over with Don around that time after he had been on sabbatical here. We became friends obviously and I used to spend time. Did he ever tell you about the skiing thing? Anyway, she was doing the 5-prime terminal sequencing stuff and I met her. I told her I'd like to do flu and since it seemed ideal for that she gave me all the enzymes and the protocols before any of the work was published and I did those flu terminal sequences. One of the things that we had done on flu in terms of RNA was show that there were two sorts of transcripts, the complete transcripts as templates and the poly A plus and that came out of the hybridization - make the hybrids, treat with RNase H or other RNases and when you did that for messenger RNA you got smaller double strands. So we had a reason for wanting to know what the terminal sequences of those RNAs were and Alan (Hay) and I did that together. I did the sequence in part from Helen and so we did the 5' and 3' terminal sequences of virion RNA and then indirectly and directly of messenger RNA and that was sort of interesting and turned out to be useful because people used the sequences for reverse transcription stuff.

SS It changed the way we thought about how RNA messages were synthesized. but let's get back to the structure.

JS What I remember most around the time when the structure came out was some draft of a paper. We weren't in the house we are in now, but he (Don) called me at home, it was about 8 o'clock at night. We had a telephone upstairs. I said hold on I will take this upstairs and get out of the way of everybody downstairs. I had this paper spread out on the bed and Anita came to bed and went to sleep and we were still on the telephone - that was way after midnight - that particular paper I remember quite well.

SS So when he called you - it must have been the day that they had essentially been able to trace everything?

JS It didn't happen like that. I was going over sufficiently and we were talking sufficiently frequently on the phone. But the tracing business was heroic, there's no doubt about that. Ian Wilson had a particular skill in tracing chains, no doubt about it.

SS Well nobody traces a chain today without knowing the sequence of the protein. Did you know the sequence?

JS Yes, we had the sequence from Colin Ward and also Fiers had sequenced it as well by then. I gave Fiers the virus and the RNA to do it with actually.

JS Colin Ward did the protein, but they didn't do it all. But Fiers by then had done the cDNA, so that was that. And that put an end to the sequencing in many ways. People rushed in to publish where the disulfides were and things like that. They got it wrong. We got it right as far as that goes, but they finished up getting more of the amino acid sequence than we did on the H3 - not a lot more but significantly more I would say of HA2.

SS In the paper you discuss fusion and the problems associated with it so some time before 1980, the fusion problem became much more important.

JS I don't exactly know when. We were in the business of collecting things that the molecule was important for but we certainly hadn't done any experiments on fusion. I think we would have finished the amino acid sequence first if we just put people on it. Mary Jane (Gething) was doing H2 and then (Joe) Sambrook came over to ICRF, which must have been about the time of the moratorium on genetic engineering and she became very attracted to doing it by DNA. They thought I was a bit of a Luddite because, literally it needed only one little push and we would have finished the amino acid sequence. But she didn't want to do it. She stopped doing it and did the RNA instead. In fact very interestingly - it says something about the difference in technology now. The amount of correction that the amino acid sequence gave to the nucleotide sequence at that stage was quite amazing.

JS So that's how it happened and we published our hemagglutinin H2 sequence, I guess, also in 1980 by nucleic acid sequencing.

SS Can you remember things that were most shocking or surprising to you when you saw the structure?

JS You asked me what hit me in the eye about HA. I thought it was very complicated. I mean Wilson had seen the receptor binding site possibility. The things I remember talking about first were the surface things, the bulges and the loops.

JS Yes. We were trying to line up any sequence changes that anybody had anywhere with likely features. We saw the helix, the long helix obviously. We didn't pay that much attention to the location of the amino terminus of HA2 - although it clearly was a peculiar fold.

JS Oh we said it was a problem of distance from here to there. I don't think there's too much that was missed out in that first paper that we knew about at the time. I think we overemphasized the antigenic sites. I even think that that was partly to make it pictorially simple, and you know, the monoclonal people were very much into talking about non-overlapping antibody reactivity. That was how they classified the antibodies that they had before they started selecting variants and sequencing and so I think the sort of sites, the A, B, C, D, E stuff was really to slot in with this non-overlapping antibody binding site business, but it isn't like that. It's like that when you come to look at the monoclonal variants but it's not like that in terms of variation in HA.

JS Well as far as neutralization goes, it seemed pretty clear to me and I think to Don too that these antibodies would probably block receptor binding. You know that's been a bit tortuous really. We still can't get to the stage of saying that neutralization simply involves inhibition of receptor binding. It certainly does for some of the antibodies. I did some work with Marcel Knossow, but that's another story I'll tell you. Some of the antibodies complex right in the site and it's clear there is no way that you can bind receptor at the same time as the antibody. Others bind outside the site and when they are IgGs then they are obviously quite big enough to block the site as well even though they are not binding in it. When they are Fabs they're not big enough, they don't neutralize and they don't block infectivity. They don't block receptor binding as far as hemagglutination goes. When they are F(ab')2 they do neutralize and this is the stage we're at now. I mean the question then is when they are F(ab')2 is there some sort of crosslinking between spikes on the surface rather than an indirect blockage of receptor binding on the basis of size? Well, it's ridiculous but we still haven't done experiments on binding to cells that are being infected in parallel with neutralization experiments. We've done things like binding to red blood cells but not binding to the cells that are infected. That's what's on at the moment. Marcel (Knossow) is on leave at the moment upstairs trying to do that with Alan Douglas.

We needed to define the receptor binding site by some other way than just seeing that similarity in structure to wheat germ agglutinin and we decided to do that by selecting a variant with non-immune horse serum. I remember telling Don we were going to do that - which he won't remember - in a cab coming back from some pub in Cambridge (Mass).

JS Horse sera were known certainly since the late '50's to contain a non-specific inhibitor of hemagglutination. So if you could get a virus that resisted that inhibition you might learn something about the receptor binding site. We were making monoclonals from the 1980's probably from 1980. The guy who has made all the monoclonals that we use is Alan Douglas, one of the technicians upstairs. When you are making monoclonals against HA, you are getting the antibodies plus sera out of the myeloma cells and you want a quick assay and the quick assay that we use is hemagglutination inhibition. Well, the serum that's in there will non specifically block hemagglutination so to get around having to get into serum free media and all that for new cells, what you do is you select a virus that is resistant to the serum and then you can do hemagglutination inhibition directly in medium which contains serum because the virus isn't affected. Then you just pick out the clones. We were doing this all the time and all we had to do was sequence the HA from resistant viruses and compare them. The difference turned out to be this residue at 226 - which is right in the site. The mutation at 226, a leu to gln change depending on whether it was sensitive to horse serum or not, defined the site. Now, at that time Paulson and his gang were doing the 2,3 - 2,6 sialic acid structures and they had sorted it out. But it turns out that horse serum was rich in 2,6 sialic acid and so the mutants that were selected recognized 2,3 - a sialic acid with 2,3 linkage rather than 2,6. That was important, that's why they are on the paper.

SS And Don said that you still don’t understand what the difference between binding to the 2,3 and 2,6 is.

JS Well, what happens is that you start developing procedures for doing binding measurements. We finished up doing it by NMR, interestingly enough, a guy called Ruigrok who was a postdoc with me did some NMR experiments here but we could never get enough NMR time to do the experiments. And people in Don's lab did them properly, Nick Sauter in particular. I don’t know whether you met Nick but he, in particular, was responsible for setting up the binding assay by NMR and doing it. And the simple fact is that you can show by hemagglutination on specifically derivatized cells, that X31, for example, binds to 2,6 and doesn't bind to 2,3 and HAM, the horse adsorption mutant selected from X31 which has a single change at 226 from leu to gln, doesn't bind 2,6 but binds 2,3. When you do the binding of sialylactose with either a 2,3 or a 2,6 linkage there's a factor of 2 difference between them. The idea is that the cooperativity of binding exaggerates that small difference in specificity so it's like a half of a half of a half. We don't know what it is that's structurally different between a 2,3 binder and a 2,6 binder because we don't have good data on the structure of the 2,3 binder. It looks as if the site comes a little closer together at the front so (John moves away from the recorder to show me a model of HA) for your information, this is 226 on the site and when that's a leucine, it sees 2,6,when it is a glutamine it sees 2,3. It looks as if when that's a glutamine, these come a little closer together here. It's subtle.

Mike Eisen (when he was a student in Don Wiley's lab) did something interesting a year or so ago in using receptor analogues which were longer, they were pentasaccharides. That was really quite interesting because in that case the helix at the top of the site - when the sialic acid there is linked 2,3 in the pentasaccharide - because the 2,3 linkage is linear - the chain comes out like that in that direction. When it's the 2,6 linkage because of the exocyclic linkage it actually bends here and the chain comes out this side so even though the rest of the chain isn't making contact with the protein, that direction of exit from the site might turn out to be important for the natural receptor - we'll see.

Fusion - what happened in a biggish way in '82 - we had started working on amantadine a long time before that, like in the late '70's. Alan (Hay) and I hadn't sorted anything out but all we said was that there was an early effect. You could show reversal and we did it with protein synthesis. So we followed the amantadine stuff and I guess it was Helenius and his group who were involved in the endocytic uptake with arboviruses (alphaviruses).

SS Now remember in 1980, you were talking about fusion in the paper, but you didn't quite know where it was happening.

JS No and the whole business of triggering, that wasn't being voiced by anybody really. I mean it wasn't sort of conceived in a way. We knew from the structure that if the hydrophobic properties of the amino terminus had to be directly involved in fusion then this thing couldn't do it. There was going to have to be some change before it could happen. But the business of triggering the change or anything like that hadn't been formulated. This was quite an interesting thing actually. You asked before if you could make HA by bromelain from any sort of strain. Well, you can, but the conditions are quite different for different strains. I had had a letter from a chap in Japan called Maeda. He said to me he had been trying to make hemagglutinin from PR8 and he hadn't had success. Could I say something about it? Well, we had made hemagglutinin from PR8 and we knew we had had some trouble. It was just the amount of reducing agent. You need to preactivate the bromelain. So I sent him this information and then I saw in FEBS Letters. It wasn't quite the sequence in which I saw it. I saw this thing in FEBS Letters from him on activation of hemolysis by low pH. I saw that around Christmas or January/February. I seem to remember this sort of date and it must have been '81. I had known by then that fusion of these viruses was being activated at low pH and I started working on the effect of incubating BHA at low pH. When I saw the paper, I thought "gee, this guy is trying to do the same stuff that I'm doing". So it was an interesting thing and I wondered when I would see something coming out. Then I discovered he worked with Ohnishi and he died in the lab. He (Maeda) was only a young man and he was clearly thinking in exactly the same way that we were thinking about the change. In fact his paper on the activation of the hemolysis is one of the ones I quote for being the first in flu and he was clearly following it up from the protein point of view because he wanted to make the soluble protein. Then he died. He had a hemorrhage or something. I discovered that from Ohnishi when I went to Japan.

JS So we started just looking at the protein. I mean it was fortunate that we could make the soluble HA and see the change in aggregation or lipid binding at low pH.

SS Is this the paper in PNAS published in 1982 (vol 79, pp. 968-972.)? I dug it out of my files and had written all over it.

JS Yes. Apart from the flotation experiment, Judy White did that, I basically did the whole lot myself.

SS But when I read that paper I was trying to read it in the context of 1982 to see whether there was anything that would have given you a hint of the big change.

JS The hint of a big change was that it became susceptible to proteolysis and HA1 floated off as a monomer. So that was a major change. Then the other - I showed that in that paper - was the formation of aggregates. But we didn't know in that paper that the formation of aggregates was due to the amino terminus exposure. We guessed that it was. But by the next year, in '83, we published the thermolytic clip to remove and solubilize the aggregates. So within 6 months or so we knew that the aggregation was because of HA2 amino terminal association.

JS Well it went fairly slowly in terms of the thermolytic thing and for a lot of reasons. There were a lot of things happening to me here in terms of taking on additional "junk". I became head of the division and then I had a series of good post docs. One of them, Steve Wharton, is still here and Rod Daniels is back again.

Ruigrok repeated all the thermolysin stuff and he was a sort of electron microscopist as well and did some electron microscopy on low pH forms as well as other stuff. We used to talk quite a lot about how to interpret the electron micrographs that we got out of low pH stuff and that we got out of thermolytic fragments of HA2 before the low pH crystal stuff. That was in the late 80's.

JS We knew that it was aggregating through HA2. We knew how big the thing was that solubilized. We had pictures of that - it was a rod with a knob on the end. We knew its length and all that stuff. So the question really was how to get to that structure from the neutral pH structure that we knew and that waswhat we spoke about all of the time.

JS Well, in fact it did go like that a bit We had gone through all the structure prediction stuff before Ruigrok left. He left in '88-'89 to go to EMBL. He's still there. We couldn't sort it out, simple as that, we had to wait until the structure was known

SS Don tells a funny story about Ian Wilson being at some meeting and somebody coming over and accusing him of having gotten the structure wrong because this region should be a helix and Don said that at the time he would have been very nervous about that. I guess Ward and Dopheide had predicted a helix.

JS They predicted it from their amino acid sequence in studies of HA2 before we knew the structure in '81 and so - you know - we refer to that in the crystal paper so we were totally aware of that. But you could predict this thing and we were aware that maybe that's another form of HA but we couldn't fit it together with the microscopy measurements and you still can't without knowing that it’s a turn. Once you know it's a turn, that's it. You see at the turn a bigger thing which is the knob in the electron microscope on the end of a fiber.

We then began a discussion about interaction of the hemagglutinin with antibodies.

JS- My feeling is that, from the point of view of how antibodies interact with viruses like influenza, the structure was very important. I don't think people had actually conceived properly at all what were antigenic sites, what was meant by a conformational determinant. I think that HA was pretty important for that and it was coupled with the escape mutant sequences and the natural variant sequences.

JS No. In fact they hadn't been done at all. There were some peptide sequences that Graeme Laver had dug out from somewhere and there were initial sequences, very few at that time. There were initial sequences of escape mutants but all the natural stuff and the routine sequencing of influenza isolates came subsequently. I mean just about then but subsequent to the paper. For example, Gerry Both in Australia had a number of papers up until about 1983. They were sequencing all viruses that had been isolated since the time of the 1968 epidemic up through '83 and so I think - I mean I don't know how quickly the penny dropped but I think quite quickly because the majority of people think we really can't interpret sequence data that they get when they don't have a structure - and it's right they can't. So I think that was important. I think the receptor binding site stuff was important for flu but you know - it was at the right tip of the molecule sticking out from the membrane - and you might have guessed that was where the thing was.

JS The good thing about it was that this group of conserved residues came together and you know that made sense in this little pocket. I think it was in the right place and then the mutant tied it down. And that was good. That was an enjoyable thing.

JS Michael Crumpton and I started it with Sarah Courtneidge, and then the immunologists got interested. Askonas' group was interested independently and a chap who you might have run across, Hans Zweerink came on sabbatical to Askonas and that must have been in 77-78, something like that. Just at that time people were trying to define the specificity of T-cell recognition. The idea had come out from the flu stuff that T-cell recognition was more cross-reactive from the strain point of view than antibody and so people were keen on trying to identify a membrane protein that was less variant than the glycoprotein. The protein that everybody jumped upon was the matrix protein which they thought was a membrane protein and that in some way could be involved in dual recognition. They were all hand waving about dual recognition and this might be dual recognition with MHC and another surface protein. I wasn't involved in doing any of that work.

What we did in influenza because of friendship with Ita Askonas and with Hans Zweerink was - we just made virus for them and gave them viruses and told them which particular strains to use and stuff like that. That continued on with Alain (Townsend) and Alain and I became friends just on the basis of giving virus and stuff. Then in the process of trying to define what virus product was recognized by T cells in a cross-reactive way, we did an experiment - he did the experiment - with viruses of different recombinants expressing different proteins and that was what tied down the fact that the nucleoprotein was what was being recognized. We used Peter Palese's recombinants and that worked out very well. I think we published in Nature (300: 655-657, 1982).

JS That was the first time that membrane proteins were not involved. The idea that you wouldn't have to be a membrane protein to be recognized by a T-cell - people were surprised. I mean Nature took it straight away - whatever that means. I remember that as being an interesting time. All of that coincided with the time I was doing the fusion stuff and the low pH stuff.

SS I think you said the other day that you really weren't focusing on fusion in 81-82. What made you start to think that that was one of the more interesting aspects of flu?

JS You mean what made me think that the hemagglutinin might be involved in fusion?

JS Yes, the fusion peptide is what did that.- that was from the sequencing. I think the main problem was that nobody really had a handle on how to make fusion happen, actually that was the block.. The idea that low pH could cause some activation of fusion was the start of it for us, really. That idea I think came from alphaviruses and I think John Lenard had described it for VSV.

SS But the paper you published in PNAS in '82, that must have changed your thinking too.

JS Yes. I mean a lot of the stuff that we've done since then has sort of followed on from that or at least was informed by that. The idea that something drastic happened that let the thing form micelles again or interact with lipid, the idea that you could solubilize it again and get a fragment of HA2, that has kept us busy, I would say.

SS There was at least 10 years between that work and the structure of the low pH form.

JS It's more than 10 years. That was '82 and the structure of TBHA2 came in 93-94. In between was largely trying to sort out what was going on, a lot of microscopy, a lot of spectroscopy, a lot of melting stuff.

SS When you did the microscopy and you had something that was too long, were you thinking that it was the same kind of structure?

JS No it was much thinner. When you just look at HA at fusion pH - the simplest sort of picture was to have HA in virus, or in virosomes and then drop the pH. What you see is that it just sort of comes apart a bit like a flower opening up.

SS And that's what many of the pictures showed as a potential mechanism for fusion.

JS Then you treated that with trypsin to get rid of the HA1 part and you finished up with these little rods still associated with the lipid which had a knob on the end. The knob of course now turns out to do the turn and we showed in '95 that both the fusion peptide and the anchor are in the virosomal membrane or in the virus membrane.

SS But when you first saw the knobs in the microscope, what did you think was going on?

JS We didn't. We tried to understand what it was because it certainly wasn't the fusion peptide. It wasn't allowing the vesicles to aggregate. It wasn't a hydrophobic thing floating out there in midair. It just wasn't clear what it was. And really the knob wasn't interpreted until the TBHA2 structure was done and then it was obvious what the knob was. Then by chance we got this monoclonal antibody which recognized the turn and it showed directly that that was the knob that we saw in the microscope.

SS Was somebody just trying to select for monoclonals, how did you get that one?

JS One of the things we did in the 80s was select variants of HA which fused at different pHs. We did that by growing virus in the presence of amantadine to jack up the pH in endosomes so that we could select viruses that could grow in the endosomes at high pH.

JS About a thousand times more than for the M2 effect. Alan Hay sorted that out - amantadine having two separate targets at least in vitro, that was pretty important. (Note that influenza mutants in which the M2 protein was resistant to amantadine were used in those experiments.)

At this point John and I talked about the time that he was the head of the Influenza Centre. Some of that discussion is included here.

JS So when I came here-I told you that Helio Pereira was head of the Division and he was also head of the World Influenza Centre. The World Influenza Centre had been started by Sir Christopher Andrewes in 1948 with WHO and interestingly enough the first director of it was Chu Chi Ming who became head of virology in China in Beijing and died last year. Laver and Webster wrote a little obituary for him in Virology (In Memoriam: Chu Chi Ming (1917-1998) W. Graeme Laver, Robert G. Webster Virology, Vol. 255, No. 1, Mar 1999, pp. f12-1) He was the "Chu inhibitor" fellow. You don't know about that but there are non specific hemagglutination inhibitors in sera. He left here in '50 to go back to China. It was a very interesting time to go back.

Alick Isaacs took over the Influenza Centre then and of course he discovered interferon and Helio Pereira took it over from Isaacs and also took over being head of division from Isaacs. Isaacs tragically died in '67.

JS No but the chap that I worked with, Derek Burke, worked with Isaacs here on interferon in the very early days of interferon so I felt as though I knew him but I never met him. And so Helio Pereira took over the Influenza Centre from him in the early 60's and then was still doing it when I arrived in '69. It was an interesting time because 1968 was the Hong Kong flu. Geoffrey Schild was in the lab. He was also very interested in surveillance and that stuff and Helio passed the Influenza Centre on to him, must have been about 1970. So he became director of the Centre - the Influenza Centre is just one lab in the Division. It's not a big deal.

JS Yes, and then at some stage in the 50's the predecessor of the CDC was in New York, something to do with Public Health in New York and they sort of became the sister organization - the New York lab. Then when it moved to Atlanta they became the influenza center for the Americas, North and South America, so it split like that and it was still split like that when Geoffrey Schild took it over in the '70's. By the time I took it over, there was the suggestion that it wouldn't be called the World Influenza Centre but be would be called an International Collaborative Centre for influenza.

Schild left to go to the National Institute of Biological Standards as head of Virology, that was in Hampstead. The Institute here started the National Institute of Biological Standards. Dale was the first Director of the Institute. He received the Nobel Prize for discovering acetylcholine. He was entranced by biological standards. He started off with insulin: How to get a standard for something which was a biological molecule? It was all by comparisons but then you had to have a standard to do your comparisons by and everybody internationally had to have a standard so that was the start of the Biological Standards. It is an important Institute now but it was spun off from this Institute in about the 1960's before I came. Part of it was still in this Institute until they got their own brand new Institute at South Mimms where they are now. Schild went to that Institute to become Head of Virology, viruses are a very important part of biological standardization. He left about '75 and I guess I was the senior person in terms of influenza virology. I wasn't head of the division, Willy Russell was head of the division. I think he thought somebody else should probably do it they should recruit somebody and it became obvious that I should do it. It wasn't that obvious that you would get a particularly good scientist just to come in to do that. It seemed to me that it would more easily be run along side another research program so that's what happened. But he was concerned a bit about me doing it and was quite interesting that WHO was concerned about me doing it. The guy in charge of the WHO virus part then which was a much more substantial piece of WHO then it is now was a guy call Cockburn who was a Brit to start and had been a senior guy in something over here, Public Health - and he came to see me to see if I'd be suitable.

JS Well, somebody that he knew would have helped (laughs). He didn't know me from Adam. Basically it was whether you had any experience with anything and he very rapidly proved that I didn't know anything about flu (more laughter). Willy Russell, who was head of the Division arranged it and I'm not sure it was ever a good idea actually although there were many enjoyable things in it for me. Peggy Pereira who was Helio's wife at that time was Head of Virology at the Public Health Service which is in Colindale should share the job with me and she did until she retired. She retired sometime in the 80's. We used to go around all over the place and it was good fun because I became very good friends with Helio Pereira. He always used to work here in the lab and was a real charmer. I knew Peggy because of that and we became good friends too. We went on separate trips to China with Helio one time and with Peggy another time. It was an enjoyable thing and she knew a lot about normal epidemiology and surveillance and I learned lot. Anyway I continued doing that until 1993-94. I did the influenza work for 19 years which were some interesting times. There was no pandemic but there was the swine flu affair and a new H1N1.

JS And also in the U.S. I attended the meetings that were in the U.S. when they made the swine flu decision. What I remember especially at that time was that everybody was really up tight in that period waiting for the swine flu to come. It was a serious pandemic when it was around in 1918 and since nobody understood what it was that was responsible for that it was feared that it could happen again. It was a very difficult position for public health decision makers to be put in.

The thing I remember about swine flu - this is not all of my interpretation and it's not from one meeting but from a number - is that people knew just as they did here that the idea would be to make the vaccine and stock pile it until there was evidence of spread of this virus internationally. That's the normal procedure before changing the vaccine strain, but then they worried what would Congress' response be to a request to make a lot of vaccine that might never be used. They figured that they would not get the financial support so they figured they had to go to the Government and say we need to give the vaccine. The risk is such that we need to give the vaccine. And that's not a ridiculous thing to do in the sense that a vaccine is not going to do any harm, at least they thought it wasn't going to do any harm. I'm sure that was the rationale that was developed at the meetings I was at. I think it was perfectly reasonable. It would have been done in this country I'm sure and any other country that felt that their health budget would stretch to accommodate it but other countries didn't have the same decision to make. There was no way their health budgets were going to be used for this and they couldn't be increased in that way. So I think the Americans had a peculiar decision to make. I think they made it from the best motives and I always thought it was a great shame that David Sencer should have been put under such incredible pressure and actually finished up resigning because of it.

SS So it was an interesting time. And one should have at least learned some lessons.

JS Well, interestingly enough, people did come around. There were books written about the decision making process and people did come around to talk about it. I don't think they ever recorded my opinion on the matter. I wasn't particularly important but that's how I remember it anyway. Walter Dowdle was the director of the influenza thing so he was my counterpart in the U.S. and we went around a lot together. Then he passed it on to Gary Noble who was also a very good friend and then he passed it on to Alan Kendal.

JS Well the main duty was to track flu, to know what's going on around the world and be in a position to know when a change in vaccine composition was required or a change in reagents for diagnosis was required. Those are the main duties still and the other responsibility is to keep the staff as up to date as possible. Alan Hay is now director. Alan took over from me in '94 so he does that. He has the same responsibility as I had.

SS Were you ever involved in making dramatic decisions about changing the vaccine?

JS Every year. We used to argue like crazy, especially with Alan Kendal. I used to think sometimes he would say black was white just for the sake of discussion. But the decision had to be made and generally it was a pretty good decision - year after year after year we had the right strain in the vaccine.

JS No, I think if you're reasonably fit, all other things being equal, there's no great reason to take the vaccine. When you take it, you've got to keep taking it and I'm not that keen on taking it all the time. If a live vaccine came along then you would have as long an immunity as you might have from (natural) infection.

JS You know there's this company that I consult for actually that is making the live vaccine in California.

JS It's John Maassab's cold adapted parental backbone and they just recombine it and make it the current hemagglutinin strain. There's nothing new under the sun. John did that in 1960.

JS Well, I think various companies did try to use it, but one of the things about influenza in terms of research on the disease is that once you're set up to do the natural experiment no influenza comes along. Numerous times people have had vaccine trials set up and the virus just doesn't come. I remember one famous one which Joe Smith (who was actually head of the National Institute of Biological Standards when Geoffrey Schild went there) had. It was a big trial of flu vaccine in post office workers some where up north and the flu virus never came but they did have a good effect of vaccination. This was like the effect of being a good employer. The people who were vaccinated came to work more frequently or had fewer absences than the people who weren't vaccinated. So I think maybe that's one of the kinds of problems that research on flu vaccines have had.

SS One of the things I was thinking about is that it must have influenced you in focussing your interest on immunogenicity.

JS On antigenicity, on the hemagglutinin, yes. But I had already made the decision to work on hemagglutinin before I took that position, but knowing these things about hemagglutinin certainly influenced what we did.

SS Except we also discussed that it was not the hemagglutinin that was inducing T-cell immunity.

JS No, I don't think T cells immunity does anything. Protection from influenza is mediated by antibody. Not to say that having an appropriate T-cell immunity can not sometimes do you some good but the natural experiment says its antibody. Let's say that you had been infected by the 1968 Hong Kong flu. All of the proteins except two in the Hong Kong virus were the same as those in the Asian H2 virus, only PB1 and the hemagglutinin changed. The virus went around the world (essentially caused a world-wide epidemic). Now, you can always say that you can't do the controls. You don't know that you didn't get some amelioration of the disease because of some pre-existing immunity. Certainly it didn't stop the infection but you can't assess the severity in natural experiments like that. What you can say is that infants don't suffer particularly badly from influenza. It's not as if when an influenza epidemic comes along that all the neonates die. I think that as far as protection goes, certainly antibody is "where it's at". It may well be that T-cell immunity is short lived for some reason and if you vaccinated fairly frequently to boost it you might have some good effect.

SS We had been talking about fusion and I want to go back to ask how you thought about it before you knew the structure of HA.

JS I find it hard to recall. Well, you didn't know enough to make a model. From the first experiments what you knew was that the thing became hydrophobic and you knew from the sequencing experiments, which were also done in '82 and '83 that the reason why it became hydrophobic was through the association with the fusion peptide. So you knew very quickly that what was happening was the fusion peptide was coming out of where it was in the neutral pH structure. What was it doing? You could imagine it was interacting with the membrane and people had shown by hydrophobic labels that it was doing that. I don't think the data are great but they're published and they fit in with what we know. One of the things that Don and I did during his sabbatical was to use hydrophobic labels to track down sites in the molecule that were interacting with the membrane and when we found out about the low pH thing, subsequently, I tried quite hard to use the same reagents to show that the fusion peptide went into membranes. The problem really is that those labels aren't that specific. Joseph Brunner has done some experiments since and gets reasonable evidence that so much of the fusion peptide interacts with the membrane and gets labeled. So the questions were: Does it interact with the membrane or does it interact with itself? Does it come out sideways or shoot out toward the membrane? I don't think we ever saw any particular advantage in drawing these things.

SS When you were showing me the electron micrographs you said it was much thinner but the idea that this structure was going to be so modified was something that was not imagined at all as far as I can tell.

JS Well, it was imagined because the high pH mutants were everywhere in the molecule, in all the interfaces throughout the length of the molecule. The interpretation of that was the thing changes everywhere.

JS Well, the details of the structure, that it formed that new structure - I mean you couldn't predict that. For me the prediction is just sort of useless. The first prediction in 1980, before we had the native structure was that HA2, the neutral pH structure, forms a helical hairpin. It has a short alpha helix which is linked to a long alpha helix that forms the center of the molecule. What the initial prediction said, just from the sequence, is that the connecting thing is helical so the whole thing gets helical. Well it wasn't. So instead of being as predicted a complete helix, it was in fact a helical hairpin, a little helix linked by an extended chain to a long helix. The thing that couldn't be predicted was the thing that turned it, that is from a little piece extended through a turn to an antiparallel helix. It's exactly the same when it comes to the low pH structure. Here you have a central coiled-coil followed by a turn into another alpha helix so you just turned the hairpin around - using different residues - you turned the hairpin round. What couldn't be predicted again was the turn, but the turn is the important thing! Either way both predictions were 50% right, but they were 50% wrong. You know there's no great fun in that.

SS What is the evidence that the structure after it's been formed in the low pH can cause fusion?

JS Well there isn't any. We know that if you express the HA2 alone in E. coli that it adopts the low pH structure. The question is: can you get that thing with the fusion peptide still on but masked in some way to form that low pH structure, then unmask it and get membrane fusion? And that we don't know. One possibility is that the final form just by being interactive with both membranes can cause fusion. Then you would say well that's some particular feature of the fusion peptide. We know that just adding fusion peptide alone to liposomes causes them to fuse, so membrane fusion could be analogous to that. It's just a matter of some way of delivering the fusion peptide to the target membrane and then they fuse.

SS I thought you were much more convinced then you seem to be now that the low pH form at the normal pH could still cause fusion.

JS No. The other alternative is that in fact what causes fusion is some transitory intermediate between the low pH form that we see and the neutral pH form. I don't think there is anything that can really say at the moment whether it is a transient intermediate or whether it's the final thing.

SS I thought that one could make an argument that the molecule that has gone through this pH change now binds lipid.

JS But those experiments aren't done in a situation in which the target membrane is also present. That would require that you could go through the change with the fusion peptide still masked. What happens in the normal situation is the virus is there to go through the change and now the fusion peptide is in its own membrane and then it can activate.

SS But in the experiments you did in the '82 paper I thought that that molecule after it had undergone the change and was purified, when you added it to lipids or membranes it then bound.

JS No. you had to mix the neutral pH molecule with lipid, then drop the pH. That was how it was done. Because if you do it before adding lipids it forms protein-protein micelles and they are really irreversible.

JS One of the things that has happened with antibodies is that in the early '80s Don's sabbatical visitor, Marcel Knossow, did the structure of the first antigenic variant and showed that the single amino acid substitution that it had was the only change in the structure from the wild type HA. Therefore the antibody must be recognizing the site which contained that amino acid substitution. So that was sort of formal proof that antibodies bind at the site of amino acid substitution and we have not come across any other instances (that differ from this conclusion). So the idea that as a result of a single amino acid substitution some gross change happened to the structure of the molecule doesn't seem likely and hasn't been seen with any of the variants that we or anybody else has selected.

What this says is that the binding is sensitive to single amino acid substitutions in the binding site. The antigenic variants that you select are single amino acid substitutions in the binding site and the positions where they are on the surface of the molecule are the same as the positions you get in natural variants. So we still continued doing those things, for example we showed that when you get a change which introduces a new glycosylation site, selected by using a monoclonal antibody, that it was a sugar chain that was blocking binding of the antibody - that was quite interesting. When Marcel went back to Paris, we agreed that he and I would look at complexes of antibodies with hemagglutinin and so we've done a number now. That's been interesting in the sense that the first complex that we did was a complex not with the intact hemagglutinin but with the HA1 fragment derived from the fusion pH form. One of the things that happens to the fusion pH form is that it becomes sensitive to protease unlike the native protein and what happens is that the globular head floats off as monomers after you digest with trypsin. So that structure, the Fab-HA1 domain structure, was the one that proved that at fusion pH, the change in the structure of the globular head only involved de-trimerization. It didn't involve any additional change in structure. We sort of knew that in a way or we knew it partly because we knew at fusion pH, the hemagglutinin continued to bind to receptor. Nick Sauter had shown that with the NMR.

SS When you say the fusion pH do you always mean pH 5 and then back to neutral pH?

JS It doesn't matter. I mean fusion pH, it's not just fusion pH 5. It varies with the temperature, so for X31 it's pH 5.6 at 37 degrees, 5.2 at 20 degrees. For different mutants it can go as high as pH 6.5 so it's just the pH at which the conformational change happens that's required for fusion and the change is irreversible. So we've done a number of antibody-HA complexes now to try to sort out what's involved in neutralization and we still don't really know.

SS That's what you said the other day and I was interested that you still don't understand it.

JS We think that in the majority of cases antibody prevents receptor binding but we have one antibody at the moment, and there will be more I have no doubt, that clearly binds distinct from the receptor binding site. None of its residues are within 15 angstroms of any of the sialic acid atoms in the binding site and everything works fine. The antibody blocks infection, the Fab doesn't. That goes along with the idea that the antibody is sufficiently long that even though it's bound outside the site, it can still sterically prevent receptor binding. The Fab is shorter, it can't prevent receptor binding so it doesn't neutralize. But, and this is what we're trying to understand at the moment, the F(ab')2 neutralizes quite well so the question is how is it doing that? Is there something about the way the F(ab')2 binds that allows it to look bigger than an Fab or is it cross linking HA to another molecule? We're trying to distinguish between those two ideas right now.

SS I wanted to end by asking you about the things that you remember that were the most interesting or exciting to you.

JS I remember two or three occasions where things just went very quickly so maybe it was because of that that they stand out. I remember shortly after I came here - the flu polymerase. I guess the trigger for doing it, I mean I wasn't going to do it, I hadn't come with any great intention of doing it, but I suppose the trigger must have been the VSV transcriptase.

JS Sure, I followed it all because of the reo work but I hadn't been intending to do anything. But, I remember talking to Ted Martin who was here at that time, in Biochemistry, and I said "I think I will see if I can detect a polymerase in flu". And it worked and it was one weekend - I did all the experiments over the weekend. He came in Monday morning and said "how did it go?" I showed him all the experiments. I titrated the magnesium, the pH and all that sort of stuff and it had all worked! I told you the other day that when I met David Bishop at some meeting and I knew all the answers. Literally nobody knew we were doing it. That was sort of interesting just because it went so quickly. I remember for some reason Bill Joklik came to this country. There was a meeting that we were both going to - an EMBO meeting or seminar - and I met him at the airport and he said: "have you done it " and I said "what?". He said, "the polymerase" and I said yes I had. Somebody in the US had just said - I don't know whether it was Spiegelman or who - had just said that it was a reverse transcriptase. And I said: "Oh no it isn't. I've done the thing". And he said "Oh, that's good."

The low pH paper I remember that quite well. I did all of that and it went remarkable quickly. It was one of those times when you can see ahead and you know which experiments to do. The advantage that we had there was that we had virus and we had protein. To be able to do protein chemistry experiments and use stained gels as an assay is a pretty important advantage and that's what we had. That worked really well unlike something we're doing at the minute with partial activation and clearly there are variables there that we don't really understand.

I remember making oligos. I mean somehow or other I had the chore of making oligos. The guy doing the sequencing mostly was Rod Daniels.

JS Both. We were priming with oligos, with labeled oligos. You couldn't buy them, there were no machines, you made them. I actually enjoyed doing that quite a bit. Also it came at a time when what I could do in the lab was changing. I was becoming Head of the Division and so I could sit at the hood and make the oligos and if somebody came and interrupted me I could put it down and go back to it again, or so come back in two minutes and put on the coupling and stuff like that. They were more "planable" experiments.

The other practical thing is - when I came here or just before I came they had hired a technician in the Influenza Centre and in those days when they hired a technician in the Division all of them had to spend time in the Influenza Centre because they learned techniques like making tissue culture cells and stuff like that. This guy, David Stevens, had just been hired and they gave him to me and he's till working with me now. I talk to David every day. We go through what he's doing and why he's doing it. So even when post docs are running around all over the place, David is there. I mean there are a lot of good people in the lab but he knows everything, knows how to interpret what our conversations are and that serves as a continuity which you wouldn't normally get in jobs like this. I also think that's one of the things about the way the flu project has gone here is that we were able to get grams of virus, grams of HA.

JS That's right and it is constant support - the long term support that projects have which really has made it possible. In fact, we get eggs every single week. We grow virus every week. We're not growing the same virus every week. I think the long term support - maybe not so much now when you might be able to express the protein - but even now you can't express HA in those sorts of amounts very easily. I think that's been a major contribution of the place, just the constancy of support which is unusual.

SS In that regard maybe we should finish up by my asking you that now that you are running the Institute how does that change the way you think about various projects? How much time do you spend on just general scientific question?

JS I would say it varies from day to day, but I spend at least 50% of my time thinking about HA.

JS I'm thinking about HA (laughs)! I think it's very lucky to be doing a job like this where you know the experimental system so well and it's set up that you can actually by sort of fleeting contacts still keep up to date. Certainly to go to somewhere else and do a job like this would have been much more difficult for me to set up again than it was to do this sort of thing here. It was one of the reasons for doing it.

SS I wondered if you feel much more responsibility now to pay attention to areas like neurobiology and developmental biology?

JS I think the main advantage in this place for me was that I didn't have to talk to any other virologists. It shows in the sort of experiments you do. I mean the idea of doing simple things like CD on HA in the early 1970's was - it might seem obvious now - but it wasn't that obvious and it wouldn't have been that obvious if you were just in a virology lab. But because your friend was a spectroscopist and he was next door to you, it becomes more obvious. The people around you are influencing what you do and Peter Bayley and I were good friends and so if the HA was going to change structure you would want to know what the structure is. And if you want to know if it's native after you've done things to it, you would have done spectroscopy on it. I mean the machine wouldn't have been in the building if you were in a virology institute. So people talk about setting up immunology institutes and virology institutes. I am not in favor of those. I am in favor of places like this where people do benefit from having people approaching topics from other positions. It's always been very important to me.