HIV/AIDS Research: Its History & Future Meeting
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Ed Berger: Okay. Thank you. That's a very good introduction because I was about to start out by saying that the HIV discoveries really came about by a highly-focused efforts from many researchers around the world. Giving my perspective on our contributions. I would say that our effort was combined with a healthy dose of fortuitous circumstances, a bit of irony, [00:00:30] and perhaps an unhealthy dose of hubris.

When I came into the field—I was not a seasoned virologist at the time I came. My history included having done my graduate work in E. coli amino acid transport and then I spent a period in my postdoc working on developmental neurobiology, which I decided it was [00:01:00] much too complicated. I needed a system  that could be manipulated, like a microorganism, but went through interesting development. I wanted something simple. I turned to Dictyostelium discoideum and did a postdoc in doing that and set up my lab at the Worcester Foundation for Experimental Biology, working on this very simple system.

During that period, I got to know very well a colleague who worked right next to me down the hall, who also worked on a very [00:01:30] simple system. I always used to show this model of the dictyostelium life cycle. I always teased this other person of always showing the same simplistic system that she worked with. Little did I know that in a few years, in 1987, I would be moving along with Harriet [Robinson] into a much more complex version of the system that she had been working on previously. [00:02:00]

When I came to NIAID/NIH in 1987 and really the stage was set for the problem that I'm going to be focusing on today. By virtue of the accomplishments in the very early periods after identification of HIV as a virus and a causative agent of AIDS, multiple groups and people, including people here, contributed to the identification of [00:02:30] the CD4 molecule itself as the primary receptor for HIV entry. (1, 2, 3)

I want to focus on one of those papers in particular, which pointed out that CD4 was not the whole story. (4) In this study, from Richard Axel's group, they made recombinant CD4 and expressed it in different cell  types and looked at their permissiveness for infection. When you put CD4 on a human cell type, it became [00:03:00] permissive for infection, but when you put that same human CD4 onto a mouse cell, it was expressed perfectly well, but the cells were not permissive for infection. They concluded that the block to infection resides as far as entry.

So, that sort of brought us to this point here where, in addition to CD4, there was some other thing going on, and it could either be an additional [00:03:30] cofactor, maybe a coreceptor, that's expressed on the human cell and not on the mouse cell, or alternatively, as similar to what Michael Malim was just saying, it could be some restriction factor in the mouse cell. That's where we were standing.

The issue was complicated, of course, by the emerging notion that different isolates of HIV-1, including isolates from the same patient [00:04:00] could have very different tropism properties for infecting different types of CD4 positive cells. There are the viruses that are also called T-cell-line-tropic, which very efficiently, infect human T cell lines, but they don't infect macrophages. There are other viruses— these viruses have also been called syncytium-inducing or rapid-high—and on the other hand, [00:04:30] there are viruses that have the ability to infect primary macrophages and very poorly infect human T cell lines. These also were known as non-syncytium-inducing or slow-low viruses. It's important to point out that all of these viruses can infect primary T cells. The distinction is TCL-tropic, not just T-cell-tropic.

With that in mind, when I entered [00:05:00] the Laboratory of Viral Diseases at NIAID, I was very interested in extending my interests, which had been in cell surface interactions during development, into cell-surface interactions associated with HIV infection. This paper had been published just before I came into the lab. (5) The people are here. Jeff [Lifson], I think, is here, who demonstrated that if you express recombinant envelope [00:05:30] in a cell, it will mediate syncytium formation when co-cultured with CD4+ T cells.

That combined with the fact that Bernie Moss (b. 1937), whose lab I joined at the time, had been developing the vaccinia system, not only as a system to study this important virus, but also to take advantage of [00:06:00] features to use it for expression of foreign proteins. Vaccinia virus has some key advantages for that. It's got a very broad cell host range, both with respect to species and cell type.

Importantly, the virus has an entirely cytoplasmic replication cycle, so all the expression that goes on is in the cytoplasm. We didn't really appreciate it at the time, but it had critical advantages for studying HIV envelope protein because, as we just heard earlier, [00:06:30] if you try to express it from a nucleus, you need Rev. With vaccinia, that's not an issue. You just put in the envelope gene and no concerns about rev or Rev responsive elements. That was a big advantage. The other was the fact that Bernie's group had been studying very extensively vaccinia promoters and defined early promoters and late promoters, which could be used in designing constructs for expression. He had also developed this hybrid bacteriophage T7 [00:07:00] polymerase system where you would infect with a vaccinia encoding T7 polymerase and put in your gene of interest linked with T7 promoter and get a very robust expression.

When I joined, I thought to study this reductionist cell-cell fusion between envelope expressing cells and CD4 expressing cells, the vaccinia system was very suitable for this. We devised a variant of the T7 system [00:07:30] where instead of putting them in the same cell to express the protein in that cell, we put the T7 polymerase in one cell, and the reporter gene, in this case, lacZ, in the other cell. (6) You just incubate those cells together for a couple of hours. In this case, you get robust production of beta-galactosidase, which you could in situ staining or just in a 96 well plate. The assay is very, very quantitative, it's very [00:08:00] sensitive, and it's very, very specific.

Importantly, we recapitulated the phenomenon, that if you express human CD4 on a human cell, the cells fuse beautifully. If you express it on a mouse cell, even though it was there, the cells didn't fuse. We recapitulated that requirement, or that distinction. It turns out envelope can be expressed on either a human cell or a mouse cell. That didn't matter. [00:08:30]

We then went on to make a couple of new findings that if you make transient cell hybrids between a mouse cell expressing CD4 and a human cell, then those hybrids were permissive for fusion. That argued in favor of the human cells having an extra cofactor rather than a restriction factor on the mouse cell.

We also showed that the envelope proteins from T-cell-line-tropic (TCL-tropic) [00:09:00] viruses show the right fusion specificity with T-cell lines but not macrophages, and vice versa for the mac-tropic (macrophage-tropic) envelopes. That left us with the notion that there are probably required cofactors, maybe coreceptors, rather than dominant-negative inhibitor restriction factors. 

That's where things stood until 1996. The tasks now for all of us [00:09:30] in the field was to identify these cofactors for the TCL-tropic and the Mac-tropic viruses.

We thought about it, "How are we going to do this?" We decided, just for technical reasons, it made sense to go after the TCL-tropic cofactor first because lots of T-cell lines like HeLa cells were highly permissive if you put CD4 onto them. We could get all kinds of reagents derived from HeLa cells, including libraries, but the first approach [00:10:00] that we thought about was maybe—Oh, I missed this. With all the activity going on, there were reports in various journals that got quite a bit of attention in the press, that had real impact on our feelings, but we decided that this was not going to discourage us too much. (7) It turned out that this particular report was not born out with subsequent studies [00:10:30] from several groups.

We went on to think about how to do this. One of the things we thought about was injecting mRNA from a human cell line—and you can get RNA libraries from HeLa cells into a CD4-expressing human cell—and see if we get fusion. The question is what system to use to do this efficiently. Here was one ironic note, which is not—Here we go. [00:11:00] Right across the street, we knew about Phil Murphy's work. Phil was working on a totally different problem. He was studying G protein-coupled receptors, but he was using xenopus oocytes for microinjection of RNA.

We thought this might be a good system to see if we could express CD4 on a xenopus oocytes: inject the RNA, and confer fusion activity. but it became clear after even just discussing the requirements for these xenopus system that didn't really [00:11:30] match with the requirements for the molecules we wanted to study. Basically, we gave up on that collaboration. 

We went ahead instead with a functional screen of a cDNA library using a library from HeLa cells and that's where we had success. Yu Feng was the predominant researcher working on this, but the whole laboratory contributed at that point. Basically, what we did [00:12:00] was to take a plasmid library from HeLa cells and transfect it into mouse cells that were expressing vaccinia-encoded CD4, and just see if that library could confer some signal of fusion as measured by beta-[galactosidase] above the background. Then, as we saw that, we would then narrow down the pool of plasmids until we went through several cycles. 

We ended up isolating a single [00:12:30] cDNA clone, and the sequence encoded what looked like a G protein-coupled receptor. It was actually a molecule that was already in the database. It was an orphan receptor. It had no known function, but its sequence showed the most closely-related molecule was the interleukin-8 receptor, which is a chemokine receptor.

The only activity we had for it was that it promoted [00:13:00] fusion between CD4 expressing cell and an envelope expressing cell. We gave it the name "fusin." Then, very soon after we presented this—We actually presented it first at a meeting quite a few months before we had the paper ready for publication and not surprisingly, several groups, including people who are in this room, Joe [Sodroski] was involved in this, identified chemokine that binds to this molecule and the chemokine [00:13:30] receptor people have a nomenclature system. (8, 9) So, very quickly, the name "fusin" was replaced with the name CXCR4. "Fusin" is no longer in the real world except, in my case, if you drive around anywhere around Bethesda or NIH, you'll see this. [laughter] I had no trouble getting this license plate. That's the only [00:14:00] place—there are some other anecdotes about that, but I'll hold her off for later. 

Okay. Here's some of the data demonstrated in that paper. (10) First of all, gain of function, in the top and the cell-cell fusion assay. If you mixed cells, non-human cells expressing CD4 and expressing Fusin, then you get a very robust fusion, but only with envelopes [00:14:30] from T-cell-line-tropic HIV, whereas the envelopes from macrophage-tropic HIV did not respond. In HIV infection, on the bottom, you could see that expression in a non-human cell, this expressing human CD4 expression of fusin render those cells permissive. And conversely, we made antibodies against synthetic peptides from fusin and in cell fusion assay. These antibodies blocked very [00:15:00] potently, but if it works for a prototypic T-cell-line-tropic virus, but not at all for macrophage-tropic virus. The same thing holds true for HIV infection.

All those data together convinced us that we have the right molecule for the T-cell-tropic cofactor. Jon Cohen had an article on this in the issue of Science. (11) We got some nice responses about that. This is a quote from John Moore in [00:15:30] that article. That elicited a whole bunch of fun things, including statements by others in various papers and in our lab, but it engendered this kind of stuff. We were having a lot of fun with Adobe Photoshop, which I was just learning how to use. It was a movie coming out around that time that gave us this idea.

So, there we were. Now, the question was, how do we go after the co-factor for the [00:16:00] mac-tropic isolates? We had such luck with this unbiased cDNA cloning strategy that I thought that was probably the best way to go. We made our own cDNA library from primary macrophages and Ghalib Alkhatib began working on that. It was starting out a lot tougher. We had never made a cDNA library before. We can't buy this the way we did for the HeLa library.

Then, another thing [00:16:30] happened and this was a major paper, which everybody here knows about. (12) That was—again, focused on a totally different problem, namely the nature of the soluble refactors released by CD8 T cells that are able to suppress HIV infection. From the Gallo lab, Paolo Lusso, they identified three proteins RANTES, MIP-1 alpha and MIP-1 beta, all chemokines as the basis [00:17:00] for that suppressive activity. So there's another chemokine connection and they had one—they actually had some demonstrations of activities against different isolates. These chemokines very potently suppressed some isolates, but they had no effect on 3B, the prototypic T-cell-line-tropic virus. 

That made us think that there's something going on here. Maybe [chuckles] this is the chemokine [00:17:30] receptor that could be involved as the coreceptor. And we knew that Phil Murphy, right across the street, had just recently cloned a chemokine receptor that is specific precisely of these three chemokines and subsequently several other groups came out with that. These papers actually came a little bit later, but we knew about the discovery of these chemokine receptors. (13, 14, 15) We could have just contacted Phil [00:18:00] and said, "Hey, let's start up a collaboration again," but I was really stuck on using this unbiased functional screen because I thought it was so nice, and Ghalib and the other people in the lab were saying, "Ed, come on." I pushed on it for a while, but finally, they got to me and they convinced me to get in touch with Phil.

So, I went across the street [00:18:30] and actually sent an email to Phil. This was March 5th of 1996. Here's what I said to him, "Hey, remember when we talked with you about xenopus? Well, that didn't work, but this will. Let's collaborate." We began that collaboration and it went remarkably fast. That was March 25th. This is March 28th. Ghalib actually used that cDNA clone [00:19:00] for CCR5 and did the various kinds of assays that I showed you before, and bingo. We got the data that we needed and that was published in June. (16)

We were not alone in this. There was tremendous competition and the sequences for CCR5, you know, they hadn't been published yet. The clones were available. Various groups got into this, and within a week's time, there [00:19:30] were five papers from five independent groups—ours is somewhere in the middle there—showing that CCR5 is the second co-factor, I should say, for the mac-tropic isolates. (16, 17, 18) Very soon thereafter, several groups showed that the gp120 actually physically binds to the chemokine receptor, to the [00:20:00] coreceptor. (19, 20 21) That allowed us to call these coreceptors rather than simply cofactors.

Also, that led to a new classification of HIV-1, this idea of calling them M-tropic or T-tropic didn't really make sense anymore. Now, the nomenclature is R5 for what we used to call mac-tropic, or X4 for what we used to call T-tropic and dual tropic are now called R5X4. [00:20:30]

This is an old story, but it led to some very, very profound molecular understandings of a phenomenon that was known from work before the coreceptor discoveries and that is that when people are initially infected with HIV-1, even if the donor happens to be harboring mac-tropic and T-cell on tropic viruses, the virus that takes hold in the newly infected individual is invariably [00:21:00] specific for macrophages. Now, it became clear that it was CCR5 specific. It was R5 HIV. In the infected person, the viral load is actually controlled mostly by a cellular immune system. The person can live for years with being in the asymptomatic state and it's only after the immune system really starts to decline and the CD4 loads start going down that you start [00:21:30] seeing viruses appear that have the capability of using CXCR4, maybe in combination with the CCR5, or using a CXR4 exclusively. This doesn't happen in all people who progress to AIDS, but certainly, the appearance of X4 using viruses is a bad sign prognostically. [00:22:00]

Science recognized two major things that happened in 1996 as deserving of the Breakthrough of the Year. Of course, the potent new drugs, combination antiretrovirals that were starting to prove so effective at suppressing HIV in infected people along with the discoveries of the HIV coreceptors.

We now have a model—this is an old [00:22:30] slide and this is just a simplistic model that the envelope, like a protein approaches the target cell, and then CD4 binding induces massive conformational changes in the envelope protein. And the work that this model I'm showing you is coming about from studies from many, many laboratories. (24) We were part of this, but many laboratories have contributed to this over the years. Those conformational changes induced either creation, or the [00:23:00] exposure of a region of gp120 that's capable of interacting with the coreceptor. The interaction with the coreceptor then triggers the triggering of gp41 and that promotes fusion between the viral membrane and the target cell membrane, and you get fusion and entry. That's the very, very simplistic model of the sequential steps for the receptor-coreceptor interactions.

Edward Alan Berger is a senior investigator at NIAID.


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A lot has happened over the ensuing years. We have drugs [00:23:30] that specifically target CCR5. Maraviroc was approved in 2007. These drugs bind within the transmembrane regions. This is a crystal structure of CCR5 bound to one of these inhibitors. (25) We know some things about how the gp120 physically interacts with the coreceptors, but we don't have a crystallographic structure yet. We know ECL-2, that second [00:24:00] extracellular loop is important, we know the first end terminal domain is very important, but the precise mode of action of how the triggering all occurs still awaits investigation.

Now, there's another aspect here that came along. I was at the International Virology Congress in Jerusalem in 1996. I think it was in August. The coreceptor discoveries had just been published [00:24:30] and were very, very exciting. I went a couple of days early and was preparing to relax a little bit before my talk. Then, a friend of mine, an Israeli friend of mine, handed me a newspaper with an amazing story and then, just a couple of hours later, another friend of mine handed me a different newspaper with a similar amazing story from a different lab.

This was findings from both the Aaron Diamond Center and the [00:25:00] University of Pennsylvania, which I was able to actually read that. I can't read it on the slide here, but basically, that there are people in the population that have the Delta 32 knockout (CCR5-Δ32). I'm not going to go into this because I'm sure all of you know about it, but that led to a molecular understanding and still, really, the only good molecular understanding of why some people are resistant to HIV infection. [00:25:30] They're homozygous for the Delta 32, which completely knocks out the expression and function of CCR5. These people appear to be mostly normal, and the main medical consequence is that they're resistant to HIV infection. There are some other consequences, but that's the major point here.

A number of studies came out later, the top two were the ones that I showed you from the Israeli newspaper. (26, 27) And then the [Stephen] O'Brien group (28) [00:26:00] and our group a little bit later (29) demonstrated through studies of cohorts of individuals, this correlation between homozygosity for Delta 32 and resistant to HIV infection and also, heterozygosity can result in a slower progression of disease.

Of course, I don't have to tell you all here about how the Berlin Patient who still is the first and still only [00:26:30] true cure of HIV infection. (30, 31, 32) This was achieved by transplantation from a donor who was homozygous for Delta 32, and it's believed, I think by most, that that contributed to the fact that this person is now capable of living a virus-free life. I'm not saying he's cured. He definitely has virus in the system, but he no longer has to take [00:27:00] antiretroviral therapies and remains healthy. Timothy Brown (1966–2020) really became an inspiration for all of us who are thinking about treating HIV and for those of us who were thinking about approaches to a cure, whether it be a functional cure or a sterilizing cure.

I mean, the cure word, most scientists would never use the C-word in public [00:27:30] with their fellow scientists, maybe it was locker room talk. But now, things are totally changed and all of you know about this. There are review articles every year. There half a dozen review articles. There are meetings every year, funding specifically towards a funding for an HIV cure. The coreceptors really, I think, played a big role in inspiring that and [00:28:00] coreceptor strategies are one set of strategies in the approaches that are being pursued to try to gain a cure for HIV infection, whether it be functional or sterilizing. (33, 34) I'm going to leave it there. I think that's the last slide and I'm happy to take any questions.

[applause] 

Anchor
qa
qa

Paul Bieniasz (moderator): Okay. We're on a bit of a tight schedule, so just two questions and keep it pithy, please. [00:28:30]

Wasif Khan: With your experience looking through the coreceptors, when the virus butts out—another thing I learned, I'm new to HIV, another thing I learned with the spike proteins are less dense than other virus. There is a lot of space in between, how much cellular, other proteins, receptors, other things, which are part of that—?

Ed: Yes. That's a good point. We've heard a little bit [00:29:00] already about receptors involved in binding. As for many of the viruses, HIV can bind to [unintelligible 00:29:10] cells. There are probably binding or attachment factors that are important in facilitating the virus binding and then, therefore, the interaction with the so-called entry receptors. There are a number of these we could talk a little more later about. [00:29:30]

Li Wu: Hi, Ed. Perhaps this is a historical question. I was wondering, in your initial discovery of the CXR4, have you tried a CD4 T cell cDNA library, before you using HeLa cell?

Ed: No. There was no reason to go with a T cell library because we and others had shown that other human cell types like HeLa cells, which are so easy to get, and the reagents, like we keep buying HeLa cell DNA library. That was the first thing [00:30:00] we went to. Presumably, it would have worked with a library from a human T cell line or even primary T cells, but it was easy to go with HeLa's because they respond so robustly and presumably had a lot of that RNA.

[00:30:12] [END OF VIDEO]


 

#structural biology

#structure-based drug design

Citations

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