Chapter 03: Initial Research with Viruses and Proteins: Slow Progress on the Gag Paul Gene

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Chapter 03: Initial Research with Viruses and Proteins: Slow Progress on the Gag Paul Gene

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Dr. Arlinghaus confirms that when he arrived at MD Anderson he "had no idea how to attack CML." He describes how he began working on viruses that cause leukemia in mice using widely studied technologies, including the Rauscher leukemia virus.Dr. Arlinghaus explains the details related to discoveries about the genomic RNA of the Rauscher leukemia virus and three key proteins associated with it.Dr. Arlinghaus explains his advances in understating the roles of genes and proteins (GAG-Paul, PR 65 GAG) in creating a leukemia particle that has a unique identity and integrity within cell systems.He also explains his discovery of the protein sequence and "stop code" of the viral protein.Next he talks about the implications of his discovery and sets it in context of other discoveries. Dr. Arlinghaus explains that he eventually received a warning after a year passed with no grants being awarded.He then explains that the NIH's Virus Cancer Program initiated in the late seventies "saved him." He talks about the acceptance of some of his discoveries.

Identifier

ArlinghausR_01_20140321_C03

Publication Date

3-21-2014

Publisher

The Making Cancer History® Voices Oral History Collection, The University of Texas MD Anderson Cancer Center

City

Houston, Texas

Topics Covered

The Interview Subject's Story - The Researcher; The Researcher; Evolution of Career; Professional Path; Overview; Definitions, Explanations, Translations; Discovery and Success; The Professional at Work; MD Anderson Impact; Understanding Cancer, the History of Science, Cancer Research

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

Disciplines

History of Science, Technology, and Medicine | Oncology | Oral History

Transcript

Ralph B. Arlinghaus, PhD:

You know, from my point of view, I had no idea – and I told Dr. Haas this – I had no idea of how to attack the problem of chronic myeloid leukemia. We didn’t know the proteins that were involved, so ---

Tacey Ann Rosolowski, PhD:

So, how did you start?

Ralph B. Arlinghaus, PhD:

Well, I wasn’t a physician so I couldn’t work with patients who had chronic myeloid leukemia. I was a scientist. So I started working on viruses that cause leukemia in mice. Not chronic myeloid leukemia. (Phone rings) Excuse me.

Tacey Ann Rosolowski, PhD:

Sure.

Ralph B. Arlinghaus, PhD:

Reminding me to make sure I see you, I think.

Tacey Ann Rosolowski, PhD:

(laughter)

Ralph B. Arlinghaus, PhD:

No, that’s something else. Okay, so — anyway, I had to bide my time while the field helped me focus my attempts on CML. So I did. I picked an area that dealt with leukemia, but in mice. And, it’s a widely studied technology. There were two famous NIH scientists, Frank Rauscher and Jim Maloney. Each of them discovered a mouse leukemia virus named after them, the Maloney leukemia virus after Jim Maloney, who I never met, and Frank Rauscher, the Rauscher leukemia virus. Both of them cause in appropriate mouse — mice strains. So ---

Tacey Ann Rosolowski, PhD:

And when was that discovery made? I mean, was that before you came to MD Anderson or ---

Ralph B. Arlinghaus, PhD:

Oh, yes.

Tacey Ann Rosolowski, PhD:

Okay. okay.

Ralph B. Arlinghaus, PhD:

Oh, yes.

Tacey Ann Rosolowski, PhD:

I’m just trying to get the timing.

Ralph B. Arlinghaus, PhD:

Yeah.

Tacey Ann Rosolowski, PhD:

Yeah. Okay.

Ralph B. Arlinghaus, PhD:

So, those discoveries were made years before I sort of landed at MD Anderson.

Tacey Ann Rosolowski, PhD:

Right. So that’s – hence, kind of gave you the opening.

Ralph B. Arlinghaus, PhD:

So I was able to get a — there were leukemia cells from the mice that were sick with Rauscher leukemia virus. And I got those cells from a colleague — one of my colleagues — I don’t even remember who gave them to me – but wrote an email — wasn’t an email then. I wrote a letter and said, and I am going to start working with Rauscher leukemia and I understand you have this cell line from these sick mice. The cells were growing culture and you could start getting to study the molecular events in these cells that grow in plastic dishes. That’s where I started. Had no relationship to chronic myeloid leukemia.

Tacey Ann Rosolowski, PhD:

. By just looking at the function ---

Ralph B. Arlinghaus, PhD:

But, as I always did even as a Ph.D. student, I made discoveries that made me — one discovery that made me famous in a small area. I always found something unique and interesting to publish. And, with those cells from Rauscher leukemia virus mice, I chose to try to understand what proteins were made in the leukemia cells that led to the production of Rauscher leukemia virus particles that were released into the media on which the cells were grown. So, in the culture media, those leukemia cells, they were Rauscher leukemia particles. I wanted to know how — what proteins were involved in creating the particles, hoping that I would identify a leukemia-causing protein for Rauscher leukemia. And after a number of years getting funded, I understood how — what proteins were being made, some of them very interesting, how they were being made. So, for example, the reverse transcriptase, have you ever heard of it? Probably not.

Tacey Ann Rosolowski, PhD:

I’ve heard of it but I don’t – I mean, as a word.

Ralph B. Arlinghaus, PhD:

Yeah.

Tacey Ann Rosolowski, PhD:

I don’t understand what it is.

Ralph B. Arlinghaus, PhD:

Reverse transcriptase was discovered by Dave Baltimore and Howard Temin as an enzyme present in these particles, like Rauscher leukemia or Maloney leukemia virus and others. And I studied in part how the reverse transcriptase in the cells from Rauscher leukemia virus infected mice, which I could grow in bottles. I studied how the reverse transcriptase was made. And of course, I discovered something interesting and it turns out that, back then, that was able — allowed me to publish a high impact paper. Now I’m at MD Anderson, so although I didn’t publish anything on CML, my first papers were on the proteins made in Rauscher leukemia virus-infected cells.

Tacey Ann Rosolowski, PhD:

What was the discovery that you made?

Ralph B. Arlinghaus, PhD:

Well, it turns out that – I have to think about how to tell you --- Rauscher leukemia virus contains what we call a genomic RNA of about 8,000 nucleotides. Others in the field about the same time I was doing my work, found out that there were three genes encoded by that 8,000 nucleotides present in the Rauscher leukemia virus particle. One of them was a protein that encoded for this — the structural entities that packaged the viral RNA, this 8,000 nucleotide viral genome, as we called it. And then, there was a second protein which was involved– I have to think of the name of the protein because it's been 20 years – it’s called the Pol gene. The Pol gene, after studying how the Pol gene proteins were made on ribosomes and found that the Pol gene was translated into two entities – the core proteins which I talked about, and then the Pol gene protein. They were fused in a protein which I called Gag-PolI initiated that term because normally, proteins that are made in cells are made as individual units so DNA polymerases are coded for by gene and DNA polymerase, and there will be a messenger RNA protein A polymerase, the messenger RNA gets on ribosomes, and the ribosome translate the coding sequence for the DNA polymerase, and you form the DNA polymerase protein. Well, the Pol protein was made as a hybrid called Gag-Pol.

Tacey Ann Rosolowski, PhD:

That’s G-A ---

Ralph B. Arlinghaus, PhD:

G-a-g. That terminology was instigated or identified or first used by David Baltimore who got a Nobel Prize for discovering reverse transcriptase, so --so, I was the first to show that the Pol gene, or the reverse transcriptase protein, was not made on ribosomes as a single but as a Gag-Pol protein.

Tacey Ann Rosolowski, PhD:

Now, what would the implications of that be? How would that be connected to abnormal function, related to leukemia?

Ralph B. Arlinghaus, PhD:

Well, at the time, we didn’t but it turns out the Gag proteins play a role in packaging the viral genomic RNA, so when the virus is assembling, the Pol — the Gag proteins sort of wrap up the viral RNA, the genome as we call it, as it encoded for these three genes that package the viral RNA. But, remember the — you don’t know, but the reverse transcriptase was encapsulated inside of that package. So, making a Gag-Pol protein allowed the Gag proteins to associate with the other Gag proteins that are made separately from – it’s complicated, I’m sorry.

Tacey Ann Rosolowski, PhD:

No, no. I’m just trying to visualize it …

Ralph B. Arlinghaus, PhD:

Okay, I know.

Tacey Ann Rosolowski, PhD:

… because it sounds like it’s making a sort of bomb.

Ralph B. Arlinghaus, PhD:

Well, Gag — the Gag — the Gag gene is — is the — I said 8,000 nucleotides form up the viral RNA. The first 3,000 nucleotides enc — encode for a precursor protein called – I’m going to lose you, I’m sorry about it — it — it encodes for Gag — for the Gag proteins which were called — and still are, Cor p15, p12, p30, and p10. Those four proteins wrap up the viral RNA and form a particle that — that’s going to be in the virus. But if you wanted to get the Pol protein in there, the reverse transcriptase, it’s very important — if you wanted to get that inside the virus particle, it makes a lot of sense to make a Gag-Pol hybrid protein. So, when you made a Gag-Pol protein, it gets packaged in the viral particle with the 15, 12, 30, and 10 Gag proteins. And then now, you’ve encapsulated not only those proteins but you’ve encapsulated the Pol protein. So, you’ve got that now inside the viral particle. Now, the third protein encoded for by the viral genome are envelope proteins. Two proteins that form spikes on the surface membrane of the particle. So the particle – if I think of it — is too simplistic– as – as a — as a ball. Inside the ball there is this core. Inside the core, there’s the viral genome packaged by these four structural proteins and the reverse transcriptase. Then that’s all packaged by a membrane which had envelope proteins – we call them envelope – surface projections that form the surface of the Rauscher viral particle.

Tacey Ann Rosolowski, PhD:

Wow.

Ralph B. Arlinghaus, PhD:

Very complicated.

Tacey Ann Rosolowski, PhD:

Yeah.

Ralph B. Arlinghaus, PhD:

So …

Tacey Ann Rosolowski, PhD:

I mean, I’m starting to get an appreciation of the — I mean, I’m — I — I’m just — my mind’s kind of being boggled at all …

Ralph B. Arlinghaus, PhD:

I’m sure it is.

Tacey Ann Rosolowski, PhD:

Well, all of the …

Ralph B. Arlinghaus, PhD:

I’ve dumped a lot on you, I know.

Tacey Ann Rosolowski, PhD:

No, no, no. And, that’s fine. But — but — but I’m trying to translate this information back to your lab, you know, the steps, the kind of breaking it down, breaking it down, how do we look at each piece, each mechanism, then figure out how it all works together.

Ralph B. Arlinghaus, PhD:

Most enzymes are made up several polypeptides and each polypeptide is encoded by a single messenger RNA and they self-assemble. That’s another story we’re not going to talk about. So, in the case of the Rauscher virus and many of the other viruses, the proteins for the 15, 12, 30, and 10 are made as a one –which I termed – polyprotein which will then slice at the appropriate sites to give you four proteins. Po — one polyprotein is made from the viral genomic RNA on ribosome to give you something which — which I named as PR 65 Gag. It’s — it’s 65,000 molecular weight protein encoded for what Baltimore termed as a Gag gene. Remember, it’s — the leukemia virus genome has got the Gag gene, the Pol gene, and the envelope gene. The envelope gene, the surface proteins; the Pol gene, the enzymatic activity, that’s involved in reverse transcription; and then the Gag proteins made on packaging the viral genome. So …

Tacey Ann Rosolowski, PhD:

So — I mean, it’s kind of amazing because it’s actually producing leukemia, in a way.

Ralph B. Arlinghaus, PhD:

Well, that’s another story now. I never did find a leukemia-causing protein …

Tacey Ann Rosolowski, PhD:

Oh, you didn’t?

Ralph B. Arlinghaus, PhD:

… made by the Rauscher leukemia virus genetic information. It wasn’t there.

Tacey Ann Rosolowski, PhD:

It wasn’t there.

Ralph B. Arlinghaus, PhD:

I only found p15, p12, p30, and p10, the four Gag gene proteins, the reverse transcriptase, and the two envelope proteins that form spikes on the surface of the particle.

Tacey Ann Rosolowski, PhD:

Now, what does that particle do?

Ralph B. Arlinghaus, PhD:

Which — which one?

Tacey Ann Rosolowski, PhD:

The — the one that’s being packaged with these three proteins.

Ralph B. Arlinghaus, PhD:

It — it packages the — the viral inside the Rauscher and the Moloney leukemia viruses. Like all retroviruses, they have two copies of the viral coding RNA – two, not one. And, it’s still being studied why there are two. But — so that 15, 12, 30, and 10 interaction of those four proteins, packages these two RNA’s inside that core protein structure which then surrounded by a membrane were the envelope proteins. Remember, the four core proteins also had the — the Gag-Pol. And Pol encodes the activity to make a — a DNA from RNA. You know, in molecular biology – DNA encodes RNA, and RNA encodes for protein. But, with the discovery of the reverse transcriptase by Dave Baltimore, Nobel laureate; Howard Temin, Nobel laureate, published a paper of the same year --I forget which year. So, they found that there’s an enzyme in these leukemia virus particles that makes DNA from RNA. Now, DNA encodes for RNA and RNA encodes for protein. But here, for the first time in the scientific universe, there’s an enzyme encoded for by these leukemia virus particles which are called retroviruses and it had an enzyme called reverse transcriptase that made DNA from RNA. And for that, Dave Baltimore, still alive; and Howard Temin – I think he is gone – got the Nobel prize — shared the Nobel prize for that. So, what I was doing was studying how the reverse transcriptase was made in these leukemia virus-infected cells, and what I became known for, because there’s a — so, this genomic RNA is at one end the ____ codes for p15, p12, p30, and p10. Then, we later found out there’s a stop codon them. That doesn’t mean much to you. But, it tells the ribosome to stop translating. Ribosomes translate the information in the RNA into the protein sequences for p15, for p12, p30, and p10. But then, there’s a — a series of two codons that say, stop putting amino acids in, there’s — this – I don’t know if you know how protein synthesis occurs but that’s it. I’m dumping a lot on you right now.

Tacey Ann Rosolowski, PhD:

That’s okay. I mean, one — one thing I’m — I’m curious about is when you — as you were beginning to assemble this knowledge of all of these details, you know, how was your mind working to think about, okay, here’s the implications. You know, here’s how I’m going to take this information and then move closer to my goal of addressing CML?

Ralph B. Arlinghaus, PhD:

Yes. Well, see, I had a big disappointment because I couldn’t find a leukemia-causing protein …

Tacey Ann Rosolowski, PhD:

Right.

Ralph B. Arlinghaus, PhD:

… in the Rauscher leukemia virus encoded proteins in the cell. So, I said either I’m stupid and I miss it, but it turns out here – and I’ll throw you a curve now --the field, not me, not Arlinghaus. The field discovered that they are two types of leukemia or cancer-causing viruses – ones that were slow-acting, and ones that were fast-acting. The fast-acting ones encoded for a cancer-causing protein. The slow-acting ones did not and cause leukemia in another way. Now, remember – so here’s another curve I’m throwing at you – when these viruses, these retroviruses that were being studied by people like me, when they enter a cell and convert the cell to an infected cell, those leukemia viruses, they make a DNA copy with reverse transcriptase. That DNA copy is integrated or inserted into the chromosome’s DNA of the cell at random. So, the slow-acting leukemia viruses like Rauscher inserted their DNA, the viral DNA, made in the core particle inside the infected cell, that viral DNA was then integrated or inserted into the cellular DNA of the nucleus of the cell and very rarely, it inserted at a place that there was a gene that could cause cancer. So, in my case, the Rauscher leukemia virus did not encode a cancer gene but encoded a — a mechanism to activate a cancer gene present in the cell that’s to be infected. But, that didn’t happen, you know, when the virus integration occurred, it didn’t happen every time because the — the viral DNA was inserted at various parts in chromosomes in the normal cell and only when it inserted nearby a — a gene we now call cancer genes, did that cause leukemia. So …

Tacey Ann Rosolowski, PhD:

Interesting.

Ralph B. Arlinghaus, PhD:

… the virus never carried a leukemia-causing protein, it carried a mechanism to activate a cellular present in the infected cell nuclei.

Tacey Ann Rosolowski, PhD:

Now, as I’m kind of — first of all, how — what’s the span of time that it took you to kind of do all of these studies after you got here at MD Anderson on this particular subject ____ (over-talking)

Ralph B. Arlinghaus, PhD:

Well, I had worked …

Tacey Ann Rosolowski, PhD:

… on the Rauscher.

Ralph B. Arlinghaus, PhD:

I — I — I worked three years on a virus much like foot and mouth disease but wasn’t. So, I had to start somewhere that I knew. And then, I wrote grants to try to get me started on working on leukemia. Once I got the cells from a that were from Rauscher leukemia virus-infected mice, I started writing grants to National Cancer Institute to be — to go — begin the — my — my long journey …

Tacey Ann Rosolowski, PhD:

Right.

Ralph B. Arlinghaus, PhD:

… to try to understand how chronic myeloid leukemia was caused. And …

Tacey Ann Rosolowski, PhD:

Now…

Ralph B. Arlinghaus, PhD:

… let’s say I reached the point where Felix Haas got tired of funding me because I got a grant to study the foot and mouth disease-like virus, but then I started writing grants on how viruses like that would cause a slow cancer. And, most of those grants were not funded. So, I — I thought that I’d reached a roadblock and I was going to fail. Even though I learned a lot about how Rauscher and Moloney leukemia virus made their proteins but I didn’t know anything about how they caused leukemia because that — that process of genetically inserting viral DNA in chromosomes wasn’t well understood for some time. So, I — I was almost — Felix Haas had me in after one year of not bringing any grants and gave me a warning. He says, “I’m not going to give you a salary increase. You’re not bringing in any money.” He was telling me…

Tacey Ann Rosolowski, PhD:

Yeah.

Ralph B. Arlinghaus, PhD:

… I’m on my last legs.

Tacey Ann Rosolowski, PhD:

I — I — obviously I want to ask you what happened next but I wanted to go back a little bit because the first thing that was striking me as you’re telling the story is how — it seems like the story couldn’t happen today. I mean, if a faculty member came …

Ralph B. Arlinghaus, PhD:

Probably not.

Tacey Ann Rosolowski, PhD:

And, it — it — it just seemed like an incredible opportunity …

Ralph B. Arlinghaus, PhD:

It took a lot of faith of Felix Haas …

Tacey Ann Rosolowski, PhD:

Right.

Ralph B. Arlinghaus, PhD:

… to give me a start, and he ran out of faith …

Tacey Ann Rosolowski, PhD:

Right.

Ralph B. Arlinghaus, PhD:

… and he …

Tacey Ann Rosolowski, PhD:

But …

Ralph B. Arlinghaus, PhD:

… almost terminated me.

Tacey Ann Rosolowski, PhD:

But, but the fact was they — they did take you on, they allowed you this freedom that seemed very unusual, and a great — an amazing opportunity for you to expand in an entirely new direction. And, I guess the other question that I had in my mind is when you arrived at MD Anderson, who were you working with? You know, who were you — who were you connecting with?

Ralph B. Arlinghaus, PhD:

I built a team of people, thanks to Felix Haas.

Tacey Ann Rosolowski, PhD:

And, who were these people that ____ (over-talking)

Ralph B. Arlinghaus, PhD:

They were the young post-doctoral fellows the same people I have working with me now.

Tacey Ann Rosolowski, PhD:

Oh, really! Wow.

Ralph B. Arlinghaus, PhD:

They’re not the same people, they’re different people. Because we’re talking many years ago. They’re off having their own lives, they’ve got their own jobs. So, I — I trained new people brainwashed them into what I think was going on in a leukemia cell. And, I never did find that leukemia protein because it didn’t exist. But then, something happened at the NIH level …

Tacey Ann Rosolowski, PhD:

Yes.

Ralph B. Arlinghaus, PhD:

… that saved me.

Tacey Ann Rosolowski, PhD:

What was that?

Ralph B. Arlinghaus, PhD:

It had — had nothing to do with me but they started what was called the Virus Cancer Program. And Jim Moloney started that program and that …

Tacey Ann Rosolowski, PhD:

I’m sorry, the Virus Cancer Program?

Ralph B. Arlinghaus, PhD:

Virus Cancer Program.

Tacey Ann Rosolowski, PhD:

Okay, thank you.

Ralph B. Arlinghaus, PhD:

And, I started writing grants to the Virus Cancer Program, talking about my mission that I was on and what I wanted to study eventually, and …

Tacey Ann Rosolowski, PhD:

What year was this that this was started? The Virus Cancer Program? What year?

Ralph B. Arlinghaus, PhD:

Oh …

Tacey Ann Rosolowski, PhD:

Just ballpark.

Ralph B. Arlinghaus, PhD:

I’m bad about this, I’m sorry.

Tacey Ann Rosolowski, PhD:

That’s okay. I’m just thinking ballpark. Must have been in the early seventies.

Ralph B. Arlinghaus, PhD:

No, late seventies.

Tacey Ann Rosolowski, PhD:

Late seventies. Okay.

Ralph B. Arlinghaus, PhD:

I remember …

Tacey Ann Rosolowski, PhD:

___ (over-talking)

Ralph B. Arlinghaus, PhD:

… I occupied most of the early seventies accumulating this data …

Tacey Ann Rosolowski, PhD:

Really.

Ralph B. Arlinghaus, PhD:

… about how — how this — the — the viral proteins were made and how they assembled in the virus particles and the — the Gag-Pol protein …

Tacey Ann Rosolowski, PhD:

Right.

Ralph B. Arlinghaus, PhD:

… which made me famous because the first time ever – and I probably didn’t make this clear – I — I predicted that there is a — a Gag-Pol protein and that when you translated it on ribosomes, you would mostly make Gag then it would stop translating. And then, it would bypass the stop codon and keep translating to give you a Gag-Pol protein. So, I proved that and published some very high impact papers in – two papers in Cell, and …

Tacey Ann Rosolowski, PhD:

You know, because the other thing that’s striking me is as you’re describing this, it is really building an entirely new field. You know …

Ralph B. Arlinghaus, PhD:

Yes.

Tacey Ann Rosolowski, PhD:

… that seems like that’s what in progress. What happened …

Ralph B. Arlinghaus, PhD:

I’m not the only one.

Tacey Ann Rosolowski, PhD:

No,

Ralph B. Arlinghaus, PhD:

A lot of us …

Tacey Ann Rosolowski, PhD:

Absolutely.

Ralph B. Arlinghaus, PhD:

.. and I’m going to tell you about an important discovery that allowed me to continue and to work directly on CML.

Tacey Ann Rosolowski, PhD:

Well, I’m …

Ralph B. Arlinghaus, PhD:

With help from the Virus Cancer Program.

Tacey Ann Rosolowski, PhD:

And, well, why don’t you tell me about that? And so, what happened — what was the next step?

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Chapter 03: Initial Research with Viruses and Proteins: Slow Progress on the Gag Paul Gene

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