The Bioinformatics CRO Podcast

Transcript of Episode 28: Bryan Cullen

Disclaimer: Transcripts may contain errors.

Grace Ratley: [00:00:00] Welcome to The Bioinformatics CRO Podcast. My name is Grace Ratley. I am the editor of the podcast and your host for today’s show. Today I’m joined by Professor Bryan Cullen, who is the James B. Duke Professor of molecular genetics and microbiology at Duke University Medical Center and the founding director of the Duke University Center for Virology. Welcome, Bryan.

Bryan Cullen: [00:00:21] Nice to be here.

Grace Ratley: [00:00:22] Perhaps we can start with talking a little bit about your research on viral epitranscriptomics and epigenomics, for which you recently published a review article. So can you explain maybe the difference between epitranscriptomics and epigenomics?

Bryan Cullen: [00:00:35] Epigenetics has been around for some time so that as you will understand, genetics is the process by which different genes are passed down from parent to a child, and the differences in the sequences can have ramifications such as hair color or skin color or eye color, or how tall you are or how fat you get those kinds of things. Now it became clear some time ago that things are inherited that are actually not in the DNA, and the word that was coined was epigenetics. And it turns out that there are modifications to the DNA or modifications more generally to chromatin, which is the DNA with histones wrapped around it, which can also be inherited. The biggest things that arise in epigenetics are changes of the methylation status for example of histone molecules, so that there are certain residues on histones that if they’re methylated, it silences the gene that’s underlying the histones and certain methylations which occur on the same histone that actually activate the gene underlying the histones on the chromatin and those can be inherited. And then there are also most important modification of DNA in this regard is the methylation of C residues in the context of the dinucleotide CPG, and that can be inherited across time. Obviously CG is GC. It seem to be on the opposite strand as well, and there is a mechanism in cells so that if one C is methylated and the DNA is replicated, then the C that comes in opposite it is also going to be methylated.

[00:02:05] And that’s a way of silencing genes long term. If you sequence the DNA, you don’t see it. You don’t see these changes in the histones. You don’t see these changes at the C residues. And nevertheless, they can have major ramifications and can be inherited from cell to cell during cell division, but also from parent to child. And there are some evidence that they can even be learned, which gets back to this idea that people can actually change their genetics during their lifetime and hand them on to their children. That has been reported for lower organisms at least. So that’s epigenetics. Epitranscriptomics is the same thing for RNA. So there are methylations of A residues, methylations of C residues, acetylations of C residues that are put on by different enzymes in the cell, and they can strongly affect the fate of the RNA after the modification has been added. So that methylation of C residues, as we showed recently increases their translation methylation of adenosine residues increases RNA stability in some settings and decreases it in others. It’s actually not understood why that would be. But regardless, these are then again changes that you can’t see in the sequence of the RNA that nevertheless affect the functionality of that RNA.

Grace Ratley: [00:03:16] And how do viruses use these two different methods to regulate their own biology?

Bryan Cullen: [00:03:22] Yeah, well, that’s complicated. So let’s talk about epigenetics first. So epigenetics can either activate expression or silence expression. And what we actually see is that cells have evolved the ability to use epigenetics to silence incoming DNA viruses. Now, viruses aren’t going to let that happen, of course. And so they’ve developed ways to either prevent the silencing or they take advantage of the silencing in order to enter a latent state from which they can reactivate at a later point. And so if they’re latent, which is to say they’re not producing any proteins, then they’re invisible to the immune system. And so they can actually subvert what really is a defense mechanism on the part of the cell to actually then remain invisible and remain able to reactivate a lytic replication cycle. Nevertheless, there are some settings in which this silencing works very well and does silence viruses. The example that we and others in particular Steve Gough at Columbia have worked on is with retroviruses, which are a kind of virus, which is an RNA virus that then gives rise to a DNA intermediate when it enters the cell. And that DNA intermediate is integrated or inserted into the genome of the host cell. And it turns out that if you block that integration, then the unintegrated DNA is epigenetically silenced and eventually destroyed.

[00:04:41] So in this case, the defense mechanism actually works. Now unfortunately the viruses have figured out that if they integrate, they avoid this mechanism because this mechanism is based on identifying small pieces of viral DNA that are not part of the genome of the host cell. So by becoming part of the genome of the host cell, you become invisible to the defense mechanism. That’s the case with retroviruses. Other related viruses that go through reverse transcription and make a DNA copy of an RNA template include hepatitis B virus. And hepatitis B virus actually encodes a protein that destroys the factors that silence the DNA. So the viruses have come to completely different ways of avoiding this defense mechanism. Herpes viruses however actually take advantage, as I alluded to earlier and can become latent. So as anybody with mouth sores will tell you, the virus lies doggo for months at a time and then reappears, causes an ulcer which releases infectious virus for a few days. And then the whole thing goes silent again. So there we have an example of a virus that’s using this defense mechanisms. Epigenetic defense mechanism actually facilitate its own spread.

[00:05:49] Epitranscriptomic, these are modifications that are added to the RNA as we discussed. It turns out that if you look at RNA viruses such as retroviruses, they have a lot more epitranscriptomic marks on the viral RNA than is typical of a cellular RNA. And that tells you right away that the viruses love it. They think it’s great because the viruses would not have it if they didn’t like it. They evolve extremely rapidly and under extreme selective pressure at all times. And so it turns out that a number of different epitranscriptomic marks can actually firstly increase the functionality of the RNA that the virus has, but then also they can disguise the RNA so that it’s not detected by innate immune factors in the cell. Now that turns out to be very advantageous to humans because that led to the discovery, that modification of RNA using epitranscriptomic marks makes it safe to give as a vaccine. And in fact the Moderna vaccine and the BioNTech-Pfizer vaccine are based on this finding that epitranscriptomic marks prevent this innate immune activation, which, you know, actually you might think would be a good thing in a vaccine, but it turns out to be quite deleterious and really makes it impossible to use.

Grace Ratley: [00:07:02] Can you maybe expand a little bit more on how we can take advantage of how viruses use these modifications to create antivirals and vaccines?

Bryan Cullen: [00:07:13] Right. Because the viruses are very dependent on epitranscriptomic marks, particularly methylations of adenosine and cytosine in RNA, that gives you the opportunity to selectively target viruses by interfering with methylation of RNA. Now, cellular mRNAs are also methylated. So this is going to have some issues in terms of messing with cellular gene expression. But in fact, there are a couple of drugs that have been developed. One is called 5 adenosine that inhibit the ability of the cell to make the substrate that’s used to methylate RNA. Without going into any details, this of course would globally inhibit RNA methylation. But it turns out that in acute diseases such as Ebola virus, for example or influenza virus, you can treat animals for a week with a drug that inhibits the methylation of RNA, and you selectively reduce the replication of these highly pathogenic viruses. There was an interesting study published where they looked at Ebola virus in mice, and when they gave this drug, they had 80% survival. And if they gave no drug, they had 0% survival. Now obviously because cellular mRNAs are also methylated, you couldn’t give this drug for months and months because that would accumulate deleterious effects. But given the high dependency of the virus on methylation of RNA for maximal gene expression, it’s possible to use something that inhibits that step for a short period of time to allow the immune system to get its act in gear and mount an adaptive immune response.

 

[00:08:50] And by reducing the peak load of the virus, you can actually then hopefully allow the animal or human to get rid of the virus completely and survive the infection. Personally, I’ve been proselytizing for this idea for a while. I don’t know that anybody has really run with it because I think people are concerned about the fact that you really wanting to target something that happens to both cellular RNAs and viral RNAs, but essentially that’s how cancer drugs work, I mean almost all cancer drugs. Chemotherapy is notorious for causing nasty side effects. And that’s because it affects normal cells as well as transformed cancerous cells. So I think the key question with these kinds of drugs that act on host targets is you need to use them for limited amounts of time during an acute phase of the disease. To go into the second part of your question, I actually just proposed the two inventors of this strategy of modifying RNA so that it’s not doesn’t activate the innate immune system, nominated them for an award. I think the work is extremely important. So the people who actually did this work are Katalin Kariko, who is now head of Research at BioNTech, which is one of the companies that developed the vaccine. And then there’s a guy called Drew Weissman, and they work together at Penn to discover that RNA modifications make RNA much more readily tolerated.

[00:10:12] So if you inject a regular RNA into a mouse, just send it off the shelf looks like a regular mRNA, nothing special about it. You get a very strong, innate immune response, high levels of interferon in the blood. The mice get really sick from their own immune system. By modifying it, you then make it invisible to the innate immune system. But it still works really well as an mRNA pumps out proteins which are then presented to the adaptive immune system. And so you get really good antibody and T-cell responses. And so I think that’s one of the most important discoveries of the last 20 years. They started working on it in 2005 and it really took off when the emergency hit with COVID. But think about it, this allows you to use RNA as a vaccine for anything. All you have to do is reprogram the little computer that’s synthesizing the RNA. And you could do flu, you could do anything you like. And not only that because the RNA is injected into cells, it’s actually presented at the cell surface, which means it’s presented in the way that a viral protein would be presented. So it should activate both the humoral and cellular arms of the immune system much more effectively than simply injecting proteins into the blood, which generally gives you a good humoral response, but not a very good cell mediated response.

Grace Ratley: [00:11:25] Yeah, I know there’s been a lot of discussion for how mRNA vaccine technology could be used to generate vaccines for a lot of these pathogens, which we haven’t been able to to treat before. So let’s move into your other research. I know one of the things that you have worked on that has been very successful is micro RNAs. Can you tell us a little bit about that?

Bryan Cullen: [00:11:48] So RNA interference was discovered, I guess it was in the late 1990s by Fire and Mello looking at C elegans nematodes and they discovered that if you injected nematodes with double stranded RNA, what resulted is the gene from which the double stranded RNA derived would be silenced. And so it took a little while to figure out what’s going on. And probably a guy called Tom Tuthill, who’s at Rockefeller, probably has more responsibility for figuring it out than anybody else. But what he figured out was that that in nematodes, there’s a mechanism in place whereby double stranded RNA is cleaved into 21 nucleotide double stranded pieces. And then one strand of that double stranded RNA is inserted into a protein complex and then acts as a guide to bring the protein complex to a complementary RNA. And so the idea is you get the antisense strand from the duplex is incorporated into this protein complex and then the complex sees the RNA. When it sees the RNA and that complementary is complete, it cleaves it. So there’s an endonuclease cleavage event which results in the RNA being degraded. Now Tom Tuthill and a couple of other guys, but initially Tom Tuthill were very interested in characterizing these small RNAs that were arising from these double stranded RNAs that they were making and introducing into cells. And so he figured out a way to sequence small RNAs, 21 nucleotides long, which nobody had ever been interested in before. I mean, why would who could care about 21 nucleotide RNAs? And when he did that, he found that he not only saw the 21 nucleotide RNAs from the double stranded RNA that he put into the cells, but he also found lots and lots of 21 nucleotide RNA that were already there.

 

[00:13:31] But it wasn’t just random pieces of 21 nucleotide RNA. They were specific pieces of RNA derived from specific locations in the genome of the cell, and there were only maybe 400 or 500 different small RNAs in the cell. So it was actually my lab that was one of the two that figured out how these microRNAs are made. So they’re actually derived by the cleavage of a stem loop structure that forms part of an RNA polymerase to capped polyadenylated mRNA like product, which is now referred to as a primary micro RNA. So that was interesting. We figured out how to make microRNA and that was actually extremely helpful because we were able to patent the process of micro RNA synthesis that has been extensively licensed by Duke to lots of people and was very nice for me and very nice for my lab in terms of generating income. But once I was doing this, I thought well these small RNAs can turn off our target, an RNA that is made by a virus or a host cell, and there are only 21 nucleotides long, so they don’t take up much room. So would viruses have come up with this idea? Would the use small RNAs like microRNAs to target host cell factors that are involved in repressing virus replication? When I thought about that, I thought what virus would be the most likely to do this?

[00:14:57] Now the thing about microRNAs is they downregulate mRNAs. They don’t touch the protein that’s made from that mRNA. Most proteins have a half life of 12, 24 hours, and most lytic viruses, poliovirus COVID-19, for example go through their entire replication cycle in about a day. And so that means that even if the virus produced a microRNA that completely destroyed an mRNA population, the protein from that mRNA population would only have gone down by maybe twofold at the end of the replication cycle. So I thought well, probably not an RNA virus then, but what viruses hang around forever and a day. And the obvious answer is herpes viruses, not just herpes simplex, but also Kaposi sarcoma virus, Epstein-Barr virus. And so I contacted scientists called Blossom Damania at Duke and asked her for some of these cell lines that she had that were duly infected with Kaposi sarcoma herpes virus, which is a virus that causes cancers in immunodeficient people and was originally only discovered as a function of HIV because fully immunocompetent individuals basically very, very rarely have Kaposi sarcoma. But if you don’t have an immune system, then it’s quite probable that you’ll get one. And then it turns out that a lot of these cells are duly infected with Epstein-Barr virus, which causes infectious mononucleosis.

[00:16:18] So I thought well this gives me two bang for my buck. So if I sequence the small RNAs in these cells, if the Kaposi sarcoma virus or Epstein-Barr virus make small RNAs, then we’ll identify them. Now I was scooped on this, so we actually came second. We were the first to report microRNAs made by Kaposi sarcoma virus. But Churchill Laboratory had been interested in looking at microRNAs that were characteristic of different tissue types. So he was looking in liver cells. He was looking in neural cells. He also looked in B cells, and B cells are what Epstein-Barr virus infects. And so he actually ended up looking I think by chance at a B cell that was infected by Epstein-Barr virus. And so he identified five Epstein-Barr virus microRNAs. And then we came in a couple of months later and we identified ten Kaposi sarcoma virus ones. But it turned out that the cell line that Tuchel’s group had sequenced small RNAs in was infected with an Epstein-Barr virus mutant that has a huge deletion, whereas the ones that we did was infected with a wild type Epstein-Barr virus. That is the one that makes people sick. And it turned out that the big deletion took out almost all the microRNAs of the virus. So we actually discovered over 20 Epstein-Barr virus microRNAs. So that was really interesting. And we initially tried to figure out what they were doing. We had some luck at the beginning.

[00:17:39] One of the really interesting things that turned out was that if you look at Epstein-Barr virus, when it infects B cells, it actually turns on several cellular microRNAs to very high levels. And one of the ones it turns on over 100 fold to make it the most highly expressed RNA in the cell. It’s called MiR-155. So Epstein-Barr virus induces B cells to grow rapidly. Kaposi sarcoma Herpesvirus also induces B cells to grow rapidly, but it doesn’t induce MiR-155. Instead, it encodes Mir-155. It has its own MiR-155. And so the two viruses that are both herpes viruses that want the cells to grow fast and to make B cells grow fast, you have to have MiR-155. One of them decided to figure out a way to turn on the cellular microRNA by over 100 fold, and the other one decided to make its own. And so that was a really interesting discovery. It was actually followed up by another group in the UK who worked on a disease of chickens. It’s actually a herpes virus that causes cancers in chickens called Marek’s disease virus. And it turns out that Marek’s disease virus which is a herpes virus, also encodes a MiR-155. And they did a very cute experiment where they knocked out the MiR-155, and the virus was still able to grow perfectly well, but it couldn’t make tumors anymore. So this MiR-155, has been around since dinosaurs. It’s only 21 nucleotides long, but it’s exactly the same since T-Rex was wandering around the earth.

[00:19:08] They took the one from the chicken and put it into the Marek’s disease virus, and it regained the ability to cause tumors. Very clean demonstration that it is in fact, the micro RNA that allows the virus to become carcinogenic. But that was one of the few instances I think, where we really could figure out what the microRNA did. So we banged our head against the wall for multiple years trying to figure out what these microorganisms were doing. And the problem is that they downregulate 200 mRNAs each. And so the question is, all right what are those 200 are important. Is one important or 5 important or 17 important? I’m certainly not all of them, because these are with exception of the MiR-155, almost all the viral microRNAs are completely different from cellular microRNAs. And the cell hasn’t conveniently sprinkled around targets for that microRNA at all. Its antiviral genes. So what the virus is probably doing is turning off 90 things that it doesn’t care about one way or the other in order to get ten things that he does care about. Trying to separate the wheat from the chaff was just very frustrating. And in fact, I would say that most of the literature out there on viral microRNAs is wrong. They haven’t really identified the targets. They’re oversimplifying. It’s all artifacts based on overexpression and things like that.

Grace Ratley: [00:20:22] What sort of methods do you think could be used to correct the wrongs of the RNA papers that have been published?

Bryan Cullen: [00:20:31] Well, what people do is you have a microRNA that you happen to be interested in for whatever reason. So we know from that microRNAs downregulate any mRNA that has a perfect complement to nucleotides 2 through 9 of the microRNA, which is called the seed sequence. Now the problem is that eight nucleotide sequences occur a lot. I mean this is every 50,000 bases you’re going to get one. And so that means something like 10% of all the RNAs in the cell have a seed sequence that could be targeted by microRNA. So what people do is they have the microRNA and they see an interesting phenotype associated with it for some reason or whatever it is. They see it overexpressed in cancer cells like MiR-155 and then you say, okay, well what is MiR-155 complementary to. And you can do that using a computer program and it will pick out all the mRNAs in the cell, which have an eight nucleotide complementarity and you’ll probably get 1000 or 800 or something like that. And you have no way to tell which ones are important. And so what you do is you do what’s I’ve heard called the shiny pebble syndrome, where you if you look in a big bucket of pebbles and one of them is really shiny, you’re going to pick that one. Because that’s that’s a cool looking pebble.

[00:21:48] And so what you do is you get this list of mRNAs that have complementarity to the microRNA and you go through a B7 62, I’d never heard of that. A6 543, never heard of that. And then you get, oh tumor suppressor. That’s got to be it. And so now what they do is they take the sequence of that that is complementary and they stick it into an indicator construct behind an indicator gene. But of course, that’s a circular argument. It’s an eight nucleotide homology, of course it’s going to be a target at some level. And so what they do is they make a massive amount of the microRNA by overexpressing it. They throw in this indicator and it goes down by four fold and they say, Oh, so you can down regulate. But that doesn’t show you that the actual mRNA is down regulated by the microRNA when the microRNA is expressed at physiological levels. Because the microRNA is able to bind to hundreds of different mRNAs. And so which of those can it actually down regulate to a sufficient degree? That’s basically what they do. And they never actually demonstrate that the physiological level of that microRNA in the relevant cell, which cancer cell is actually down regulating the whatever tumor suppressor or whatever. And if you got rid of the microRNA or you increase the expression of the tumor suppressor, that you would actually reverse the phenotype. They almost never see that, almost never see that.

Grace Ratley: [00:23:07] So what is your approach then?

Bryan Cullen: [00:23:08] We gave up on microRNAs. One of the things that you have as a professor is you have people come through your lab all the time. And I’ve tended to have a lot more postdocs than graduate students, although I’ve had some really good graduate students as well that are professors at universities now. But what happens is the postdocs come into your lab and they spend 4 or 5 years there and they’re working on problem X. And then they go off to another university. And what are they going to work on? Well, they want to work on problem X because that’s what they’ve been working on for the last 4 or 5 years. That’s what they know how to do. And so that puts you as an established investigator into a difficult situation because you don’t really want to directly compete with your own offspring, so to speak. And so what I’ve done over the years is that I basically change everything I work on about every ten years. And so we only started working on epitranscriptomics in 2018 and we’ve only been working on epigenetics since 2019.

[00:24:03] And so the other stuff that we were doing before that has gone away with people. I’m going to be retiring in 2 or 3 years and that will solve the problem in a permanent sense. But I’m not going to worry about epigenetics and transcriptomics being a problem, but it’s an issue that everybody faces and some PIs are really nasty about it. They’ll bring a postdoc into their lab and give them a project to work on, and then when they’re going to leave, they won’t even let them take their own reagents with them. They say, No, those reagents are made in my lab. They belong with me, and you don’t even get to take them, which I think is extraordinarily reprehensible. I mean, I don’t know how anybody gets a postdoc done that once or twice, but I’ve always felt that it was important to let people take things along. So there are several postdocs of mine out there working on viral microarrays with greater or lesser success.

Grace Ratley: [00:24:51] Well speaking of postdocs, I know that you did not complete your postdoc. So let’s talk a little bit about your training as a scientist. So I guess we’ll start from the beginning. What got you into science when you were a kid?

Bryan Cullen: [00:25:05] I was interested in a lot of things, but I was interested in things that were black and white more than I were in things that were gray. I liked my answers to be yes or no, not well, maybe and twice on Wednesdays or something. But I was very interested in things like archeology, astronomy. And like a lot of kids, I was thinking that would be really cool being an astronaut. And then as you get older and you start reading the fine print, you realize, well, that’s not such a good job actually. You spend your entire life and you maybe go to the moon once. I mean, that’s not fun. So I always did really well at science. I graduated top of my class in high school and I grew up in industrial northern England in a town called Bradford, which was rapidly declining as the industries which had supported it were being lost. So I was not totally aware of all the possibilities that existed. The school I went to was actually quite good, but the concept of giving people advice on their career had not penetrated through to the British educational system at that point. So nobody in my family had ever been to university, but I thought, Well, I definitely want to go.

[00:26:11] And I actually got a full scholarship to Warwick University, which has gone on since then to become one of the top universities in the UK. It’s ranked like number five or something like that now. And at the time it had a I thought a very forward looking department called Biological Sciences, which really wasn’t so much a biology department or a biochemistry department, which a lot of them were in those days. But actually it was really interested in what was going to become molecular genetics. And they were very interested in pathogens. And so I became interested in that. And then when finishing my third year in in England, a bachelor’s degree only takes three years. My parents told me that they were going to emigrate to the United States, to New York specifically. So what I ended up doing was I went to graduate school at University of Birmingham in England, at the medical school there. And the reason I went there was because they had a Department of Virology, which is really unusual and I wanted to do virology. But I didn’t end up staying there to do a PhD. I ended up deciding to take a master’s degree after a year and moved to the United States. Because I don’t know whether this is still true, but at that time if you were less than 22 years old, you could emigrate to the United States as a dependent child, which meant that I had to leave within a year of going to Birmingham. Otherwise, I wouldn’t have been able to go to the United States at all.

[00:27:30] So the concept was really to go to the United States, work there as a tech for a couple of years. And then move back to England, which I felt very happy as a Briton. And I liked the UK and do my PhD and unfortunately I didn’t do much research on this. So I got to the United States and I got a really great job as a tech in a research institute called the Roche Institute of Molecular Biology, which actually had a Nobel Prize winner on the staff and three National Academy members and some really good virologists. And I got a job there and learned vast amounts actually working there. And then after a few years, I got married to an American woman, but she was okay with the idea of going back to the UK. So I applied back to the UK. At that point, I was a British citizen. And they said, Well, yes you’re a British citizen, but in fact we can’t give you a scholarship because you’re not a resident. And I was like, Well nobody told me this before. So an opportunity came up, in fact, at Hoffmann-La Roche, where the Roche Institute was, because they were setting up a Department of Biotechnology and I had become an expert at cloning and was considered a cloning whiz in those days before PCR.

[00:28:39] And there were a lot of tricks that you had to do. You had restriction enzymes and that was about all you had. And all you had was pBR322, which is a low copy number plasmids, so much lower amounts of DNA than you get these days. Every plasmid had to be isolated through caesium gradient centrifugation. So it was extremely hard work, but I was good at it. And they decided to set up a Department of Biotechnology at Hoffmann-La Roche. And they asked me if I’d like to come on board. And I said, Well I mean, what’s in it for me? And they said, tell you what, we will sponsor you for a PhD. And when you finish the PhD, we guarantee you your own lab here at Roche. So I said, Well, that’s good, I’ll work with that. So they said, Well, go find a university that’s not too far away. That’ll take you because we need you here 40 hours a week to do your regular job. So you’ll have to do your PhD basically in your spare time. So eventually what was then the University of Medicine and Dentistry of New Jersey is now part of Rutgers University in Newark, said they would take me on and they would allow me to use my work at Roche as my project.

[00:29:42] So there was a lot of commuting involved because I had to go to attend all the classes, of course in Newark. So I was driving back and forth between Nutley and Newark two or three times a day to go to class. And then we also had to demonstrate stuff for the medical students and so forth and teach them lab. But that was the hardest I’ve ever worked in my life. And I managed to get my PhD in two and a half years and it wasn’t a cheap PhD. I actually got seven first author papers out of it, including a nature article that then was my PhD and they gave me a job that they promised me and they asked me to work on something called Interleukin-2. There are lots and lots of interleukins now, but in the old days it started with one and two and three. Now there’s like 40 of them. I don’t know. But interleukin-2 is one that was discovered by Bob Gallo, and it’s required in culture to make T cells grow. And so the idea was that I was going to generate cell lines that produced large levels of interleukin-2 that then might be a drug.

[00:30:39] They said, well you can work on that, but you can also work on another project that might interest you. And my PhD project had actually been on avian retroviruses, particularly avian leukosis virus. And at that point HIV was discovered. So I got my PhD in 87. HIV was sequenced in 88, and I said, Well, I know how to work with retroviruses. I’ve been working with retroviruses for years when I work on HIV. So I started working on HIV. It was an odd thing because I was doing it in my spare time sort of thing. My lab was very small. I had a technician initially and that was it. Later on I got a postdoc, but I was working on this one issue, which was this protein called Tat that HIV one makes. And it had been proposed that it regulated the translation of HIV RNAs. And when I did some work on it, I discovered it was actually a transcription factor. And so in 1986, I submitted a paper to cell, which was very unusual and that I was the only author. So I’d done every single experiment in the manuscript and written it, put it all together, sent it off. And I remember I was on vacation with my wife, so we went on vacation right after I submitted it. In those days, we didn’t have computers. And so I got a fax from Hoffmann-La Roche that had originally come from Sal saying that they were going to accept the paper for publication. So I knew at that point that that was going to really kick start my career.

[00:32:03] So the next thing that happened was somebody from Duke approached me. Actually, I was approached by several people at several different places. But one of the people at Duke approached me and they said, well, we have a Howard Hughes investigator position available. In those days, universities were assigned Howard Hughes investigators, and they could then recruit somebody. That doesn’t work that way anymore. But in those days they did. And so how would you investigate? A position came with about $800,000 a year in research funds. And so I looked at that. I’d never been to North Carolina before in my life and came down and thought, Well, this looks nice. I took the job. And within a year we had actually figured out how the RF protein of HIV worked. So we published two nature and two cell papers on that. I was very lucky because I brought the postdoc with me from Duke, a guy called Yogi Huber, a German guy. And then I recruited a really talented English postdoc called Michael Milam. In 1990, the three of us plus one tech, we had 19 papers out of the lab when I was an assistant professor. It was actually funny because I got a phone call from from Sal and they wanted me to be an editor on Sal, and I wasn’t even tenured yet. So I was put on the editorial board of Sal. Turned out to be a really bad idea actually because I was one of only two people on the editorial board of Sal, who was a virologist. And so only 10% of the manuscripts that go to Sal are accepted. And they all assumed that I was responsible for the rejection. I got me a lot of really negative vibes from a lot of colleagues who were more senior than me out there in the field. So it was good for the ego, but it really didn’t help my career, although it certainly impressed Duke and they moved my tenure along real quick after that. And since then I’ve been at Duke. I’ve looked at a number of jobs elsewhere. There was one at Rockefeller a few years ago that was tempting, but my wife said that she would absolutely refuse to move to New York City.

[00:33:54] So that kind of went out the window. What’s interesting is that most of the top universities around the country are either in the middle of nasty areas in cities like Johns Hopkins, for example, which historically was in a not very nice area. And that was also true of Yale, although it’s gotten a lot better. It was also true of Penn and Chicago. Generally, what you have to do is live way out of town and have this big long commute. But then secondarily, most of the top universities around the country are in really expensive places. They’re in places like San Diego or San Francisco or Boston or whatever. I remember one offer from a California school, and they basically couldn’t give me much more money than I was making at Duke because it’s a state position. And so they have strict rules about how much they can pay you. So they would have given me maybe $20,000 more to move there, but I would have had to take on an extra million dollar of mortgage for a house that would be about half the size of the one I had. I thought, well, does this really make any sense? So here I am still and say getting ready to retire. But yeah, it’s been interesting watching this area grow and change over the years, especially Durham. Nobody went to downtown Durham when I got here in 1986, and now it’s sort of this vibrant hub and everybody wants to live there, which is curious.

Grace Ratley: [00:35:13] Yeah, I’m a huge fan of North Carolina, having done my undergrad over at UNCW. I totally respect your decision to stay there, but I am curious what your plans are for after you retire. Are you thinking of maybe moving back to England or staying in the Durham area, being a professor emeritus?

Bryan Cullen: [00:35:34] We actually built a house about five years ago in Chapel Hill. We used to live in Durham before that. It’s on a four acre lot in the forest, so it’s only six miles from Duke. It’s a single floor house, very appropriate for our life going forward as we get older. And really a beautiful place. I love it. We spent so much time on planning it and working with the architect and the builder to make it just perfect. I can’t imagine leaving this place any other way, but in a big box I do quite a few other things than just work for Duke. I do quite a lot of consulting and expert witness work, so right at this moment in time, I’m an expert witness on three different patent litigation lawsuits. That actually keeps me busy some of the time and is very interesting. I mean, they’ve all been very quiet recently because all the courts are shut. But that will resume in the relatively near future. I like working with these really high quality lawyers. They’re people who are from the very top law schools that have clerked on the Supreme Court. And they’re really, really bright, but they’re bright in a different way than scientists are bright. Their minds work in a somewhat obtuse way, but it’s really entertaining to watch. And I actually really enjoy being cross-examined by really bright lawyers from the other side. It’s a real game of intellectual cut and thrust.

[00:36:55] When you’re up there on the witness stand for eight hours or whatever, with three lawyers going after you. It’s quite an entertaining. I do like to travel and I do serve on several advisory boards for universities and things like that. So that’ll presumably keep going when I’m emeritus. I mean, part of the problem for me has been it’s become much more difficult to get funding. I lost my Howard Hughes investigator position a few years ago, so that made life a lot more difficult. Then I had to get all my money I had to come from NIH. And so you spend a lot of your time writing grants and actually getting postdocs and graduate students. Graduate students, not so much, but postdocs is a challenge in North Carolina. Most European postdocs, for example, want to go to the big cities. They want to go to San Francisco or Boston or New York or what have you. You can get some people from universities in the South or the Midwest, who think Durham is a cool place, really like basketball or whatever. Those individuals will come. But not so many people from the Northeast or the West I think. You’re only as good as the people in your lab. If you don’t have good people, then you’re not going to make any progress, that’s for sure.

Grace Ratley: [00:38:03] So as we wrap up here, I just wanted to ask one more question. From your many years working in science, what is the most important piece of advice that you could give to young scientists, early career scientists, or students who are interested in entering the field of science?

Bryan Cullen: [00:38:23] Stay nimble on your feet. I mean, I think there’s a tendency for people to keep doing what they’ve been doing. And eventually you just run into the ground if you do that. So there are labs out there that are still working on Lambda Phage or whatever because that’s what they did when they were 25 and they’re still doing it at 55. Try to move with what’s out there. So when something like CRISPR-CAS comes along, jump on it immediately and something like epitranscriptomics comes along, jump on it immediately and something like RNA comes along, jump on it immediately. You’ve got to go with what’s hot. That doesn’t mean that you have to work on Ebolavirus because Ebolavirus was important for a year. And I don’t think it necessarily means you have to work on COVID at this point in time. But for these incrementally game changing technologies like CRISPR-Cas and RNA and epitranscriptomics. I think there is a lot of potential for high profile, high citation work. That in the end is going to allow you to move forward and become successful.

[00:39:23] The other thing I would say is think about patents. I mean, I’ve probably made almost as much money from patents as I have from being paid by the university. That’s because I was very aware of things that could be patented. Obviously if you’re working on fruit flies, the chances of you getting a patent that’s going to be of use to anybody, a relatively low. So if you can think in terms of working in areas that have applied potential and after all, that is about human health, which is something important as well. That’s an area that you might want to consider taking into account. Because if you can, it doesn’t have to be a drug. It can be a process or a method or something. If you do discover something important like that, then that can make a big difference to your long term financial welfare, which if you’re just living off a faculty salary, isn’t necessarily going to be that great, especially if you’re in an expensive area like San Francisco or something.

Grace Ratley: [00:40:16] Well, thank you for that advice and thank you for coming on our podcast. I have really enjoyed talking with you. You have such a wonderful, varied experience working in science and a lot of awesome insight into emerging technologies. Yeah, thank you for coming on.