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Luis P. Villarreal: We Need a Nonlinear Language for Life

03/17/2015 01:59 pm ET | Updated May 17, 2015

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Having just published a book on the origin and synthesis of life, I was fascinated to learn that I'd missed interviewing one of the field's most provocative scientists -- virologist Luis Perez Villarreal -- who takes a "virus-first" perspective to life. But Villarreal, founding director of the Center for Virus Research, University of California at Irvine, says he acknowledges that he is a bit of an outsider in the sense that his virus-first perspective is somewhat dismissed by traditional biologists. Nevertheless, Villarreal's thinking draws applause in Europe and he is among the first experts the American mainstream media consults when viruses like Ebola and HIV loom large in the news.

But part of the reason why such viruses linger in the news, Villarreal says, is the fact that we are spinning our wheels by regarding evolution as a linear process when it is clearly nonlinear. In fact, Villarreal thinks we need a new language to deal with the dynamic of viruses, which exist as nonlinear collective entities, i.e., consortia. He considers viruses active organisms that are not only the biggest component of the biomass but comprise 10% of the human genome. Villarreal goes as far as saying that "all living habitats . . . have and must operate in a virosphere" -- Ex virus omnia (from virus everything) is his motto.

Luis Villarreal is a native of East Los Angeles, which he describes as a neighborhood where collectives (gangs) emerge spontaneously. But his family moved around during his childhood because of his father's professional interests and commitments -- as a musician recording with "Los Caballeros Trio," as a mechanic, and as a tortilla machines salesman. Villarreal says his father also liked to buy, fix-up, and sell the family's house -- one after another -- and that he learned a valuable lesson in flexibility from those years.

Science was, of course, an early interest. Two of his life-long passions have been high performance motorcycles and Latin dancing (he says his feet now feel it).

Aside from Villarreal's current role in the virus center at UC-Irvine, he is a professor of molecular biology and biochemistry at the university. He has developed many science education programs for minority students. Villarreal is also the author of two books: Viruses and the Evolution of Life and Origin of Group Identity. In July 2014, he co-organized a major conference on viruses in Salzburg, Austria -- "DNA Habitats and RNA Inhabitants" -- with philosopher of science Günther Witzany.

Among Luis Villarreal's distinctions are: SACNAS Distinguished Scientist; Distinguished Alumnus Award from California State University, Los Angeles; National Science Foundation Presidential Award for Excellence in Science, Mathematics and Engineering Mentoring.

He is a Fellow of the American Society of Microbiology and a member of the Society for Advancement of Chicanos and Native Americans.

Villarreal's PhD is in biology from the University of California, San Diego -- his mentor was the late John Holland, one of the principal scientists to experimentally evaluate viral quasispecies. Villarreal's BS degree is in biochemistry from California State University, Los Angeles. His postdoc was in virology at Stanford University where he worked with Nobel laureate Paul Berg.

Our interview follows.

Luis Villarreal: In decades of research and teaching of virology I've heard stories about the 'going away' of viruses, beginning in the 1970s. That was when we eradicated smallpox in the world. The polio vaccine campaign had tremendous strides. Measles, and so forth were on the wane. Many predicted that studying virology would soon be a historic topic. Then HIV made its presence known.
Many million human deaths later we accept that viruses don't go away. They just keep coming. This evolutionary reality now has us scrambling. HIV, this little virus of 10,000 nucleotides of RNA, has defeated the combined technologies of the planet since it emerged decades ago, and we still don't have an effective vaccine or way to prevent its transmission. This just demonstrates the massive evolutionary power these entities have.

Suzan Mazur: It's clear to me from reading your papers that you have issues with neo-Darwinism.

Luis Villarreal: The issues first came up when I began looking at quasispecies in the early 1970s. I was a researcher in the lab of the late John Holland, where there was real enthusiasm for studying evolution theory in the context of actual virus measurements, because the whole concept derived from thinking about RNA viruses.

I wasn't interested really in evolutionary theory at a deep level. I was interested in persistence, and for this the Modern Synthesis didn't seem to be working. I just assumed that the theory didn't apply or work in the specific situations we were studying involving persistence, such as VSV, rabies and measles. But the more I got into the research, neo-Darwinism appeared to be working less and less. After I left that area of RNA virus research and began investigating SV40/polyoma virus with Paul Berg at Stanford, that's when it really struck me that a shift in evolutionary thinking was required -- because I realized that these were viruses that make their living by being persistent and stable entities that are essentially part of the host. The theory we had at the time to explain that relationship did not make any sense to me. And that's when I started on my current path.

Suzan Mazur: Neo-Darwinism made no sense.

Luis Villarreal: Right. In terms of selfish individual types, runaway replicators, and that whole set of related concepts.

Suzan Mazur: You open your recent New York Academy of Sciences paper on viruses by saying: "All living habitats (including prebiotic ones) have and must operate in a virosphere (a network of infectious genetic agents)". That's pretty sweeping.

Luis Villarreal: Yes, I stick with that statement. I will argue with anybody on that point.

I'd like to define what I mean by a virus, which is a molecular parasite dependent on its host for its maintenance and replication. A virus can also infect another virus, which then becomes its host. Fundamentally, this definition is symbiotic because it has embedded in it dependence on others.

Suzan Mazur: You've also noted that "most experimental paradigms seek to eliminate or have ignored viruses" and you've advocated that evolutionary science now "adopt a virus-first perspective."

I want to focus on the science in our interview, but obviously, politics comes into play here. Would you talk about that?

Luis Villarreal: Absolutely. That whole issue of politics--

Suzan Mazur: What kind of resistance do you see among evolutionary scientists to accepting a virus-first perspective?

Luis Villarreal: There are actually relatively few formally-trained evolutionary scientists who will accept the virus-first perspective. I've encountered a couple. I'm immersed in a population of traditional evolutionary biologists here at Irvine. They just dismiss it. They don't even want to think about it. So it's not a prevailing perspective. But it's one that I think much better explains the foundations of observations, especially now that we have metagenomics informing us about the character of genetic entities -- how lineages and genomes change.

Suzan Mazur: Scientists don't want to take the time to study it?

Luis Villarreal: It's some of that. They very much believe in their theory, and the theory comes first and the observations come second. This is an issue with Manfred Eigen, for example.

Eigen has developed a nice theory and career based on this approach. When this happens the science becomes not very available or open to experimental correction. Of course, if it weren't for Manfred Eigen perhaps we wouldn't have pursued the path of viruses the way we did. But Eigen's book published last year, which covers the history of quasispecies thinking, etc., with in-depth mathematical model and evaluations, is completely missing references to a generation and a half of experimental results virologists have accumulated -- many of which are not really terribly consistent with what Eigen is arguing.

Esteban Domingo and colleagues in Spain have written a really fantastic review of the last 20 years of virus experimentation. But Eigen doesn't refer to any of it in his book. It's what can happen when a scientist believes their own theory too much.

Suzan Mazur: It should be evidence first.

Luis Villarreal: Which is the perspective that I adopt.

Suzan Mazur: While we're on the subject of politics, I follow you all the way to the end of your New York Academy of Sciences paper "Force for ancient and recent life: viral and stem-loop RNA consortia promote life" -- which you call your "gangen" paper because of your perspective of viruses as consortia -- until I get to the concluding paragraphs where you make reference to the work of Nilesh Vaidya and Niles Lehman. I interviewed Vaidya at Princeton two years ago about his work on "recombination events to produce autocatalytic sets," as he described it to me, and I ran that interview online. Vaidya has since his 2012 Nature paper on that subject with Niles Lehman redirected his research interests. It is unclear why.

Luis Villarreal: I can't actually answer that, but I have suspicions, and they're social in nature. When you make an experimental inference that confronts fundamentally these well-entrenched views - it makes a career very difficult. I got to a position in my career where I was able to extricate myself from a lot of those consequences. Somebody just starting out would be hammered and out of business.

Suzan Mazur: Lehman's work had been supported by the late Harry Lonsdale for several years. Lonsdale's reviewers cited lack of results in 2014, one noting: "[E]ven if apparently recombined sequences are detected, it will be difficult or impossible to trace their genesis."

And regarding your mention of a so-called "autocatalytic ribozyme" in your NYAS paper with reference to Lehman's research - Nobelist Jack Szostak has told me this about autocatalytic sets:

"Autocatalytic sets is one of those concepts where the people who came up with the original idea, like Stuart Kauffman, rather than admit being wrong kept changing their story until it was basically the same concept everybody was already working on.

The original idea was that there would be large numbers of compounds where one would help another to replicate, and that one could help some other one to replicate, and that somehow out of this huge population of interacting molecules autocatalytic replication would emerge.

In my opinion that was never chemically realistic. Now you see people talking about non-enzymatic RNA replication and calling that autocatalytic sets. If that's what you want to call it, that's fine. But it seems like the concept has lost all meaning."

Luis Villarreal: I've had some conversations with chemists along these lines, which gets into a distinct domain of thinking. They drift into more complicated catalytic outcomes where you just don't have a linear relationship of A to B to C. A to B to C is typically how chemistry is taught. To deny the existence of the nonlinear is not an effective way of thinking about things.

Suzan Mazur: Vaidya references Kauffman's concept.

Luis Villarreal: That may be, but my point is more fundamental. In order to create a system that has participants that interact and can form an identity as a group, it has to have these characteristics I've described or outlined in the paper that you and I have been discussing, the NYAS "gangen paper," which is consistent with a lot of these attempts to get catalytic RNA to function in a population.

Suzan Mazur: So you're saying more traditionally-trained chemists just don't get it, they want to see the molecules.

Luis Villarreal. Yes, I would say that. In fact, you might want to talk to Vera Kolb at the University of Wisconsin. She's a professor of organic chemist and is interested in origin of life issues and has been active in astrobiology. Vera was trained along traditional lines but thinks in more complex ways with respect to how origin of life occurred.

Suzan Mazur: Nick Hud, a Georgia Tech chemist who is trying to find the simplest molecules needed to start life -- a precursor to RNA -- says he's got "a molecular assembly that is perhaps one bond away from making it a polymer - a covalent polymer." Hud told me, "[There is a long history of people proposing autocatalytic cycles and thinking that such cycles are central to starting life, but then not getting much to work in the lab."

Luis Villarreal: It' a difficult transition to the nonlinear because it's not how we traditionally do these kind of experiments.

The cooperative stem-loop RNA business has had some successes experimentally. Some of those observations I incorporated in the NYAS paper.

Suzan Mazur: Corrado Spadafora, who's found evidence of a germline crossing of the Weismann barrier in his experiments, now questions whether a Weismann barrier even exists. He supports your thinking, telling me this:

"([E]ndogenous) retroviruses have contributed to shape our genome, are constitutive components of it and are currently playing crucial roles in fundamental processes such as embryogenesis, tumorigenesis and, in the long run, evolution."

And František Baluška at the University of Bonn's Institute of Cellular & Molecular Botany has noted:

"Infection, especially the viral one, but also bacterial and fungal infections, followed by symbiosis, is proposed to act as the major force that drives biological evolution toward higher complexity."

Baluška told me viruses "manipulate adjacent cells to form cell-cell adhesions."

Animal biologists bristle at the idea of cell-cell adhesion linked to viral infection and viruses, one animal biologist cynically describing them to me as "intelligent viruses."

Luis Villarreal: When people use terms like "intelligent viruses" -- it is one thing to have a competence, code competence, communication competence that viruses essentially must have in order to function, but if that transitions into a concept of intelligence, that becomes problematic for me.

Suzan Mazur: Do viruses have an effect at only the microscale or is their effect apparent at the mesoscale and even macroscale?

Luis Villarreal: Well, what is the effect of the Ebola outbreak on the human culture and human condition? Ebola is a specific example to think about because the bats have acquired a very unlikely bit of information from Ebola itself, and they did so with the help of another virus, probably an endogenous retrovirus, because it turned that RNA into a DNA and deposited it into the chromosome -- which is what had to have happened. It's not something that a virus can do by itself. It has to have cooperated in a rather complicated way. But my point is that as a consequence, the adaptions that must occur are at all these different levels -- in the context of the immune system, in the context of the culture, in the context of the behavior -- all of this is affected by these events.

Suzan Mazur: But in terms of form. Spadafora mentions embryogenesis and you make some reference in a previous paper to the embryo.

Luis Villarreal: The systems biologists have immersed themselves in this issue quite a bit because they are characterizing all the transcripts that have occurred in the early stages of development -- 1, 2 cell stages. If you just look at the math, at the amount that is being expressed or transcribed without consigning meaning to it, you see that on a very large scale these parasitic sequences, these endogenous viruses, virons and all of these retroposons are very highly expressed at these points of development and commitment. Some of these are entities that were acquired in a lineage specific way so that you see them in primates or you see them in chimps or you see them in humans. You don't see them necessarily in sister species. So it's clear that some editing events mediated by infectious agents have occurred at the very earliest stages of commitment and differentiation. They are occurring on a significant scale. These are not minor numbers. They're quite abundant.

Suzan Mazur: So it affects morphology.

Luis Villareal: Absolutely, yes. But in ways we are just beginning to tease apart. We don't really understand how, for example, the large mass of non-coding RNAs is doing what it's doing.

Suzan Mazur: If your gangen hypothesis is correct, which is that gangs, i.e., "RNA collective ensembles coupled with the emergence of endonuclease, ligase, cooperative catalysis, group identity and history markers (RNA) are needed for life to emerge" -- what happens to the current thinking?

Luis Villarreal: It means that sphere of logic is not sufficient to understand this collective. Because if you think about how the collective must operate, in fact, it becomes even a problem of language.

You can always trace a linear link between any two participants or any three or four or five participants in a collective. But if it has a conditional character to it. . . If you think of alu retroposon RNA in the human brain and it has a particular activity or function and a particular neuron, and it's doing a specific activity of connecting to some other cell, but then you see in another state, say during learning and memory where it is doing something different and can actually oppose the activity that you saw in the first state -- this means an individual entity has multiple activities. And that's the character of a consortia, which means you now have to start thinking in terms of how the population works and not by just these linear nodes that represent the end reactions. Because they have a conditionality associated with them.

Suzan Mazur: You've noted that during evolution 3.3 gigabases of DNA, which is the current size of our genome, was once retrovirus, that there are about 330,000 solo long terminal repeats of retroviruses now in human DNA. You've also said these long terminal repeats (LTRs) "contributed to reregulating about 1,500 genes needed for the placenta to emerge." In terms of percentages how much of today's human genome is virus and how are you able to measure this?

Luis Villarreal: The definition of what is virus is a little bit fuzzy. The LTRs are clearly identified and categorized so that they themselves don't present too much of a problem as having originated from a viral event. The 330,000 number is one anybody can substantiate by going to the primary sequence and with a recognition algorithm can pull out the LTRs. The problem is the collection of this data goes through a process of masking. This stuff is considered of no interest, or as junk to most people who are doing genomics. The actual collection of the primary data tends to be masked, so you don't even see it. So in order for you to look at this stuff and get a sense of it, you, yourself, have to go directly to the primary sequence data -- which is all this compiled, complicated stuff and apply analyses that can deal with the junk sequence. It's a conundrum just from an experimental perspective.

Suzan Mazur: Is it possible to say what percentage of the human genome is virus?

Luis Villarreal: Depending on how you word that -- for example, in terms of retroviruses and LTRs, the number is about 10%.

Suzan Mazur: Same as the percentage of virus in a mouse.

Luis Villarreal: But it's a different 10% in a mouse. It doesn't overlap that well with the human 10%. In the mouse you have significantly greater numbers of more intact endogenous retroviruses. So when you breed mice these endogenous retroviruses tend to become active and transmissible. When they first started breeding BalbC mice, within that first year the mice began to develop tumors and die in the breeding colonies. So mice are a little different than humans in that respect. Mice are colonized by viruses that are much closer to being able to make transmissible endogenous viruses.

Suzan Mazur: In my conversation with Ricardo Flores, he told me that a viroid-like entity is the most likely candidate as the first replicon on Earth, rather than virus because viroids don't need protein but viruses do. He said Theodor Diener who discovered viroids first proposed the idea.

Luis Villarreal: Right.

Suzan Mazur: Flores also told me that virus and viroid are not related, they have separate evolutions. From your paper, I get the sense that you don't agree.

Luis Villarreal: I've had this conversation with him. Carlos Briones, a colleague of Flores, has made the more fundamental argument that it's really the stem-loop structure -- and that's related to my NYAS gangen paper -- that is the functional entity even within a viroid. If look at these stem-loop structures, you see that they're crucial and central to the identity and function of all RNA viruses. And if you look at the simplest of all RNA viruses, it's basically one sequence of these complicated, interacting stem loops.

Even though I don't disagree for the most part with what Flores said about RNA viruses and viroids having distinct lineages -- it's not absolutely true because we do have an example of the hepatitis situation where you have a viroid that encodes a capsid protein that does behave like a viroid but it also behaves like a virus. So we can see examples of entities that cross those two domains.

Suzan Mazur: So you do think that a viroid-like entity is the most likely candidate for the earliest replicon on Earth. But did it arise on Earth or did it come to Earth via comet? What are your thoughts?

Luis Villarreal: The viroid-like entity seems more basal. What you're really talking about is an infectious, transmissible nucleic acid that doesn't necessarily encode something. But whether it arose on Earth or came via comet -- at that level I'm not sure you can conclude one way or another whether a seed of our RNA came on a comet because you do see chemistries occur on these comets -- this is something you might want to ask Vera Kolb at the University of Wisconsin -- that might be indicative of that ability to put together precursor molecules to make this consortia.

Suzan Mazur: You conclude your NYAS paper by saying that the stem-loop RNA consortium became a resident of DNA, it created a stable habitat. When you say stable, you mean there is creative and destructive action going on simultaneously?

Luis Villarreal: What I'm asserting is the fundamental importance of the ability of entities to persist not just to replicate. For me persistence is a big deal. The persistence of Ebola in a bat, for instance, is not an accident. It's a deep evolutionary relationship associated with the origin of bats. We often dismiss that by saying: Oh, that's just the reservoir species -- as if that relationship happens willy-nilly all the time. It's a much more fundamental event when an entity figures out how to persist and permanently figures out how to colonize its host.

Suzan Mazur: You're saying the persistence has to do with toxic versus antitoxic forces.

Luis Villarreal: Yes, but in the case of Ebola, for instance, it is the ability of the virus to kill. That's pretty toxic. But it doesn't kill bats, it kills other populations. That relationship is the one that creates group behavior.

Suzan Mazur: But the toxic and antitoxic co-exist.

Luis Villarreal: They are inseparable. Linear logic fails here. A virus itself will spit out entities that regulate themselves and are subfunctional. They're considered defective, junk by themselves. That's what we see occupying the genomes of eukaryotes -- bits and pieces of regulatory viral chunks like the LTRs. It's also the stuff that seems to be orchestrating, for example, how a placenta works.

Suzan Mazur: Do we need a paradigm shift? How do you suggest moving forward in evolutionary science?

Luis Villarreal: That's a much more difficult question for me now than you might expect, because it has to do with how we even think and communicate. If living systems work by these processes that are consortial and complex, then our very language and logic is a problem in terms of how we apply it to understand what's going on. I don't have a solution.

In a sense our brains are entities adapted to complex thinking. When we think socially, we have this immediate gestalt reaction to a circumstance. All kinds of data are being dealt with, some of it contradictory. We end up with a sense or understanding of what we're experiencing. But in formal thinking and logic, that's not how we operate. We use -- it's a system 1 and a system 2 brain, basically.
System 1 behaves by this consortia of information and has both positive and negative, contradictory views that it uses quite comfortably. System 2 behaves linearly and logically.

Suzan Mazur: It seems European scientists have accepted these ideas more than American scientists.

Luis Villarreal: I agree with that. I think the consortia concept is also built socially into the Japanese culture and society, and Japanese scientists may be increasingly interested in the research.

Politics is inherent in human behavior because humans have group identity that's based on cognitive input, what we believe. Information we accept. It's built into how our brains function. Negative reactions to a belief basically confront group identity that the person has.

Suzan Mazur: Have you had dialogue with scientists synthesizing life in the lab?

Luis Villarreal: No. But I do look at their literature and it's apparent they're excluding viruses.

Suzan Mazur: Freeman Dyson spoke about parasites and origin of life in my interview with him.

Luis Villarreal: I've read a lot of the earlier stuff that Freeman Dyson wrote. It seems quite congruent with how I think about things.

Suzan Mazur: It's disappointing if we've got decades of compelling evidence about viruses and evolution, why it's not being responsibly considered by the scientific establishment, particularly in America.

Luis Villarreal: These shifts usually take generations. How can evolutionary science not be talking about viruses when genomes are so heavily colonized by these entities? It's because scientists are human beings, and it's an inherent tendency of human cognition to defend what beliefs it has come to accept whether acquired by learning neo-Darwinian thinking or acquired by other social or cultural practices.

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