Molecular cut and paste
A combination of cheap DNA synthesis, freely accessible databases, and our ever-expanding knowledge of protein science is conspiring to permit a revolution in creating powerful molecular tools, suggests William McEwan, Ph.D., a virologist at the MRC Laboratory of Molecular Biology, Cambridge, U.K., in this excerpt from the new book Future Science: Essays From The Cutting Edge, edited by Max Brockman.
This afternoon I received in the post a slim FedEx envelope containing four small vials of DNA. The DNA had been synthesized according to my instructions in under three weeks, at a cost of 39 U.S. cents per base pair (the rungs adenine-thymine or guanine-cytosine in the DNA ladder). The 10 micrograms I ordered are dried, flaky, and barely visible to the naked eye, yet once I have restored them in water and made an RNA copy of this template, they will encode a virus I have designed.
My virus will be self-replicating, but only in certain tissue-culture cells; it will cause any cell it infects to glow bright green and will serve as a research tool to help me answer questions concerning antiviral immunity. I have designed my virus out of parts—some standard and often used, some particular to this virus—using sequences that hail from bacteria, bacteriophages, jellyfish, and the common cold virus. By simply putting these parts together, I have infinitely increased their usefulness. What is extraordinary is that if I had done this experiment a mere eight years ago, it would have been a world first and unthinkable on a standard research grant. A combination of cheap DNA synthesis, freely accessible databases, and our ever expanding knowledge of protein science is conspiring to permit a revolution in creating powerful molecular tools.
Nature is already an expert in splicing together her existing repertoire to generate proteins with new functions. Her unit of operation is the protein domain, an evolutionarily independent protein structure that specializes in a particular task, such as an enzymatic activity or recognition of other proteins. We can trace the evolutionary descent of the protein domains by examining their sequences and grouping them into family trees. We find that over the eons of evolutionary time the DNA that encodes protein domains has been duplicated and combined in countless ways through rare genetic events, and that such shuffling is one of the main drivers of protein evolution.
The result is an array of single and multidomain proteins that make up an organism’s proteome. We can now view the protein domain as a functional module, which can be cut and pasted into new multidomain contexts while remaining able to perform the same task. This modular capability immediately lends itself to engineering: we don’t have to go about finding or artificially evolving a protein that performs our chosen task; we merely combine components that together are greater than the sum of their parts.
I’m interested in the defense mechanisms within cells — mechanisms that specifically recognize and disable intracellular pathogens. This type of defense is considered separate from the two main branches of immunity that are more intensely studied: the evolutionarily ancient “innate” immune system and the vertebrate-specific “adaptive” immune system. Innate immunity is the recognition of conserved features of pathogens—for example, the detection by specialized cells, such as macrophages, of the sugary capsule that surrounds many bacteria. Adaptive immunity works by fielding a huge diversity of immune recognition molecules, such as antibodies, and then producing large quantities of those that recognize nonself, pathogen-derived targets.
The newly discovered kind of immunity on which I work, sometimes termed “intrinsic immunity,” shares features with innate immunity but tends to be widely expressed, instead of residing just within “professional” immune cells, and is always “on.” In other words, every cell in an organism is primed and ready to disable an invading pathogen. The intrinsic immune system is at a strategic disadvantage, as its targets are often fast-evolving viruses that can rapidly mutate to evade recognition. Unlike the adaptive immune system, which can quickly generate a response to an almost infinite diversity of targets, the intrinsic immune system must rely on rare mutations and blind selection over evolutionary time to compete with its opponents.
So far, the study of the intrinsic immune system has been dominated by its interaction with retroviruses. The retroviruses, an ancient affliction of vertebrates, violate the central dogma of biology—that DNA makes RNA makes protein: they are RNA viruses able to generate DNA copies of themselves and insert this Trojan-horse code into the host’s genome. Almost one-tenth of the human genome is the defunct relic of this sort of infection.
Within the past 7 million to 12 million years, a comparatively recent member of the retrovirus family, the lentivirus, has emerged and spread slowly through the branches of the mammalian family tree. The oldest known traces of lentivirus have been found in the genome of rabbits, but current infections occur in horses, cats, ruminants, and primates. Lentiviruses arrived in humans in the form of HIV, as several cross-species transmission events from other primates. Only one of those viral transmissions— from chimpanzee to human, sometime in the late nineteenth or early twentieth century—has adapted to its new host in such a devastating manner, the virus being HIV-1 M-group, which causes AIDS and currently infects 33 million people worldwide.
One of the major players in intrinsic immunity is TRIM5, a four-domain protein that is expressed in virtually every cell in the human body. By virtue of one of its domains—the RING (which stands for Really Interesting New Gene) domain—TRIM5 has an enormously high turnover rate; that is, each of its molecules is degraded within about an hour of the cell’s having synthesized it. By virtue of another of its domains, it can recognize and engage retroviruses soon after their entry into the cell. As a result, incoming viruses can be degraded along with TRIM5 and thus made noninfective.
A classic arms-race situation has developed, wherein TRIM5 has tried to maintain its ability to recognize the rapidly evolving retroviruses, placing the gene under some of the strongest Darwinian selection in the entire primate genome. However, HIV-1 seems to have the upper hand at the moment: the human TRIM5 variant only marginally reduces the replication of HIV-1. Could this be one of the failures in human immunity that has permitted such a dramatic invasion by this pathogen? And what does human TRIM5 need to do in order to gain the upper hand? Or, to ask a bolder question, what can we do to it to engineer resistance to the disease?
One surprising answer is provided by protein-domain fusions in other primate species. A fascinating thing has happened in South American owl monkeys: TRIM5 has been fused with a small protein that HIV-1 depends on for optimal replication. The resulting fusion protein is called TRIMCyp and can reduce the replication of lentiviruses by orders of magnitude, essentially rendering the owl monkeys’ cells immune to the virus. Almost unbelievably (and it amazes me that espousers of Intelligent Design aren’t onto this), this feat of genomic plasticity has happened twice: versions of TRIMCyp have also been described in the unrelated macaque lineage. Since no wild populations of owl monkeys or macaques have been shown to harbor lentiviruses, it is difficult to say whether TRIMCyps have been selected specifically to combat lentiviruses, but there remains the intriguing possibility that TRIMCyp has helped lead to the current lentivirus-free status of one or both of these species.
So how can we benefit from gene fusions in other species? The first lesson is that by splicing together domains from seemingly unrelated proteins, unexpected and useful products can be generated. Researchers have already generated human TRIMCyps, which would avoid the immune-system rejection that introducing an owl monkey gene would produce. My colleagues and I have also engineered a feline TRIMCyp that prevents replication of the feline immunodeficiency virus in tissue-culture systems. However, in a clinical setting TRIMCyp must be expressed within cells to be useful as an antiviral, and the only effective means of achieving this is through gene therapy to alter the target cell’s genetic material. In a neat twist of roles, the best means we have of doing this is with a modified retroviral vehicle, or vector, to introduce a stretch of engineered DNA into the genome.
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