Selection FTW!
posted by saurabh in Biology, Health! |Next time someone asks you what studying evolution has ever done for humankind, you can tell them: “It cures AIDS, Biyotch!”
Whether that’s true is unclear at the moment, but still, this is insanely cool stuff. The Cre/lox system is a well-known tool in the molecular biologist’s napsack. It’s a very simple system that developed in the bacteriophage* P1. Many phage have the same basic strategy for replication that HIV does - integrate yourself into the host genome. The host then goes through repeated cycles of replication, copying the phage DNA in the process. At the right time, it expresses Cre recombinases, which recognize the two identical “loxP”† sites flanking the inserted phage genome, neatly excise the intervening viral DNA and stitch together the flanking sequences.
Previous work has suggested that Cre can be tweaked to recognize some slightly different DNA sequences. Some folks at the Max Planck Institute in Germany decided to push this to the limit. HIV-1, being a retrovirus, has a similar insertion strategy. In fact viral integration is the key problem with HIV - once the damn thing is in your genome, it’s hard to get it out. But if Cre could be altered so that it worked on HIV’s flanking sequences (called LTRs - long terminal repeats), then it could be used to recognize and excise HIV-1 sequence, killing it dead. Hey-presto!
So, they found sites within the HIV LTRs bearing some similarity (50%) to the 34-bp loxP sites. Unfortunately, Cre couldn’t recognize those sites at all. So they met it halfway, starting with some intermediate Cre COULD recognize (stealing their figure, here):
LoxLTR is the HIV-1 sequence they’re trying to recognize; the others are variants based on this sequence that share more identity with the original loxP site. By creating versions that Cre could at least partially recognize, they were able to select for better recognition, and gradually coax it towards recognizing the site of interest (involving 126 cycles of evolution in toto). In the end they had an enzyme they called Tre that was capable of excising HIV from the genome:
To examine whether Tre can excise the provirus from the genome of HIV-1–infected human cells, we produced loxLTR containing viral pseudotypes that were used to infect HeLa cells. A virus particle-releasing cell line was cloned and stably transfected, either with a plasmid expressing Tre or with the parental control vector. The respective cell pools were monitored with respect to recombinase activity and virus production. All assays performed demonstrated the efficient deletion of the provirus from the infected cells without obvious cytotoxic effects.
As they say in the biology labs: fucking hardcore.
Original paper, smoke if you got ‘em.
* Virus that preys on bacteria.
† lox = locus of crossover.
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maybe they could add it to yogurt
Saurabh, I wish you would science-blog more often.