Next time you belt a beer or munch a slice of bread, consider this: You are in fact swallowing millions of molecules of a close chemical cousin to HIV, the AIDS virus. Those common comestibles contain yeast Saccharomyces cerevisiae which in turn deploys a gene called Ty and its protein, Ty.

Those initials stand for “transposon of yeast,” said molecular biologist and geneticist Jef Boeke, at the Johns Hopkins School of Medicine in Baltimore.

“We have seen very similar behavior of the HIV reverse transcriptase in vitro,” Boeke observed, “to the Ty1 reverse transcriptase in vitro.”

“HIV the human immunodeficiency virus is a family member of the retroviruses,” Boeke explained. “They’re RNA viruses that copy their genome to DNA, using the enzyme reverse transcriptase. Then the DNA copy goes into the genome of the host cell, and subsequently is transcribed to make progeny HIV particles. That’s how the AIDS virus multiplies. So-called retro-elements,” he continued, “include retroviruses, but also include a much larger world of other mobile elements that use reverse transcriptase to replicate. They’re found in everything from E. coli to elephants.

“Among the best characterized of those, called the retrotransposons,” Boeke went on, “is the Ty1 gene of Saccharomyces cerevisiae. It’s a DNA sequence 6 kilobases long, and it makes an RNA from one end to the other. This is very much the way HIV makes an RNA from its 10-kb DNA, from one end to the other. And then that Ty1-made RNA is translated into two proteins called gag and pol. These are just like HIV’s two most important genes, gag and pol, which are the nuts and bolts of its retroviral multiplication machinery.

“You can think of the Ty1 protein as a very stripped-down version of HIV,” Boeke proposed, “and retrotransposons, which we think predated retroviruses, are retrotransposons with an attitude.

“One distinction is that the Ty1 gene transposes meaning that it makes a new copy of itself in the same cell. It’s not infectious; it doesn’t go from cell A to cell B. HIV, of course, and retroviruses, are infectious; hence the distinction between retrotransposons and retroviruses. So basically, one of the main goals of my research here in this laboratory has been to study Ty1, and maybe learn something about nasty retroviruses.”

Manganese Manipulation A Clue To Drug Design

Boeke, who is also a professor of oncology at Hopkins, is senior author of a paper in the April 2002 issue of the journal Molecular Cell. Its lead author is molecular biologist Eric Bolton, a graduate student.

“Our finding is summarized in the paper’s title,” Boeke told BioWorld Today: “Inhibition of reverse transcription in vivo by elevated manganese ion concentration.”

“That means, essentially,” he explained, “by manipulating the concentration of manganese in the cytoplasm of cells, one can interfere with reverse transcription. If we could do this in HIV-infected cells,” he pointed out, “we might have a novel way to interfere with reverse transcription, and thereby curb HIV multiplication. So that’s the article’s big picture, its bottom line. Based on that, we’re extrapolating and predicting that this will be the case. And we’re currently pursuing that drug development possibility very actively in the laboratory.

“So over the years.” he recalled, “we’ve been hunting mutations in host genes that block the ability of Ty1 to move around by reverse transcription. We have a library of mutants of this type in the freezer, and Eric Bolton pulled one of them. It turned out that that mutation was in another yeast gene called PMR1. Its acronym stands for Plasma-membrane-related ATPase.’ PMR1 encodes a protein that’s located in the cytoplasm’s Golgi apparatus. One of the things that it does in that organelle is pump calcium and manganese out of the cytoplasm; they ultimately leave the cell.

“So in a really neat experiment,” Boeke recounted, “Eric got two different mutants of the PMR1 gene, one that was selectively crippled to interfere with manganese transport, and the other with calcium transport. Lo and behold, the one defective in calcium transport retrotransposed its Ty normally, as it does in the wild type, whereas the mutant, defective in manganese transport, behaved just like a knockout of the PMR1 gene, which also blocks transposition. This showed that the manganese levels are probably critical.

“When we titrated in tiny amounts of manganese, we saw a very drastic inhibition of the reverse transcriptase in vitro. And this really had never been documented before. We showed it also for the reverse transcriptase,” he said. “For HIV, one reverse transcriptase paralleled the effect we saw for Ty1’s reverse transcriptase. One of the great things about working with yeast is you can do the genetics of the host cells much more easily than you can with mammals.

“From which we conclude that PMR1’s human homologue might in principle be a good target for drug development,” Boeke suggested. “We’re working on that now, and we’ll see what happens. “That’s pretty much the sum total of our data.”

PMR1 In Crosshairs Of New Compounds

“So generically we are engineering yeast to hunt for compounds that might interfere with the human PMR1 protein,” he observed. “There are lots of ways that compounds can block transporters and channels by essentially interacting with the surfaces that these ions would normally pass through or bind. So in general, these are good targets for compounds, because there are lots of examples for channels and transporters that are interfered with by drugs.”

Boeke said, “HIV’s ability to adapt, and generate resistance to therapeutic drugs, means that current treatments like AZT, which target reverse transcriptase directly, generally stop working as time goes by. Finding new drugs or a new class of drugs is needed to help keep the AIDS virus at bay. Our new work suggests that targeting a cell’s manganese transporter could be an effective way to stop HIV from replicating, without targeting its reverse transcriptase directly.

“The yeast that was missing PMR1 appeared fine,” he noted, “which suggests that targeting the manganese transporter in humans may be relatively safe. Whether it will have therapeutic benefit is not yet known, but the mantra of HIV therapy,” Boeke concluded, “is to reduce the number of virus copies in the patient.”