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The carefully designed gene would act as a "Trojan horse" that clinicians would deliver via gene therapy to sabotage the virus within infected cells, says Charles Craik, Ph.D., professor of pharmaceutical chemistry at UCSF.

Craik leads a research team that tested the approach on HIV-infected cells growing in the laboratory. The researchers found that the AIDS virus, HIV, uses the faulty protein chain encoded by the gene to make a defective version of a much-needed enzyme called HIV protease.

Because of this sabotage, the enzyme does not perform its task, and the ability of the virus to make new, infectious virus particles is drastically reduced as a result, Craik and his colleagues report in the Oct. 1 issue of the Proceedings of the National Academy of Sciences.

"It may be possible to fight the spread of HIV within infected individuals by inserting a defective version of a gene encoding this crucial enzyme into vulnerable cells," Craik said. "The virus would then use this blueprint to its own detriment."

HIV protease normally cuts the raw material of HIV into distinct proteins needed to assemble new virus particles. The newest available class of HIV-fighting drugs, the protease inhibitors, are small molecules that obstruct the cutting part of the enzyme and prevent it from cutting out new virus parts.

"These drugs appear to have some efficacy in combination with earlier developed anti-viral therapies," Craik said. "But to further increase the effectiveness of AIDS treatment, trans-dominant inhibitors could also be used in combination with small-molecule HIV protease inhibitors," Craik suggested. Alternatively, several HIV enzymes could be targeted simultaneously through combined gene therapy with trans-dominant inhibitors, he said.

The new strategy can be used to target proteins made up of two or more parts, Craik explained. HIV protease itself consists of two protein parts, identical halves that pair to form the complete enzyme.

To implement their new anti-HIV strategy, the researchers insert a gene encoding a genetically engineered, defective version of the protease half into cells susceptible to HIV infection. Activation of this gene leads to production of the defective protein. When a real HIV protease half pairs with one of Craik's defective impostors, called "trans-dominant inhibitors," the result is a protease enzyme that does not do its job.

Statistically, if equal numbers of the normal and the trans-dominant inhibitor protein halves were available for protease formation, and if these halves paired off indiscriminately, a working version of the enzyme would be expected to result from one-quarter of these pairings. Only protease formed from two of the true HIV-derived protein halves would be expected to work normally.

But in an elegant protein-engineering feat, Craik's team succeeded in designing trans-dominant inhibitors that were 30 times more likely to pair off with a normal HIV protease half than with another trans-dominant inhibitor. As a result, even fewer "wild-type" HIV protease halves combined to form a working enzyme.

The fast-mutating HIV has been generating drug-resistant strains with alarming regularity, but because of the unique design of the trans-dominant inhibitors it is improbable that HIV will be able to mutate and change its shape in a way that would permit the virus to elude the clutches of a drug made using this strategy, according to Craik.

"It's extremely unlikely that HIV would become resistant to the defective gene," Craik said. "The protease gene of HIV has only very rarely been observed to mutate in the region encoding the protein surfaces that make up the interface between the paired protein halves. In contrast, outside this region, the protease gene has been observed to mutate quite easily.

"Attempts by HIV to escape from the trans-dominant inhibitors through mutation of the HIV protease gene would lead the virus to make protein chains that are shaped in ways that just won't function well, even when one of these HIV-encoded protein halves pairs up with an identical HIV-encoded twin," Craik said.

Craik describes use of the trans-dominant inhibitor as "a case in which one bad apple -- the genetically engineered, defective protein -- can spoil a whole bunch." The unavailability of working HIV protease that results causes the virus to make a multitude of incomplete progeny that are non-infectious and which do not reproduce.

Craik and colleagues, including Fiona McPhee, a postdoctoral fellow, and Andrew C. Good, a postdoctoral fellow in the laboratory of Irwin D. Kuntz, Ph.D., professor of pharmaceutical chemistry, used computer models to predict which changes would result in defective mutant protease halves that would prefer to attach to the wild-type protease half.

They selected the three best-looking inhibitors for the Trojan horse treatment of HIV-infected cells. All dramatically reduced the ability of HIV to infect cells growing in laboratory cultures, and all kept the virus from replicating in already infected cells.

CONTACT: University of California San Francisco Jeffrey Norris, 415/476-2557

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