A team from Imperial College London and Harvard University report solving the 3-D crystal structure of a retroviral integrase enzyme bound with viral DNA. The work is published in Nature in a paper titled “Retroviral intasome assembly and inhibition of DNA strand transfer.”
Integrase is an essential retroviral enzyme that binds both termini of linear viral DNA and inserts them into a host cell chromosome, explains lead author, Peter Cherepanov, Ph.D., and colleagues. However, to date, the structure of full-length retroviral integrase, either separately or in complex with DNA, has been lacking. Moreover, the researchers add, while clinically useful inhibitors of HIV integrase have been developed, their mechanism of action remains speculative. The structural organization of the enzyme active site, which is believed to adopt its functional state only after viral DNA binding, is also unknown.
The four-year study by the researchers has now obtained diffracting crystals of the full-length integrase enzyme from the prototype foamy virus in complex with its cognate viral DNA. The structural data has shown that the retroviral intasome—the minimal functional complex involving viral DNA and integrase—comprises an integrase tetramer tightly associated with a pair of viral DNA ends. All three canonical integrase structural domains are involved in extensive protein–DNA and protein–protein interactions.
The binding of strand-transfer inhibitors essentially displaces the reactive viral DNA end from the active site, disarming the viral nucleoprotein complex, the scientists suggest. “Our findings will allow the generation of reliable HIV-1 integrase and integrase strand inhibitor pharmacophore models, which will be invaluable for the development of next-generation strand-transfer inhibitors.”
The project achieved results that had previously thwarted many other researchers, Dr. Cherepanov commented. The final success was only achieved following more than 40,000 trials, from which just seven crystals were grown, only one of which was of sufficient quality to allow determination of the 3-D structure. “We knew that the project was very difficult and that many tricks had already been tried and given up by others long ago,” he admits. “Therefore we went back to square one and started by looking for a better model of HIV integrase, which could be more amenable for crystallization.”