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X-ray diffraction has led to a breakthrough in our understanding of an important aspect of the human immunodeficiency virus (HIV). The structural results lay bare a problem a solution to which had eluded scientists for more than two decades. It has the potential to one day produce better treatments for HIV/AIDS. Peter Cherepanov and Stephen Hare, Saumya Shree Gupta, Eugene Valkov of Imperial College London working with Alan Engelman at Harvard University, Massachusetts have studied crystals of the integrase enzyme found in retroviruses, including HIV. Integrase is critical to the biochemical cut and paste the virus uses to splice its genetic information into the host cells' DNA. Over the last twenty years or so, various research teams have tried and failed to work out the detailed three-dimensional structure of full-length integrase bound to viral DNA. Given that modern antiretroviral drugs for HIV work by blocking integrase, it is important that scientists learn more about how these small molecules function to deactivate viral replication. The new work, which was funded by the UK's Medical Research Council and the US National Institutes of Health, takes a big step towards a clearer understanding of the HIV enzyme. Despite its acute importance for antiretroviral drug discovery and decades of rigorous research, the complete structure of integrase, either as a separate protein or in the context of the functional intasome, is lacking," the researchers explain. "Accordingly, the structural organization of the enzyme active site, which is believed to adopt its functional state only after viral DNA binding, is unknown." Over a period of four years, the researchers carried out more than forty thousand trials, out of which they were able to grow just seven kinds of crystals of an integrase enzyme. Only one of these was of sufficient quality to allow determination of the three-dimensional structure. The team grew a crystal of a type of integrase borrowed from a little-known retrovirus called Prototype Foamy Virus (PFV). Based on their knowledge of PFV integrase and its function, they were confident that it was sufficiently similar to its counterpart in HIV to represent a valid model. Cherepanov explains just how intense was the process of crystallising and studying integrase: "It is a truly amazing story. When we started out, we knew that the project was very difficult, and that many tricks had already been tried and given up by others long ago. 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. Despite initially painstakingly slow progress and very many failed attempts, we did not give up and our effort was finally rewarded." The integrase crystals and crystals soaked in solutions of the integrase-inhibiting drugs Raltegravir (Isentress) and Elvitegravir were all analysed using the synchrotron at the Diamond Light Source in South Oxfordshire. The diffraction data represent the first observations of how these two antiretroviral drugs bind to and inactivate integrase. Intriguingly, the team found that the retroviral integrase has quite a different structure to that which had been predicted based on earlier research. Having the integrase structure to hand now means that researchers can begin to fully understand how existing drugs that inhibit integrase are working, how they might be improved, and how to stop HIV developing resistance to them.
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