Background: Despite progress in the development of combined antiretroviral therapies (cART), HIV infection\r\nremains a significant challenge for human health. Current problems of cART include multi-drug-resistant virus\r\nvariants, long-term toxicity and enormous treatment costs. Therefore, the identification of novel effective drugs is\r\nurgently needed.\r\nMethods: We developed a straightforward screening approach for simultaneously evaluating the sensitivity of\r\nmultiple HIV gag-pol mutants to antiviral drugs in one assay. Our technique is based on multi-colour lentiviral\r\nself-inactivating (SIN) LeGO vector technology.\r\nResults: We demonstrated the successful use of this approach for screening compounds against up to four HIV\r\ngag-pol variants (wild-type and three mutants) simultaneously. Importantly, the technique was adapted to Biosafety\r\nLevel 1 conditions by utilising ecotropic pseudotypes. This allowed upscaling to a large-scale screening protocol\r\nexploited by pharmaceutical companies in a successful proof-of-concept experiment.\r\nConclusions: The technology developed here facilitates fast screening for anti-HIV activity of individual agents from\r\nlarge compound libraries. Although drugs targeting gag-pol variants were used here, our approach permits\r\nscreening compounds that target several different, key cellular and viral functions of the HIV life-cycle. The modular\r\nprinciple of the method also allows the easy exchange of various mutations in HIV sequences. In conclusion, the\r\nmethodology presented here provides a valuable new approach for the identification of novel anti-HIV drugs.
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