A novel HIV-1 integrase mutation pattern, L74F V75I, which conferred resistance to first-generation integrase strand transfer inhibitors (INSTIs), was identified in a clinical case with virological failure under a raltegravir-based regimen. Addition of L74F V75I to N155H or G140S Q148H increased resistance levels to the second-generation INSTIs dolutegravir (Ͼ385-and 100-fold, respectively) and cabotegravir (153-and 197-fold, respectively). These findings are important for the development of an accurate system for interpretation of INSTI resistance and the rational design of next-generation INSTIs.KEYWORDS dolutegravir, drug resistance mechanisms, human immunodeficiency virus, integrase, integrase strand transfer inhibitor I ntegrase strand transfer inhibitors (INSTIs) constitute the latest class of available antiretroviral agents exhibiting potent antiretroviral effects in vitro and vivo (1-8). The first-generation INSTIs raltegravir (RAL) and elvitegravir (EVG) display broad crossresistance, whereas second-generation INSTIs of carbamoyl pyridine analogues, dolutegravir (DTG) and cabotegravir (CAB), demonstrate superior activity against firstgeneration INSTI-resistant HIV-1 variants (4, 9-13). Although a DTG resistance mutation, R263K, has been reported, and its resistance mechanism has been well studied (10,12,14,15), other potential DTG resistance mutations and their mechanisms are not fully understood.In a clinical case (Fig. 1A), the plasma HIV-1 RNA level rebounded to 2,900 copies/ml 1 year after starting antiretroviral therapy (ART) that included tenofovir disoproxil fumarate (TDF), emtricitabine (FTC), and RAL. However, no known INSTI resistance mutations were identified based on major drug resistance mutation lists (IAS-USA drug resistance mutations list [16] and the HIV Drug Resistance Database at Stanford University [HIVDB]) at time points 2 and 3 (Fig. 1B). Clinical samples were obtained from the fresh plasma of a patient attending the outpatient clinic of the National Hospital Organization Nagoya Medical Center. The Institutional Review Board approved this study , and written informed consent was obtained from this patient. To identify novel mutations associated with RAL resistance in the clinical isolates, we constructed infectious HIV-1 clones with cDNA fragments of the integrase (IN)-coding region derived from the clinical isolates and performed phenotypic resistance assays using TZM-bl cells as previously described (8, 17). Briefly, viral RNA was extracted from