Nucleoside analogue reverse transcriptase (RT) inhibitors (NRTIs) are major antiviral agents against hepatitis B virus (HBV) and human immunodeficiency virus type-1 (HIV-1). However, the notorious insoluble property of HBV RT has prevented atomic-resolution structural studies and rational anti-HBV drug design. Here, we created HIV-1 RT mutants containing HBV-mimicking sextuple or septuple amino acid substitutions at the nucleoside-binding site (N-site) and verified that these mutants retained the RT activity. The most active RT mutant, HIV-1 RT7MC, carrying Q151M/G112S/D113A/Y115F/F116Y/F160L/1159L was successfully crystallized, and its three-dimensional structure was determined in complex with DNA:dGTP/entecavir-triphosphate (ETV-TP), a potent anti-HBV guanosine analogue RT inhibitor, at a resolution of 2.43 angstrom and 2.60 angstrom, respectively. The structures reveal significant positional rearrangements of the amino acid side-chains at the N-site, elucidating the mechanism underlying the differential susceptibility of HIV-1 and HBV against recently reported 4'-modified NRTIs. (C) 2019 Elsevier Inc. All rights reserved.