Nucleoside analogs have broad clinical utility as antiviral drugs. Key to their systemic distribution and cellular entry are human nucleoside transporters. Here, we establish that the human concentrative nucleoside transporter 3 (CNT3) interacts with antiviral drugs used in the treatment of coronavirus infections. We report high-resolution single-particle cryo-electron microscopy structures of bovine CNT3 complexed with antiviral nucleosides N⁴-hydroxycytidine, PSI-6206, GS-441524 and ribavirin, all in inward-facing states. Notably, we found that the orally bioavailable antiviral molnupiravir arrests CNT3 in four distinct conformations, allowing us to capture cryo-electron microscopy structures of drug-loaded outward-facing and drug-loaded intermediate states. Our studies uncover the conformational trajectory of CNT3 during membrane transport of a nucleoside analog antiviral drug, yield new insights into the role of interactions between the transport and the scaffold domains in elevator-like domain movements during drug translocation, and provide insights into the design of nucleoside analog antiviral prodrugs with improved oral bioavailability.

核苷类似物作为抗病毒药物具有广泛的临床用途。它们的全身分布和细胞进入的关键是人类核苷转运蛋白。在这里，我们确定人类浓缩核苷转运蛋白 3 (CNT3) 与用于治疗冠状病毒感染的抗病毒药物相互作用。我们报告了牛 CNT3 与抗病毒核苷N ⁴ -羟基胞苷、PSI-6206、GS-441524 和利巴韦林复合的高分辨率单粒子冷冻电子显微镜结构，全部处于向内状态。值得注意的是，我们发现口服生物可利用的抗病毒药物 molnupiravir 将 CNT3 阻滞在四种不同的构象中，使我们能够捕获载药的外向态和载药的中间态的冷冻电子显微镜结构。我们的研究揭示了核苷类似物抗病毒药物膜转运过程中 CNT3 的构象轨迹，对药物易位期间转运域和支架结构域之间的相互作用在药物易位期间类似电梯结构域运动中的作用产生了新的见解，并为设计核苷类似物抗病毒前药，具有改善的口服生物利用度。