Transient receptor potential mucolipin 2 and 3 (TRPML2 and TRPML3), as key channels in the endosomal-lysosomal system, are associated with many different cellular processes, including ion release, membrane trafficking and autophagy. In particular, they can also facilitate viral entry into host cells and enhance viral infection. We previously identified that two selective TRPML agonists, ML-SA1 and SN-2, that showed antiviral activities against dengue virus type 2 (DENV2) and Zika virus (ZIKV) in vitro, but their antiviral mechanisms are still elusive. Here, we reported that ML-SA1 could inhibit DENV2 replication by downregulating the expression of both TRPML2 and TRPML3, while the other TRPML activator, SN-2, suppressed DENV2 infection by reducing only TRPML3 expression. Consistently, the channel activities of both TRPML2 and TRPML3 were also found to be associated with the antiviral activity of ML-SA1 on DENV2 and ZIKV, but SN-2 relied only on TRPML3 channel activity. Further mechanistic experiments revealed that ML-SA1 and SN-2 decreased the expression of the late endosomal marker Rab7, dependent on TRPML2 and TRPML3, indicating that these two compounds likely inhibit viral infection by promoting vesicular trafficking from late endosomes to lysosomes and then accelerating lysosomal degradation of the virus. As expected, neither ML-SA1 nor SN-2 inhibited herpes simplex virus type I (HSV-1), whose entry is independent of the endolysosomal network. Together, our work reveals the antiviral mechanisms of ML-SA1 and SN-2 in targeting TRPML channels, possibly leading to the discovery of new drug candidates to inhibit endocytosed viruses.

瞬时受体电位粘蛋白 2 和 3（TRPML2 和 TRPML3）作为内体-溶酶体系统中的关键通道，与许多不同的细胞过程相关，包括离子释放、膜运输和自噬。特别是，它们还可以促进病毒进入宿主细胞并增强病毒感染。我们之前确定了两种选择性 TRPML 激动剂 ML-SA1 和 SN-2，它们在体外对 2 型登革热病毒 (DENV2) 和寨卡病毒 (ZIKV) 显示出抗病毒活性，但它们的抗病毒机制仍然难以捉摸。在这里，我们报道了 ML-SA1 可以通过下调 TRPML2 和 TRPML3 的表达来抑制 DENV2 复制，而另一种 TRPML 激活剂 SN-2 通过仅降低 TRPML3 表达来抑制 DENV2 感染。一致地，还发现 TRPML2 和 TRPML3 的通道活性与 ML-SA1 对 DENV2 和 ZIKV 的抗病毒活性有关，但 SN-2 仅依赖于 TRPML3 通道活性。进一步的机制实验表明，ML-SA1 和 SN-2 降低了依赖于 TRPML2 和 TRPML3 的晚期内体标记物 Rab7 的表达，表明这两种化合物可能通过促进从晚期内体到溶酶体的囊泡运输然后加速溶酶体来抑制病毒感染。病毒的降解。正如预期的那样，ML-SA1 和 SN-2 均不抑制单纯疱疹病毒 I 型 (HSV-1)，其进入与内溶酶体网络无关。总之，我们的工作揭示了 ML-SA1 和 SN-2 在靶向 TRPML 通道中的抗病毒机制，可能导致发现抑制内吞病毒的新候选药物。