Fares Perla, Duhaini Mariam, Tripathy Suvranta K, Srour Ali, Kondapalli Kalyan C
Department of Natural Sciences, University of Michigan-Dearborn, Dearborn, Michigan, USA.
Department of Natural Sciences, University of Michigan-Dearborn, Dearborn, Michigan, USA.
J Biol Chem. 2025 Feb;301(2):108144. doi: 10.1016/j.jbc.2024.108144. Epub 2024 Dec 26.
Endocytosis is a prominent mechanism for SARS-CoV-2 entry into host cells. Upon internalization into early endosomes (EEs), the virus is transported to late endosomes (LEs), where acidic conditions facilitate spike protein processing and viral genome release. Dynein and kinesin motors drive EE transport along microtubules; dynein moves EEs to the perinuclear region, while kinesins direct them towards the plasma membrane, creating a tug-of-war over the direction of transport. Here, we identify that the luminal pH of EEs is a key factor regulating the outcome of this tug-of-war. Among the known endosomal pH regulators, only the sodium-proton exchanger NHE9 has so far been genetically linked to severe COVID-19 risk. NHE9 functions as a proton leak pathway specifically on endosomes. We show that limiting acidification of EEs by increasing the expression of NHE9 leads to decreased infectivity of the SARS-CoV-2 spike-bearing virus in host cells. Our investigation identified the EE membrane lipid phosphatidylinositol-3-phosphate (PI3P) as a link between luminal pH changes and EE transport. Normally, as EEs mature, PI3P depletes. However, in cells with high NHE9 expression, PI3P persists longer on EEs. PI3P plays a pivotal role in the recruitment of motor proteins and the subsequent movement of EEs. Consistently, we observed that NHE9-mediated alkalization of EEs hindered perinuclear movement. Specifically, EE speed and run length were negatively impacted, ultimately leading to EEs falling off microtubules and impairing the delivery of viral cargo to LEs. NHE9 thus offers a unique opportunity as a viable therapeutic target to impede SARS-CoV-2 host cell entry.
内吞作用是严重急性呼吸综合征冠状病毒2（SARS-CoV-2）进入宿主细胞的主要机制。病毒内化进入早期内体（EEs）后，被转运至晚期内体（LEs），在晚期内体中，酸性条件促进刺突蛋白加工和病毒基因组释放。动力蛋白和驱动蛋白沿着微管驱动早期内体运输；动力蛋白将早期内体移向核周区域，而驱动蛋白则将它们导向质膜，从而在运输方向上形成了一场拉锯战。在这里，我们发现早期内体的腔内pH值是调节这场拉锯战结果的关键因素。在已知的内体pH调节剂中，到目前为止，只有钠-质子交换体NHE9在基因上与严重的2019冠状病毒病风险相关。NHE9在内体上特异性地作为质子泄漏途径发挥作用。我们发现，通过增加NHE9的表达来限制早期内体的酸化，会导致携带SARS-CoV-2刺突蛋白的病毒在宿主细胞中的感染性降低。我们的研究确定早期内体膜脂质磷脂酰肌醇-3-磷酸（PI3P）是腔内pH变化与早期内体运输之间的联系。通常情况下，随着早期内体成熟，PI3P会耗尽。然而，在高表达NHE9的细胞中，PI3P在早期内体上持续存在的时间更长。PI3P在驱动蛋白的募集以及随后早期内体的移动中起关键作用。一致地，我们观察到NHE9介导的早期内体碱化阻碍了核周移动。具体而言，早期内体的速度和运行长度受到负面影响，最终导致早期内体从微管上脱落，并损害病毒货物向晚期内体的递送。因此，NHE9作为一个可行的治疗靶点，为阻止SARS-CoV-2进入宿主细胞提供了独特的机会。
