Acidic pH of early endosomes governs SARS-CoV-2 transport in host cells

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.