Hydroxychloroquine blocks SARS-CoV-2 entry into the endocytic pathway in mammalian cell culture

Abstract

Hydroxychloroquine (HCQ), a drug used to treat lupus and malaria, was proposed as a treatment for SARS-coronavirus-2 (SARS-CoV-2) infection, albeit with controversy. In vitro, HCQ effectively inhibits viral entry, but its use in the clinic has been hampered by conflicting results. A better understanding of HCQ’s mechanism of actions in vitro is needed. Recently, anesthetics were shown to disrupt ordered clusters of monosialotetrahexosylganglioside1 (GM1) lipid. These same lipid clusters recruit the SARS-CoV-2 surface receptor angiotensin converting enzyme 2 (ACE2) to endocytic lipids, away from phosphatidylinositol 4,5 bisphosphate (PIP 2 ) clusters. Here we employed super-resolution imaging of cultured mammalian cells (VeroE6, A549, H1793, and HEK293T) to show HCQ directly perturbs clustering of ACE2 receptor with both endocytic lipids and PIP 2 clusters. In elevated (high) cholesterol, HCQ moves ACE2 nanoscopic distances away from endocytic lipids. In cells with resting (low) cholesterol, ACE2 primarily associates with PIP 2 clusters, and HCQ moves ACE2 away from PIP 2 clusters—erythromycin has a similar effect. We conclude HCQ inhibits viral entry through two distinct mechanisms in high and low tissue cholesterol and does so prior to inhibiting cathepsin-L. HCQ clinical trials and animal studies will need to account for tissue cholesterol levels when evaluating dosing and efficacy.

Regulation of neuroinflammation by astrocyte-derived cholesterol

“…We previously showed the cholesterol released from astrocytes requires apoE 17 . We have also showed that purified apoE3 and 10% fetal bovine serum (FBS, an external source of cholesterol) can recapitulate the effects of high extracellular cholesterol 17,21,22 . To confirm microglia activation by apoE-mediated cholesterol uptake, we cultured HMC3 cells with apoE3 with/without FBS and measured TNFα production using an enzymelinked immunosorbent assay (ELISA) for TNFα.…”

“…And studies on cultured cells will need to consider high and low cholesterol environments. In order to recapitulate a disease state, cholesterol will likely need to be elevated 21,22 . Lastly, if the cytokines generated from microglia have access to the same astrocytes that produce their cholesterol, the process would be autocatalytic and produce a chain reaction that would escalate neuroinflammation.…”

Cholesterol Regulation of Membrane Proteins Revealed by Two-Color Super-Resolution Imaging

“…Elevated cholesterol is a key cellular component important to many metabolic and neurological diseases, including Alzheimer’s, cardio-vasculature, diabetes, viral infections, and chronic inflammation [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 ]. A gap in understanding exists for how cholesterol affects many diverse biological functions.…”

“…Phosphatidylinositol 4,5 bisphosphate (PIP 2 ) is also a membrane-signaling lipid [ 14 , 15 , 16 , 17 , 18 ], and forms charged clusters separate from GM1-containing clusters [ 19 , 20 , 21 , 22 , 23 ] ( Figure 1 A). The distance between GM1 and PIP 2 clusters ranges from 42 nm in muscle cells [ 22 ] to ~200 nm in lung and kidney cells [ 7 ]. PIP 2 clusters are largely insensitive to cholesterol [ 19 , 20 , 22 , 23 ].…”

“…PIP 2 also dramatically regulates protein function through protein–lipid interactions, including gating ion channels and trafficking membrane proteins [ 13 , 14 , 15 , 16 , 17 , 35 , 36 , 37 , 38 , 39 ]. Here we use these biophysical properties and recent findings from super-resolution imaging [ 7 , 22 , 40 , 41 , 42 , 43 ] to establish basic models for proteins moving nanoscopic distances in the membrane. We define cholesterol-regulated proteins (CRPs) and their activation mechanism by CRP localization.…”