Abstract
Cholesterol 25-hydroxylase (CH25H) is an interferon (IFN)-stimulated gene that shows broad antiviral activities against a wide range of enveloped viruses. Here, using an IFN-stimulated gene screen against vesicular stomatitis virus (VSV)-SARS-CoV and VSV-SARS-CoV-2 chimeric viruses, we identified CH25H and its enzymatic product 25-hydroxycholesterol (25HC) as potent inhibitors of SARS-CoV-2 replication. Internalized 25HC accumulates in the late endosomes and potentially restricts SARS-CoV-2 spike protein catalyzed membrane fusion via blockade of cholesterol export. Our results highlight one of the possible antiviral mechanisms of 25HC and provide the molecular basis for its therapeutic development.
Highlights
Cholesterol 25-hydroxylase (CH25H) is an interferon (IFN)-stimulated gene that shows broad antiviral activities against a wide range of enveloped viruses
To identify potential IFNstimulated genes (ISGs) effector proteins that act to block coronavirus (CoV) at the entry or egress stages of the replication cycle, we utilized replication-competent chimeric vesicular stomatitis virus (VSV) eGFP reporter viruses decorated with either fulllength SARS-CoV spike (S) protein or SARS-CoV-2 S in place of the native glycoprotein (G) [14]
We examined a series of early events and excluded possible effects of 25HC on: 1) ACE2 surface levels; 2) S cleavage by TMPRSS2; 3) lipid raft localization, stained by a fluorophoreconjugated cholera toxin subunit B; 4) plasma membrane fluidity, stained by 6-dodecanoyl-2-dimethylamino naphthalene (Laurdan)
Summary
(37); 5) endosomal pH; and 6) its ability to directly bind to recombinant SARS-CoV-2 S protein (SI Appendix, Fig. S4 C and D). Statistical significance is from pooled data of the multiple independent experiments (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001). We hypothesized that SARS-CoV-2 depends on endosomal trafficking to establish active replication Consistent with this hypothesis, ectopic expression of Rab and Rab dominant-negative mutants but not the wild-type proteins significantly decreased VSV-SARS-CoV-2 infection (Fig. 4B and SI Appendix, Fig. S4G). The antiviral activity of 25HC and ICZ depended on cholesterol accumulation in the endosomal/lysosomal compartment because the inhibition was markedly diminished in serum-free media that is devoid of cholesterol (SI Appendix, Fig. S5C).
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