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Authors:Ruochen Zang  et al. 

Journal/ Pre-Print:BioRxiv 

TagsAutophagy/Lysosome, Cell Biology, Molecular biology, Therapeutics, Virology 

Research Highlights 

  1. Overexpression of the interferon stimulated gene Cholesterol-25 hydroxylase (CH25H) inhibits SARS-CoV2 replication by production of 25-hydroxycholestreol (25HC) 

  1. 25HC inhibits membrane fusion initiated by the SARS-CoV2 spike protein 

  1. The FDA approved drug Itraconazole, which raises endosomal cholesterol levels, inhibits SARS-CoV2s replication in vitro 

Summary 

The authors overexpress 57 known interferon-stimulated genes (ISGs) in HEK293 cells expressing ACE2, and then infect with vesicular stomatitis virus (VSV) expressing the SARS-CoV2 spike proteinThey find that overexpression of Cholesterol 25-hydroxylase (CH25H), as well as its enzymatic product 25-hydroxycholesterol (25HC), reduce viral replication. These finding are then validated in fully infectious model of SARS-CoV2. By over expressing the SARS-CoV2 spike protein in 293 cells the authors show that CH25H expression and 25HC reduce cell-cell fusions. This suggests that 25HC may prevent SARS-CoV2 virions from fusing to cellular membranesFinally, Itraconazole an FDA approved drug - is shown to inhibit SARS-CoV2 replication in vitroThis demonstrates that cholesterol transport maybe an effective new drug target against SARS-CoV2 

Impact for SARS-CoV2/COVID19 research efforts  

Understand the virology and/or cell biology of SARS-CoV2/COVID19 

  • Highlights the need for Spike-protein membrane fusion (known) and how this may be blocked by the ISG CH25C (novel) – although provides no evidence of mechanistic details (just suggestions made). 

  • Sheds light on the role/importance of endosomal trafficking in SARS-CoV2 viral replication 

  • Uncertain if trafficking of cholesterol is directly or indirectly responsible for phenotype observed. I.e., does 25HC perturb a shared trafficking pathway or does cholesterol build-up itself have an antiviral effect? 

  • Emphasis on dual layered impact; blocking viral-host membrane fusion, and inhibition of replication through cholesterol accumulation 

Inhibition of SARS-CoV2/COVID19 transmission 

  • Block infection and replication using therapeutic dose of 25HC 

Treat of SARS-CoV2/COVID19 positive individuals 

  • Block infection/replication using therapeutic dose of 25HC 

  • CH25H expression & 25Htreatment did not induce type I or type III IFN expression, enhancing its therapeutic potential (less unwanted side effects) 

  • Itraconazole isan  FDA approved drug so it may prove efficacious in preventing SARS-CoV2 replication.  

Study Type  

  • In vitro study 

Strengths and limitations of the paper 

Novelty:  

  • Same group highlighted CH25H as one of the ISGs most highly induced by type I/III IFN in a SARS-CoV-2 primary human intestinal enteroid model 

  • 25HC was previously reported to have broad antiviral activity on both enveloped and non-enveloped viruses, but its role in SARS-CoV-2 infection is novel 

  • 10.1038/srep07242 

Standing in the field: 

  • The ISG screen identified molecules already known to restrict SARS-CoV2 replication, and some were investigated previously by other groups: 

  • GLIT 

 Appropriate statistics:  

  • Yes (exception: Fig. 4I) 

  • Student's t test 

  • Pairwise ANOVA 

  • EC50  Non-linear Regression 

  • Figure 4I: Testing of Itraconazole in fully replicative model, no statistics. 

Viral model used: 

Replication competent chimeric constructs of SARS-COV and SARS-CoV-2 and vesicular stomatitis virus (VSV-SARS-CoV) and (VSV-SARS-CoV-2). Fully replicative SARS-CoV-2 (2019-nCoV/USA-WA1/2020 strain) and an mNeonGreen SARS-CoV-2 reporter virus.  

Translatability: 

  • Very fundamental at this stage, lot more research required and mechanistic questions need answering 

  • However, itraconazole is an FDA approved drug, so it may prove efficacious in preventing SARS-CoV2 replication in clinical settings, while above questions are investigated 

Main limitations:  

  • A CH25C knock-out, or other loss-of-function model, is lacking. 

  • Location at which 25HC works remains unclear throughout paper. 

  • Details for mechanism by which 25HC blocks viral replication are limited. 

  • Almost all of the work is done in recombinant vesicular stomatitis virus (VSV) expressing the SARS-CoV2 envelope protein. This complicates findings as VSV is also restricted by CH25H. Despite this some small amount of data is presented on single round WT SARS-CoV2 infection. Unfortunately, only one of these panels is convincing due to missing controls or statistical tests.  

  • Find that serum free media prevents CH25H mediated inhibition of SARS-CoV2s replication but cannot explain why.