CH25H,
IFIH1,
IFITM1,
IFITM2,
IFITM3, and
LY6E were cloned into pLX304 lentiviral vector with a C-terminal V5 tag. Human ACE2 was cloned into pDEST-mCherry vector with an N-terminal mCherry tag. TMPRSS2 and TMPRSS4 plasmids were used as previously described (18 (
link)). Single-guide RNA against
CH25H or
NPC1 (
SI Appendix, Table S1) was cloned into
lenti-CRISPR_v2 vector (Addgene, #52961).
CH25H point mutations were introduced by
QuikChange II site-directed mutagenesis (Agilent, #200524) using primers in
SI Appendix, Table S1. GFP-tagged Rab5 and Rab7 constructs were used as reported previously (64 (
link)). Codon-optimized SARS-CoV-2 S was a kind gift from Nevan Krogan at the University of California, San Francisco, CA (65 (
link)). pCAGGS-SARS-CoV S was a kind gift of Paul Bates at the University of Pennsylvania, Philadelphia, PA (66 (
link)). pMIG-WEEV-IRES-GFP plasmid was generated by Zhuoming Liu in the S.P.J.W. laboratory at the Washington University School of Medicine in St. Louis, St. Louis, MO. PM-GFP and VSV-G plasmids were obtained from Addgene (#21213 and #12259, respectively). pCAGGS-FAST-p10 from pteropine orthoreovirus was generated in the Kobayashi laboratory at the Osaka University, Japan (67 (
link)).
pEGFP-N1 and
pCMV-TdTomato were obtained from Clontech.
Zang R., Case J.B., Yutuc E., Ma X., Shen S., Gomez Castro M.F., Liu Z., Zeng Q., Zhao H., Son J., Rothlauf P.W., Kreutzberger A.J., Hou G., Zhang H., Bose S., Wang X., Vahey M.D., Mani K., Griffiths W.J., Kirchhausen T., Fremont D.H., Guo H., Diwan A., Wang Y., Diamond M.S., Whelan S.P, & Ding S. (2020). Cholesterol 25-hydroxylase suppresses SARS-CoV-2 replication by blocking membrane fusion. Proceedings of the National Academy of Sciences of the United States of America, 117(50), 32105-32113.
