The lipid moiety of brincidofovir is required for in vitro antiviral activity against Ebola virus

•We established robust in vitro assays to screen compounds against Ebola.•We found brincidofovir to inhibit Ebola infection in multiple cell lines.•The lipid moiety of brincidofovir was associated with this antiviral activity.•The phosphorylation of cidofovir was not required for activity.

Brincidofovir (BCV) is the 3-hexadecyloxy-1-propanol (HDP) lipid conjugate of the acyclic nucleoside phosphonate cidofovir (CDV). BCV has established broad-spectrum activity against double-stranded DNA (dsDNA) viruses; however, its activity against RNA viruses has been less thoroughly evaluated. Here, we report that BCV inhibited infection of Ebola virus in multiple human cell lines. Unlike the mechanism of action for BCV against cytomegalovirus and other dsDNA viruses, phosphorylation of CDV to the diphosphate form appeared unnecessary. Instead, antiviral activity required the lipid moiety and in vitro activity against EBOV was observed for several HDP-nucleotide conjugates.