Screening of Botanical Drugs against Lassa Virus Entry

Abstract

Lassa virus (LASV) belongs to the Old World Mammarenavirus genus (family Arenaviridae). At present, there are no approved drugs or vaccines specific for LASV. In this study, high-throughput screening of a botanical drug library was performed against LASV entry using a pseudotype virus bearing the LASV envelope glycoprotein complex (GPC). Two hit compounds, bergamottin and casticin, were identified as micromolar range inhibitors of LASV entry. A mechanistic study revealed that casticin inhibited LASV entry by blocking low pH-induced membrane fusion. Analysis of adaptive mutants demonstrated that the F446L mutation, located in the transmembrane domain of GP2, conferred resistance to casticin. Furthermore, casticin antiviral activity extends to the New World (NW) pathogenic mammarenaviruses, and mutation of the conserved F446 also conferred resistance to casticin in these viruses. Unlike casticin, bergamottin showed little effect on LASV GPC-mediated membrane fusion, instead inhibiting LASV entry by blocking endocytic trafficking. Notably, both compounds showed inhibitory effects on authentic lymphocytic choriomeningitis virus. Our study shows that both casticin and bergamottin are candidates for LASV therapy and that the conserved F446 in LASV GPC is important in drug resistance in mammarenaviruses. IMPORTANCE: Currently, there is no approved therapy to treat Lassa fever (LASF). Our goal was to identify potential candidate molecules for LASF therapy. Herein, we screened a botanical drug library and identified two compounds, casticin and bergamottin, that inhibited LASV entry via different mechanisms.

Screening of Botanical Drugs against SARS-CoV-2 Entry

“…The SARS-CoV-2 strain used in this study is a clinical strain of SARS-CoV-2 (nCoV-2019BetaCoV/Wuhan/WIV04/2019). The pseudotype VSV bearing the S protein of SARS-CoV-2 S (GenBank QHD43416.1) and MERS-CoV S (GenBankNC_019843.3) were generated as previously reported (41, 42). Briefly, 293T cells transfected with pcDNA3.1-SARS-CoV-2 S (ct19), pcDNA3.1-SARS-CoV-2 S (wt), or pcDNA3.1-MERS-CoV S (ct16) for 48 h were infected with pseudotype VSV (described below), in which the G gene was replaced with the luciferase gene, at an MOI of 0.1 for 2 h. The culture supernatants were harvested 24 h later, centrifuged to remove cell debris, and stored at −80°C.…”

Identification of residues in Lassa virus glycoprotein 1 involved in receptor switch

“…Anti-GP2 LASV antibodies were produced in our laboratory. (25, 26).…”

“…To quantify the efficiency of membrane fusion, a dual-luciferase reporter assay was performed as described previously (25)(26)(27). Briefly, 293T cells in a 24-well plate were co-transfected with plasmids expressing T7 RNA polymerase (pCAGT7) and pCAGGS-LASV GPC.…”

“…The copyright holder for this preprint this version posted May 1, 2021. ; https://doi.org/10.1101/2021.04.30.442226 doi: bioRxiv preprint Plasmid pVSVΔG-eGFP (Addgene plasmid #31842) was modified to pVSVΔG-Rluc to generate LASV GPC pseudotyped viruses (LASVpv). LASVpv were produced as described previously (25)(26)(27)(28). Briefly, 293T cells were transfected with the envelope LASV GPC-pCAGGS plasmid or its mutant derivatives using PEI.…”

Screening and Identification of Lassa Virus Endonuclease-targeting Inhibitors from a Fragment-based Drug Development Library

“…We have focused on developing entry inhibitors against LASV (11)(12)(13). Fragment-based drug discovery (FBDD) can identify early lead candidates for treatment, provide a backbone for drug optimization, and facilitate elucidation of the mechanism underlying the infection (14).…”