Alpha-ketoamides as broad-spectrum inhibitors of coronavirus and enterovirus replication

Linlin Zhang; Daizong Lin; Yuri Kusov; Yong Nian; Qingjun Ma; Jiang Wang; Albrecht von Brunn; Pieter Leyssen; Kristina Lanko; Johan Neyts; Adriaan de Wilde; Eric J. Snijder; Hong Liu; Rolf Hilgenfeld

For enterovirus (except CVB3) infection experiments, human rhabdomyosarcoma cells (RD; for EV-A71; BRCR strain) and HeLa Rh cells (for EV-D68 and human rhinoviruses) were grown in MEM Rega 3 medium supplemented with 1% sodium bicarbonate, 1% Lglutamine, and fetal calf serum (10% in growth medium and 2% in maintenance medium). For HCoV-229E (a kind gift from Volker Thiel (Bern, Switzerland)), culture and infection experiments were carried out as described. 60 For MERS-CoV or SARS-CoV infection experiments, Vero, Vero E6, and Huh7 cells were cultured as described previously. 61, 62 Infection of Vero and Huh7 cells with MERS-CoV (strain EMC/2012) and SARS-CoV infection of Vero E6 cells (strain Frankfurt-1) at low multiplicity of infection (MOI) were done as described before. 61, 63 All work with live MERS-CoV and SARS-CoV was performed inside biosafety cabinets in biosafety level-3 facilities at Leiden University Medical Center, The Netherlands. Viral replicons. The DNA-launched SARS-CoV replicon harbouring Renilla luciferase as reporter directly downstream of the SARS-CoV replicase polyprotein-coding sequence (pp1a, pp1ab, Urbani strain, acc. AY278741), in the context of a bacterial artificial chromosome (BAC) under the control of the CMV promoter, has been described previously (pBAC-REP-RLuc). 33 Apart from the replicase polyprotein, the replicon encodes the following features: the 5'-and 3'-non-translated regions (NTR), a ribozyme (Rz), the bovine growth hormone sequence, and structural protein N.