Geneart

SMT DNA sequences were codon-optimized for expression in E. coli and artificially synthesized by GeneArt (ThermoFisher Scientific) or through the Department of Energy Joint Genome Institute (DOE JGI) DNA Synthesis Science Program. SMT sequences from DOE JGI were obtained in the IPTG-inducible plasmid pSRKGm-lacUV5-rbs5^{70 (link)} in E. coli TOP10. SMT sequences from GeneArt were subcloned into pSRKGm-lacUV5-rbs5 by sequence and ligase independent cloning (SLIC)^{71 (link)} and transformed into electrocompetent E. coli DH10B (Invitrogen) using a MicroPulser Electroporator (BioRad). Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA). PCR was performed according the to the manufacturer’s protocol using Phusion DNA Polymerase (New England Biolabs). DNA fragments were purified using the GeneJET Gel Extraction Kit (ThermoFisher Scientific). Plasmid DNA was isolated using the GeneJET Plasmid Miniprep Kit (ThermoFisher Scientific). Plasmid DNA was sequenced to confirm promoter and SMT sequences by ELIM Biopharm (Hayward, CA) using the following primers: 5′-AATGCAGCTGGCACGACAGG-3′ (forward) and 5′-CCAGGGTTTTCCCAGTCAC-3′ (reverse).

E1E2 sequence of isolate H77, (Genbank accession no. AAB67037) was a gift from Dr Jean Dubuisson [34 (link)]. E1E2 sequences from the isolates UKN4.11.1, UKN5.15.7 and UKN6.5.340 (accession no. AY734986, EF427672 and AY736194 respectively) [35 (link),36 (link)] were synthesized by GeneArt (Invitrogen) and cloned into the pcDNA3.1 Zeo(+) vector (Invitrogen).
From plasma of local chronically infected patients, AMS E1E2 sequences were isolated using the Boom extraction method [37 (link)]. In brief, RNA was purified from stored plasma, cDNA was synthesized and the sequences of E1 and E2 were obtained (AMS.1b.2, AMS.2b.21, AMS.3a.26 and AMS.4d.8 see Genbank accession no. KR094962, KR094963, KR094964 and KR094965).
To determine which amino acids of E2 are crucial for antibody binding, the H77 E1E2 sequences were synthesized with single alanine mutations. 25 E2 mutants were generated either by GeneArt (Invitrogen), by using primers coding for the specific mutation (Biolegio) or by use of the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent). If the residue of interest was alanine, it was substituted by a glycine.
To generate HCVpp viruses 2 plasmids are needed next to a vector expressing the E1E2 sequences: the phCMV vector containing the MLV gag/pol and the phCMV packaging vector encoding the Luciferase gene (both are a gift from Dr Jean Dubuisson).

Full-length P. somniferum PsTyDC1 native coding sequence was synthesized by Integrated DNA Technologies (IDT, Coralville, IA, USA). Codon optimization of PsONCS3 and TfNCS nucleotide sequences^{60 (link)} for expression in E. coli was assisted by Codon Optimization OnLine (COOL)^{61 (link)}, resulting in the coding sequences shown in Supplementary Table 9, and the selected sequences were synthesized by IDT. The native sequence of full-length P. somniferum NMCH isoform 1 (PsNMCH-I1) was also synthesized by IDT.
Native coding sequences of full-length PsPDC1, full-length Ps2HCLL, and N-terminal truncated PsPDC2 were synthesized and cloned into pBAD-DEST49 (LifeSensors Inc., Malvern, PA, USA) via the Gateway cloning system by GeneArt (Invitrogen, Waltham, Massachusetts, USA). Native coding sequences of full-length EcNMCH, AtATR2, and P. somniferum CPR-like (PsCPR-L) were synthesized and subcloned into the pMA vector by GeneArt (Invitrogen). Native coding sequences of full-length PsTyDC6 and N-terminal truncated PsPDC1-IX1 were synthesized and cloned into pTYB21 (NEB) by GenScript (Piscataway, NJ, USA).

DNA encoding the N-terminal secretory leader sequence from CD5 (residues 1–24) upstream of either HuACE2 (residues 19–615) or RaACE2 (residues 19–165), followed by a short linker FLAG-epitope tag and C-terminal Histidine₆ was synthesised by GeneArt (Invitrogen). ACE2-Fc constructs include a GS4-linker and fragment of human IgG1 Fc (residues 104–330) between ACE2 and FAG-epitope. To generate a FLAG-free HuACE2 fusion, the FLAG-epitope tag in the HuACE2 construct was replaced with a HA tag. Constructs encoding soluble RBD proteins comprise of DNA encoding the N-terminal secretory leader sequence from CD5 (residues 1–24) upstream of Wuhan-Hu-1 SARS-CoV-2 RBD (residues 319–541), or RBD mutants indicated in Results, followed by a short linker HA-epitope tag and C-terminal Histidine₆ and were synthesised by GeneArt (Invitrogen). All other reagents were as described previously [11 (link),24 (link)].