Table 1.
Primers used to introduce nucleotide substitutions in the E gene of pBAC-OC43FL (St-Jean et al., 2006) for recombinant HCoV-OC43 virus production with amino acid modifications within the E protein (Section A). Primers used to verify sequences of the full HCoV-OC43 E and M gene of recombinant viruses (Section B). Bold and underlined sections represent newly introduced nucleotide substitutions. Underlined section in mutant E-PBM 82–84 represents previously introduced nucleotide substitutions.
|
A – Primers for site-directed mutagenesis | ||
|---|---|---|
| Recombinant Virus | Primer Name | Primer Sequence |
| rOC/Estop | OC-E-stop | 5’-CT GAT GCT TAT CTT TGA GAC ACT GTG TGG-3’ |
| rOC/E-TMQ17A | OC-E-TM-Q17A | 5’-GTG TGG TAT GTG GGG GCA ATA ATT TTT ATA GTT GCC-3’ |
| rOC/E-PBMD82A | OC-E-PBM-D82A | 5’-CA GTC CTT GAT GTG GCC GAC GTT TAG GTA ATC-3’ |
| rOC/E-PBMV84A | OC-E-PBM-V84A | 3’-GT TTG GAT TAC CTA AGC GTC ATC CAC ATC AAG-5’ |
| rOC/E-PBMD82A-V84A | OC-E-PBM-D82V-V84A | 5’-CTT GAT GTG GCC GAC GCT TAG GTA ATC CAA AC-3’ |
| B – Primer for sequence verification | ||
| Primer Name | Primer Sequence | |
| OC-ns5-116-E-For | 5’-GTA GAG TTC CTA GTC ATG CTT G-3’ | |
| OC-E-222-For | 5’-GAT GTA AAA CCA CCA GTC CT-3’ | |
| OC-M-127-E-Rev | 5’-ACA TAC TGC GAC TTG TAT AGC C-3’ | |
| OC-M-172-For | 5’-ATT TTG TGG CTT ATG TGG CCC-3’ | |
| OC-M-241-E-Rev | 5’-CTA TAG AAA GGC CAA GAT ACA C-3’ | |
| OC-M-439-For | 5’-GTC ACA ATA ATA CGC GGC CA-3’ | |
| OC-M-514-Rev | 5’-CCT TAG CAA CAG TCA TAT AAG C-3’ | |
| OC-N-60-Rev | 5‘-CAT TAC CAG AAC GAT TTC C-3’ | |
| GAPDH-For | 5’-CGG AGT CAA CGG ATT TGG TCG TAT-3’ | |
| GAPDH-Rev | 5’-AGC CTT CTC CAT GGT GGT GAA GAC-3’ | |