TABLE 2.
Plasmids used in this study
| Plasmid or 3′UTR chimera | Characteristics | Sequence of 5′ regiona |
|---|---|---|
| pHK1A | pHK0A replaced SCoV TRS core sequence (SCoV 67-72)b with MHV TRS core sequence (MHV 65-71)c | SCoV 1-66/MHV 65-71/SCoV 73-264 |
| pHK4A | pHK0A replaced SCoV SL1, SL2, and SL3 (SCoV 1-72) with their MHV counterpart (MHV 1-68) | MHV 1-68/SCoV 73-264 |
| pHK5A | Plasmid A replaced MHV 5′UTR SL1 (MHV 1-40) with SCoV 5′UTR SL1 (SCoV 1-35) | SCoV 1-35/MHV 41-209 |
| pHK8A | Plasmid A replaced MHV 5′UTR SL4 (MHV 67-139) with SCoV 5′UTR SL4 (SCoV 73-130) | MHV 1-66/SCoV 73-130/MHV 140-209 |
| pHK11A | Plasmid A replaced MHV sequence (MHV 140-209) between SL4 and start codon of ORF1 with its SCoV counterpart (SCoV 136-264) | MHV 1-139/SCoV 136-264 |
| pHK12A | Plasmid A replaced MHV TRS core and flanking sequence (MHV 59-73) with SCoV 5′UTR SL3 (SCoV 58-72) | MHV 1-58/SCoV 58-72/MHV 74-209 |
| pHK18A | Plasmid A replaced MHV 5′UTR SL2 (MHV 42-56) with SCoV 5′UTR SL2 (SCoV 42-57) | MHV 1-41/SCoV 42-57/MHV 57-209 |
| pLP0F | Plasmid F containing a frameshift mutation at nt 398 of nsp12 (RdRp) | |
| p0A-1 | pGEM-T vector carrying SCoV 5′UTR with BsmBI site at 5′ and 3′ terminals | |
| p0A-2 | PCR-XL-TOPO carrying BamHI fragment of plasmid A | |
| p0A-3 | p0A-2 deleted MHV 5′UTR by inverse PCR with primers containing BsmBI sites | |
| p7077 | p0A-3 ligated with BsmBI fragment from p0A-1 | |
| pHK0A | Plasmid A replaced with the BamHI fragment of p7077 by restriction fragment exchange, which ultimately replaced MHV 5′UTR (MHV 1-209) with SCoV 5′UTR (SCoV 1-264) | SCoV 1-264 |
| pB36 | T7 transcription vector encoding an MHV A59 DI RNA consisting of 467 nt of the 5′ end of the genome connected in frame, via a 48-nt linker, to the entire N gene and 3′UTR, followed by a poly(A) tail of approximately 115 residuesd | |
| p0G-1 | pGEM-T vector carrying SCoV 3′UTR and 15 nt of poly(A) tail | |
| p0G-2 | pB36 deleted MHV 3′UTR by inverse PCR with primers containing BsmBI sites | |
| p0G-3 | p0G-2 ligated with BsmBI fragment from p0G-1 | |
| pMF1G | G plasmid replaced with the NheI and PacI fragments of p0G-3 by restriction fragment exchange, which ultimately replaced MHV 3′UTR (MHV 31034-31357) with SCoV 3′UTR (SCoV 29389-29751) | SCoV 29389-29751 |
a
The SCoV or MHV origin of sequences in the chimeric portion of the plasmids are given.
b
Indicates SCoV sequence relative to the SCoV genome reported under GenBank accession no. AY278741 (27).
c
Indicates MHV sequence relative to the MHV genome reported under GenBank accession no. NC_001846 (39).
d
See reference 14.