TABLE 2.
Plasmids used in this study
| Plasmid | Description |
|---|---|
| Plasmid Fa | PCR-XL-Topo vector containing a 6.9-kb DNA fragment of MHV cDNA (bp 15755-22739 on the MHV-A59 genome) |
| pWtNsp15-PG | pGEM-T (Promega, Madison, WI) containing a PCR product corresponding to a 3.4-kb BamHI-HpaI fragment in plasmid F of MHV cDNA |
| p1F-PG | pWtNsp15-PG containing an H262A mutation in mNsp15 |
| pHK1Fb | Plasmid F containing the mNsp15 H262A mutation |
| p2F-PG | pWtNsp15-PG containing a G275A mutation in mNsp15 |
| pHK2F | Plasmid F containing the mNsp15 G275A mutation |
| p3F-PG | pWtNsp15-PG containing an H277A mutation in mNsp15 |
| pHK3F | Plasmid F containing the mNsp15 H277A mutation |
| p4F-PG | pWtNsp15-PG containing a D324A mutation in mNsp15 |
| pHK4F | Plasmid F containing the mNsp15 D324A mutation |
| pcD-mNsp15 | pcDNA1 (Invitrogen, Carlsbad, CA) containing mNsp15 coding sequence |
| pcD-sNsp15 | pcDNA1 containing sNsp15 coding sequence |
| pcD-GFP-sNsp15 | pcDNA1 containing GFP conjugated to the N terminus of the mNsp15 coding sequence |
a
One of seven plasmids used in the MHV-A59 reverse-genetic system (30).
b
The BamHI-HpaI fragment containing the H262A mutation in p1F-PG was excised and religated into BamHI-HpaI-digested plasmid F to construct pHK1F. Plasmids pHK2F, pHK3F, and pHK4F were constructed using p2F-PG, p3F-PG, and p4F-PG, respectively, with the same protocol as that for the construction of pHK1F.