TABLE 1.
Primers used for generating BHV-1 BAC, BHV-1gEAm453, and BHV-1gEAm453R
| Purpose and primer | Sequence (restriction endonuclease) |
|---|---|
| BAC insertiona | |
| gB-for | 5′-CGAGGAATTCGATGCGCGCGCCCGGC-3′ (EcoRI) |
| gB-rev | 5′-TTCCTTAATTAAGGGGCGCCCTGCCGTGC-3′ (PacI) |
| UL26-for | 5′-TTCCTTAATTAAGTTTGGCGCGCGGTGG-3′ (PacI) |
| UL26-rev | 5′-TACCAAGCTTGTGCGGCCTCGGCGCAC-3′ (HindIII) |
| Mutagenesisb | |
| gEAm453-for | 5′-GCAGCCCTCGCCGTTCGGGTGTGCGCGCGCCGCGCAAGCTAGAAGCGCACCTACGACATCaggatgacgacgataagtaggg-3′ |
| gEAm453-rev | 5′-CGGGCCCGAAGGGGTTGAGGATGTCGTAGGTGCGCTTCTAGCTTGCGCGGCGCGCGCACACcaaccaattaaccaattctgattag-3′ |
| gEAm453 rescue-for | 5′-GCAGCCCTCGCCGTTCGGGTGTGCGCGCGCCGCGCAAGCCAGAAGCGCACCTACGACATCaggatgacgacgataagtaggg-3′ |
| gEAm453 rescue-rev | 5′-CGGGCCCGAAGGGGTTGAGGATGTCGTAGGTGCGCTTCTGGCTTGCGCGGCGCGCGCACACcaaccaattaaccaattctgattag-3′ |
Primers used for amplification of BHV-1gB and UL26 sequences for the construction of the BAC insertion vector. The underlined sequence indicates the restriction site, and bold letters indicate modified bases to accommodated the restriction site.
Oligonucleotide sequence used for point mutation using the two-step Red-mediated mutagenesis system (32) to construct the BHV-1gEAm453 mutant and BHV-1gEAm453 rescue viruses. BHV-1 gE-specific sequences are shown in uppercase letters; the italicized and italicized-underlined sequences are complementary to each other in inverse orientation. Bold letters indicate the changed nucleotide to introduce the stop codon or to rescue the original sequence in the BHV-1gE cytoplasmic tail. Nucleotides in lowercase indicate the pEPkan-S-specific sequence (for details, see Materials and Methods and reference 32).