TABLE 1.
Nucleotide sequence of mutagenesis primersa
| Primer B |
Primer C |
||
|---|---|---|---|
| Target | Sequenceb | Target | Sequenceb |
| Δ(E165/S169) | GCTGAACATGGGATTCCTAGGACCC* | Δ(E165/S169) | CTAGGAATCCCATGTTCAGCGCAGG* |
| GCTGAACACGGGATTCCTAGGACCCC** | CTAGGAATCCCGTGTTCAGCGCAG** | ||
| Δ(G170/P174) | CATCACATCACTTCTCGTGTTACAGGC | Δ(G170/P174) | ACACGAGAAGTGATGTGATGTTCTCCATG* |
| ACACGAGAAGTGATGTGATGTTCTCCG** | |||
| Δ(L175/L178) | CCTAGGACCCCAGGCGGGGTTTTTC | Δ(L175/L178) | ACCCCGCCTGGGGTCCTAGGAATCCTG |
| Δ(Q179/G181) | TCTCGTGTTATTTTTCTTGTTGACAAGAATC | Δ(Q179/G181) | ACAAGAAAAATAACACGAGAAGGGGTC |
| Δ(F182/L185) | ACAGGCGGGGACAAGAATCCTCACAATACC | Δ(F182/L185) | GGATCTTGTCCCCGCCTGTAACAC |
| Δ(T186/R187) | TTTCTTGTTGATCCTCACAATACCGC | Δ(T186/R187) | TTGTGAGGATCAACAAGAAAAACCCC |
| Δ(I188/I191) | GTTGACAAGACCGCAGAGTCTAGACTCG | Δ(I188/I191) | GACTCTGCGGTCTTGTCAACAAGAAAAACC |
| Δ(I157/S169) | CTTCTCGAGGGGATTCCTAGGACCCC | Δ(1157/S169) | CTAGGAATCCCCTCGAGAAGATTGACG |
| S(L175/L178) | CCTAGGACCCGCTGCCGCGGCACAGGCGGGGTTTTTC | S(L175/L178) | ACCCCGCCTGTGCCGCGGCAGCGGGTCCTAGGAATCCTG |
| S(F182/L185) | ACAGGCGGGGGCTGCCGCGGCGACAAGAATCCTCACAATACC | S(F182/L185) | GGATTCTTGTCGCCGCGGCAGCCCCCGCCTGTAACAC |
| S(T186/R187) | CTTGTTGGCAGCAATCCTCACAATACCGC | S(T186/R187) | AGGATTGCTGCCAACAAGAAAAACCCC |
| S(I188/I191) | GTTGACAAGAGCCGCCGCAGCACCGCAGAGTCTAGACTCG | S(I188/I191) | GACTCTGCGGTGCTGCGGCGGCTCTTGTCAACAAGAAAAACC |
| V177A | ACCCCTTCTCGCGTTACAGGCGGGG | V177A | CCGCCTGTAACGCGAGAAGGGGTCCTAG |
| L184A | GGGGTTTTTCGCGTTGACAAGAATCCTCAC | L184A | GATTCTTGTCAACGCGAAAAACCCCGCC |
| I188A | GTTGACAAGAGCCCTCACAATACCGCAG | I188A | GTATTGTGAGGGCTCTTGTCAACAAGAAAAACC |
| I191A | GAATCCTCACAGCACCGCAGAGTCTAGACTC | 1191A | GACTCTGCGGTGCTGTGAGGATTCTTGTCAAC |
| I188A I191A | AGAGCCCTCACAGCACCGCAGAGTCTAGACTCG | I188A I191A | CGGTGCTGTGAGGGCTCTTGTCAACAAGAAAAACC |
Primers B and C correspond to forward and reverse primers, respectively (see “Plasmids and mutagenesis” in Materials and Methods). Deletions (Δ) are inclusive, and residues are numbered according to their respective positions relative to the first N-terminal amino acid of the L protein. In mutations preceded by an S, alanines were substituted for all amino acids in the range specified. For the unique and double substitutions, we used the classical writing convention to indicate the mutation.
The same oligonucleotides were used for mutagenesis in pSVSX and pSV12SX S− plasmids with exception of those that include the initiation codon (M164) of the S protein, for which designed primers were used with the pSVSX vector (*) and the pSV12SX S− vector (**).