TABLE 4.
Construction of plasmids to express μNS point mutants
| Mutationa | Primer (5′ to 3′)b
|
|
|---|---|---|
| Forward | Reverse | |
| C561S | CAAGTCAGCTCAATCATCTAGCTTGGATATCTATC | GATACATATCCAAGCTAGATGATTGAGCTGACTTG |
| H569Q | GATATGTATCTGCGACAACACACTTGCATTAATGG | CCATTAATGCAAGTGTGTTGTCGCAGATACATATC |
| H570Q | GATATGTATCTGCGACACCAAACTTGCATTAATGG | CCATTAATGCAAGTTTGGTGTCGCAGATACATATC |
| C572S | GATATGTATCTGCGACACCACACTTCCATTAATGGTC | GACCATTAATGGAAGTGTGGTGTCGCAGATACATATC |
| H576Q | CCACACTTGCATCAATGGTCAAGCTAAAGAAGATG | CATCTTCTTTAGCTTGACCATTGATGCAAGTGTGG |
a
Desired mutation at the indicated amino acid position of μNS. Amino acid residues are written in single-letter code as follows: wild-type residue, position number, and mutant residue.
b
In each primer, the nucleotide change to give the desired amino acid change is double underlined. For the C561S mutant, this change also caused loss of a BfaI restriction enzyme site useful for screening. For the other mutants, an additional nucleotide change was made to cause loss of a restriction enzyme site (single underline); the lost site is DdeI for H569Q, H570Q, and C572S and MseI for H576Q.