An efficient system for site-directed mutagenesis to make various mutants of the env gene of human immunodeficiency...

We developed an efficient system of site-directed mutagenesis for the envelope (env) gene of human immunodeficiency virus type 1 (HIV-1). To make a template plasmid for mutagenesis, pS+B/MluI, two independent selection markers, i.e. a unique restriction site, MluI, and an in-frame termination codon, were introduced into the region encoding the V3 domain of the env gene of an HIV-1 strain, NL4-3, which had been cloned in the pUC118 plasmid. When the env gene of the pS+B/MluI plasmid was mutated successfully using mutagenic primers such as synthetic oligonucleotides or PCR-amplified DNA fragments longer than 1.5 kbp, the plasmids became resistant to digestion with MluI and competent env genes were formed by suppression of the in-frame termination. Various site-directed mutants of the env gene of HIV-1 were accurately constructed in a short time even in the absence of proper restriction sites by this system. The system of site-directed mutagenesis we reported here will be a useful method to analyze the functions of variable genes like the env gene of HIV-1 precisely and rapidly.