Construction of infectious molecular clones of HIV-1 containing defined mutations in the protease gene

A DNA clone of HIV-1 containing the full-length infectious viral sequence was cleaved at a unique Nco I restriction site within the viral genome, and DNA fragments containing the 5' and 3' portions of the HIV genome were subcloned into separate plasmid vectors. The 5' 'half-virus' construct was further modified by incorporating a class IIS restriction site, Esp3I, near the 3' end of the protease gene of HIV. This site, in combination with a natural ApaI site near the 5' end of the protease gene, creates a convenient cassette shuttle vector in which the protease coding region can be easily replaced. Recombinant viruses containing protease genes either altered by site-directed mutagenesis or amplified from clinical or laboratory isolates can be reconstructed. The DNA fragment containing the protease gene is first subcloned into the 5' half-virus shuttle vector plasmid. Infectious recombinant virus is subsequently recovered by cotransfecting 5' and 3' half-virus plasmids linearized at their common Nco I sites into mammalian cells. This method was successfully applied to constructing viruses containing various substitutions in protease.