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 seque nce was cleaved at a unique Nco I restriction site within the viral ge nome, and DNA fragments containing the 5' and 3' portions of the HIV g enome were subcloned into separate plasmid vectors. The 5' 'half-virus ' construct was further modified by incorporating a class IIS restrict ion site, Esp3I, near the 3' end of the protease gene of HIV. This sit e, in combination with a natural ApaI site near the 5' end of the prot ease gene, creates a convenient cassette shuttle vector in which the p rotease coding region can be easily replaced. Recombinant viruses cont aining protease genes either altered by site-directed mutagenesis or a mplified from clinical or laboratory isolates can be reconstructed. Th e DNA fragment containing the protease gene is first subcloned into th e 5' half-virus shuttle vector plasmid. Infectious recombinant virus i s subsequently recovered by cotransfecting 5' and 3' half-virus plasmi ds linearized at their common Nco I sites into mammalian cells. This m ethod was successfully applied to constructing viruses containing vari ous substitutions in protease. (C) 1994 Academic Press, Inc.