Yeast two-hybrid assay for examining human immunodeficiency virus proteaseheterodimer formation with dominant-negative inhibitors and multidrug-resistant variants

The yeast two-hybrid assay was used to study the dimerization of engineered and naturally occurring variants of human immunodeficiency virus (HN) prot ease (PR) monomers, Defective monomers that were previously shown to exhibi t a dominant-negative (D-N) effect in cultured mammalian cells were tested for their ability to interact in the two-hybrid assay. Similarly, monomers with dimer-interface substitutions and monomers harboring in vivo selected mutations that confer multidrug resistance (mdr) in an AlDS patient were te sted for interaction in yeast, Dimer formation between wt monomers with cat alytic aspartates was not detected in yeast, whereas the dimerization of PR monomers harboring the acid active site substitution D25N was readily demo nstrated. The use of inactive monomers harboring the D25N substitution as a genetic background for studying additional HIV PR mutations allowed for th e probing of interactions between monomers with mdr-associated mutations wi th those based on the HIV-1 HXBBR sequence. The HTLVIII/HIV-1 HXB2R clone h as been the basis for a large number of HIV-related plasmids, primers, anti bodies, and other specific reagents throughout the HN research community. T he results of our assay suggest that HXB2R-based D-N PR inhibitors associat e with variant monomers based on the recently obtained nucleotide sequence from an AIDS patient with a multidrug-resistant virus. These results furthe r encourage the use of D-N PR inhibitors as antiviral agents which may comp lement existing small-molecule combination therapies. (C) 2000 Academic Pre ss.