Reverse Two-Hybrid Screening to Analyze ProteinProtein Interaction of HIV-1 Viral and Cellular Proteins

HIV-1 replication involves a complex network of multiple protein–protein interactions. HIV-1 viral proteins exhibit both homomeric interactions among themselves and heteromeric interactions with other viral or cellular proteins. Identification and characterization of these protein–protein interactions have provided a wealth of information about the biology of the virus. Precise information about the residues involved in interaction is valuable in understanding the functional significance of these interactions, and can be determined relatively easily for proteins whose three-dimensional structure is known. However, the lack of three-dimensional structural information for several host proteins makes it harder to carry out detailed biochemical and functional studies. Reverse-two-hybrid system, a variation of the yeast-two-hybrid system can be used to genetically isolate mutants of a protein that are defective for specific protein–protein interactions. The strategy is to create a library of random mutations in one of the interacting partners and from among this library, screen for those that are defective for interaction using yeast two-hybrid system. In this review, we will describe a method to efficiently generate a library of random mutations and to further screen this library using the simple color scheme of using LacZ as a reporter gene. Once the mutants are isolated, they are tested in other biochemical systems and can be subjected to further functional and virological studies.