Abstract
Background: Development of drug resistance to current therapies for human immunodeficiency virus type 1 (HIV-1) infection has lead to the need for additional strategies for drug treatment. The HIV-1 regulatory and structural proteins represent drug targets that have not yet been exploited. Finding inhibitors of the interaction of these proteins with other viral and/or cellular proteins is a novel approach to HIV-1 drug discovery. Methods: The HIV-1 nucleocapsid (NC), viral protein R (vpr) and Gag-p6 genes were cloned into pACT and pBIND from the Checkmate mammalian two hybrid system (Promega) using standard molecular biology techniques. This system allows for the in vitro detection of protein-protein interactions by using a luciferase reporter gene. To detect the interaction of NC with vpr or Gag-p6, cells were co-transfected with the resulting NC and vpr clones or NC and p6 clones. 48 hours post transfection cells were washed, lysed and monitored for luciferase activity. Results: The interaction of NC with vpr and p6 was readily detectable. Luciferase expression reached levels 30 fold higher than those in control wells. Adaptation and optimization of the assay for use on a robotic platform is in progress. Initial screening of 3000 compounds will be presented. Conclusion: The intracellular binding of NC to vpr and Gag-p6 has been demonstrated. The system used is adaptable to many HIV-1 proteins and can be used for high-throughput screening to find inhibitors of these proteins. With viral resistance limiting current therapies, such compounds could prove useful in treating HIV infected individuals.
Introduction
Interactions between proteins play critical roles in cellular regulatory events, such as transcription, signal transduction pathways and enzyme-mediated metabolic effects. Identification, verification, characterization and manipulation of discrete protein:protein interactions within eukaryotic cells can be quite difficult and time-consuming. The introduction by Fields and co-workers of an in vivo yeast genetic screening method provided a means to accelerate the rate of progress in the identification of novel protein:protein interactions (1,2). The method, known as the yeast two-hybrid system, is a clever adaptation based upon the structural nature of certain eukaryotic transcription factors. Some transcription regulatory factors, such as yeast GAL4, which was first used in the yeast two-hybrid system, possess separate functional portions of the protein, or domains, that bind DNA and activate transcription (1,3-5). These protein domains can be expressed individually from recombinant vectors, and functional transcriptional activation that is specified by the DNA binding moiety can be reconstituted. The DNA binding domain and the transcriptional activation domain, which are produced by separate plasmids, are closely associated when one protein ("X") fused to a GAL4 DNA binding domain interacts with a second protein ("Y") fused to a GAL4 transcriptional activation domain. The physical interaction between proteins X and Y brings the two functional regions of the GAL4 protein in close proximity and enables activation of gene expression when placed in the proximity of a target promoter. Usually, one protein is known (X or Y), and the system is used to find a protein that interacts with the known protein. Newer applications of the two-hybrid system include assessing levels of protein:protein interactions.
The CheckMateTM Mammalian Two-Hybrid System is a modification of the original yeast system (6,7). In the mammalian-based system, reconstituted transcription factor activity comes from two different protein domains that are expressed from two separate vectors. The DNA binding domain of GAL4 protein and the activation domain of herpes simplex virus type 1 VP16 protein provide functional transcriptional activation from RNA polymerase II basal promoters with upstream GAL4 binding sites (8). In the CheckMateTM System, five GAL4 binding sites are positioned upstream of the firefly luciferase gene (luc+)(a,b), providing a sensitive and quantitative reporter system for functional assessment of reconstituted GAL4:VP16 activity. Mammalian cells are cotransfected with the firefly luciferase reporter vector, pG5luc, along with vectors that express the GAL4 DNA binding domain and VP16 activation domain. In the absence of interacting fusion partners to GAL4 and VP16, the level of transcription from the GAL4 element promoter is low, and consequently the luciferase expression is low. Cotransfection of mammalian cells with vectors coding for GAL4-X and VP16-Y, in which "X" and "Y" are interacting protein domains, results in close physical association of GAL4 and VP16 domains. The GAL4 promoters are functionally activated with concomitant increases in the luciferase reporter activity.