A high-throughput yeast two-hybrid protocol to determine virus-host protein interactions

Abstract

The yeast two-hybrid (Y2H) system is a powerful method to identify and analyze binary protein interactions. In the field of virology, the Y2H system has significantly increased our knowledge of structure and function of viral proteins by systematically assessing intraviral protein interactions. Several comprehensive approaches to determine virus-host interactions have provided insight into viral strategies to manipulate the host for efficient replication and to escape host-derived countermeasures. To expand our knowledge of intraviral and virus-host protein interactions, we here present a Y2H protocol that is well suited for high-throughput screening. Yeast mating followed by liquid handling in a 96-well format as well as fluorescent readout of the reporter system provides a highly standardized and fully automated screening situation. The protocol can either be applied to screen complex host cDNA libraries or protein pairs arrayed for cross-testing. The ease of use, the cost-effectiveness as well as the robotic handling allows for extensive and multiple rounds of screening providing high coverage of protein-protein interactions. Thus, this protocol represents an improved "deep" screening method for high-throughput Y2H assays.

Fig. 1

The array-based yeast two-hybrid (Y2H) screening allows the cross-testing of a single protein against a defined set of possible interaction partners. The haploid yeast strain carrying the bait vector is propagated under selective conditions in synthetic minimal medium lacking Tryptophane (SD-W), while yeast cells of opposing mating type transformed with the various prey vectors are grown arrayed in 96-well microtiter plates (MTP) in SD medium lacking Leucine (SD-L). The combination of the bait yeast strain with the multiple prey strains in complete medium (YPDA) over night leads to mating, resulting in diploid cells carrying both bait and prey vectors, which are selected by growth in SD-L/W medium. The reporter gene activity is examined in SD medium lacking additionally Histidine (SD-L/W/H) and supplemented with 4-Methylumbelliferyl-α-d-galactopyranoside (4-MUx) and 3-Amino-1,2,4-triazole (3-AT) in a fluorescence plate reader (excitation 365 nm, emission 448 nm). The interaction of the bait protein fused to Gal4 DNA binding domain with the prey protein fused to Gal4 activation domain leads to the reconstitution of Gal4 transcription factor and subsequently expression of HIS3 and MEL1 reporter genes. Expression of the reporter genes allows growth in a Histidine-free environment as well as the hydrolysis of 4-MUx to fluorescent methylumbelliferone, whose fluorescent activity is a measure of protein-protein interaction. Usually 3-AT is added in increasing concentration as competitive inhibitor of the leaky expression of the HIS3 reporter gene

Fig. 2

The library-based yeast two-hybrid (Y2H) screening is applied to test an individual bait protein for interaction against a complex prey library, usually consisting of cDNA of a certain tissue type and pretransformed in yeasts. Haploid yeast strains of opposing mating type carrying either bait or prey vectors are propagated under selective conditions in synthetic minimal medium lacking Tryptophane (SD-W) or Leucine (SD-L), respectively. The mating process to gain diploid cells containing both bait and prey vector is performed in complete medium (YPDA + 20 % Polyethyenglycol) under gentle shaking. Afterwards, the yeast culture is spread on 96-well microtiter plates (MTP) and incubated under selective conditions (SD-L/W). The reporter gene activity is then examined in SD medium lacking additionally Histidine (SD-L/W/H) and supplemented with 4-Methylumbelliferyl-α-d-galactopyranoside (4-MUx) and 3-Amino-1,2,4-triazole (3-AT ) in a fluorescence plate reader (excitation 365 nm, emission 448 nm). The interaction of the bait protein fused to the Gal4 DNA binding domain with a prey protein fused to the Gal4 activation domain leads to reconstitution of the Gal4 transcription factor and subsequently expression of HIS3 and MEL1 reporter genes. The reporter genes allow growth in a Histidine-free environment as well as the hydrolysis of 4-MUx to fluorescent Methylumbelliferone, whose fluorescent activity is a measure of protein-protein interaction. The optimal 3-AT concentration as a competitive inhibitor to leaky reporter gene HIS3 expression was defined in a previously performed prescreen. To identify the prey genes that resulted in a positive outcome, a colony PCR of the respective diploid cells is carried out. The agarose gel electrophoresis reveals those hits, where only one prey gene is present. These PCR products then undergo sequencing analysis and their gene ID is identified using an alignment tool like NCBI’s BLAST (http://blast.ncbi.nlm.nih.gov/)