Virus Host Protein Protein Interaction Screens

As the third component of our integrative approach, we have been systematically examining protein variation and interactions associated with MD resistance. Specifically, we have screened for MDV-chicken protein-protein interactions using a two-hybrid screen. Briefly describing the method, the two-hybrid system screens a cDNA (prey) library that has been fused to the activation domain (AD) of a tran-scriptional activator to identify proteins that interact with bait (protein of interest) that is fused with the DNA-binding domain (BD). As the AD and BD do not need to be physically connected to promote transcription, if the two fusion proteins interact, a reporter gene is expressed. Our hypothesis was that some chicken proteins that interact with MDV proteins are involved in the immune response and genetic resistance to MD. Thus, we could utilize the two-hybrid system to quickly identify interacting (and interesting) proteins, which when combined with genetic mapping would identify positional candidate genes for MD resistance.

Our initial MDV bait highlights the success we have achieved (Liu et al., 2001b). We initially chose MDV SORF2 gene as bait since SORF2 overexpression in the RM1 strain may account for the reduced virulence compared to its parental JM/102W strain (Jones et al., 1996). Using the yeast two-hybrid system and a splenic cDNA library, growth hormone (GH) was found to specifically interact with SORF2 (Liu et al., 2001b). It is critical to confirm the two-hybrid results, as this method is known to have a high false-positive rate. Thus, to corroborate the detected interaction, in vitro protein binding assay using GST-fusion proteins was performed to confirm direct binding of GH to SORF2. Our results showed that, while SORF2 protein was not retained by GST protein alone, SORF2 could be retained by GST-GH fusion protein presumably due to the presence of GH. This result was in agreement with the result of the yeast two-hybrid system assay and indicated that the interaction between SORF2 and GH is a direct and specific protein-protein interaction without other intermediary factors (e.g., yeast proteins) involved.

Having confirmed the SORF2-GH interaction, we treated the GH gene (GH1) as a candidate gene for MD resistance. To see if GH1 had a genetic basis, an association study was conducted in our MD resource population derived from commercial White Leghorn lines. GH1 variation was significantly associated (P < 0.01) with a number of MD-associated traits in MHC B2/B15 chicks (Liu et al., 2001b). Furthermore, to provide some functional information support, our DNA microarray results indicate that GH is differentially expressed between MD resistant (Line 6) and susceptible (Line 7) chicks following MDV challenge (Liu et al., 2001a).

Thus, the combined results of a specific MDV-chicken protein interaction, differential expression of GH between MD resistant and susceptible chickens, and association of GH1 with MD disease-related traits and selected lines for MD resistance, all strongly suggested that GH1 is a MD-resistance gene. This conclusion is supported by reports demonstrating that GH modulates the immune system in many species (e.g., reviews by Gala, 1991; Auernhammer and Strasburger, 1995), and GH1 alleles change in chicken strains in response to selection for MD resistance (Kuhnlein et al., 1997). Most importantly, it exemplifies the power of combining genetic and molecular approaches to identify positional candidate genes for QTL.

While effective, the number of baits that could be screened by an investigator would be limited using the yeast two-hybrid system. Fortunately, two-hybrid systems based in Escherichia coli have become commercially available. With the complete sequence of the MDV genome (Lee et al., 2000; Tulman et al., 2000), it became feasible to conduct a systematic screen of the relevant MDV genes for interacting chicken partners; there are ~100 different MDV genes and proteins (Lee et al., 2000; Tulman et al., 2000; Liu et al., 2006). We screened all the MDV genes that are considered unique to serotype I (virulent) strains, and all potential MDV-host protein interactions were tested by an in vitro binding assay to confirm the initial two-hybrid results. As a result, eight new MDV-chicken protein interactions were identified (Niikura et al., 2004). More importantly, genetic mapping and association analyses of the encoding chicken genes revealed that LY6E [lymphocyte complex 6, locus E, aka, stem cell antigen 2 (SCA2) and thymic-shared antigen 1 (TSA1)] is another MD-resistant gene (Liu et al., 2003) and suggest that BLB, the gene for MHC class II (3 chain, is a strong positional candidate gene (Niikura et al., 2004).

Was this article helpful?

0 0
How To Bolster Your Immune System

How To Bolster Your Immune System

All Natural Immune Boosters Proven To Fight Infection, Disease And More. Discover A Natural, Safe Effective Way To Boost Your Immune System Using Ingredients From Your Kitchen Cupboard. The only common sense, no holds barred guide to hit the market today no gimmicks, no pills, just old fashioned common sense remedies to cure colds, influenza, viral infections and more.

Get My Free Audio Book


Post a comment