title: Analysis of vaccinia virus-host protein-protein interactions: validations of yeast two-hybrid screenings.
abstract: Vaccinia virus, a large double-stranded DNA virus, is the prototype of the Orthopoxvirus genus, which includes several pathogenic poxviruses of humans, such as monkeypox virus and variola virus. Here, we report a comprehensive yeast two-hybrid (Y2H) screening for the protein-protein interactions between vaccinia and human proteins. A total of 109 novel vaccinia-human protein interactions were detected among 33 viral proteins. To validate subsets of those interactions, we constructed an ORFeome library of vaccinia virus strain WR using the Gateway plasmid cloning system. By co-expressing selected vaccinia and host proteins in a variety of expression systems, we found that at least 17 of the Y2H hits identified between vaccinia and human proteins can be verified by independent methods using GST pull-down assays, representing a 63% validation rate for the Y2H hits examined (17/27). Because the cloned ORFs are conveniently transferable from the entry vectors to various destination expression vectors, the vaccinia ORFeome library will be a useful resource for future high-throughput functional proteomic experiments.
authors.affiliation: Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, FL 32610, USA.
authors.forename: Leiliang
authors.initials: L
authors.lastname: Zhang
authors.forename: Nancy Y
authors.initials: NY
authors.lastname: Villa
authors.forename: Masmudur M
authors.initials: MM
authors.lastname: Rahman
authors.forename: Sherin
authors.initials: S
authors.lastname: Smallwood
authors.forename: Donna
authors.initials: D
authors.lastname: Shattuck
authors.forename: Chris
authors.initials: C
authors.lastname: Neff
authors.forename: Max
authors.initials: M
authors.lastname: Dufford
authors.forename: Jerry S
authors.initials: JS
authors.lastname: Lanchbury
authors.forename: Joshua
authors.initials: J
authors.lastname: Labaer
authors.forename: Grant
authors.initials: G
authors.lastname: McFadden
mesh_terms: D054884:Host-Pathogen Interactions / Q000502:physiology*; D006801:Humans; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D025941:Protein Interaction Mapping / Q000379:methods; D040901:Proteomics / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D020798:Two-Hybrid System Techniques; D014615:Vaccinia / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D014764:Viral Proteins