title: Application of a split luciferase complementation assay for the detection of viral protein-protein interactions.
abstract: Intraviral protein-protein interactions are critical for virus survival in the host. Discovery of such interactions is important to understand molecular mechanisms of viral replication and pathogenesis. The development of a cell-based assay that can be employed to examine systematically viral protein interactions is described. The method, known as the split luciferase complementation assay (SLCA), is based on the principle that N- and C-terminal domains of luciferase alone do not emit luminescence; however, if fused to interacting proteins the two non-functional halves can be brought into close enough proximity through a specific protein-protein interaction to restore the functions of the enzyme and emit detectable light. The well-studied influenza B polymerase acidic protein (PA) and basic protein 1 (PB1) interaction was used as a model system to develop the assay. Consistent with previous studies, a strong PA-PB1 interaction was demonstrated in the assay. The PA-PB1 interaction was also disrupted by single amino acid mutations in the N-terminal domain of PB1 that is responsible for binding PA. The described SLCA is highly specific and easy to perform, and thus may be useful for studying protein-protein interactions in viral diseases.
authors.affiliation: Department of Veterinary and Biomedical Sciences, South Dakota State University, Brookings, SD 57007, United States.
authors.forename: Qiji
authors.initials: Q
authors.lastname: Deng
authors.forename: Dan
authors.initials: D
authors.lastname: Wang
authors.forename: Xiaoxiao
authors.initials: X
authors.lastname: Xiang
authors.forename: Xiaofei
authors.initials: X
authors.lastname: Gao
authors.forename: Philip R
authors.initials: PR
authors.lastname: Hardwidge
authors.forename: Radhey S
authors.initials: RS
authors.lastname: Kaushik
authors.forename: Thorsten
authors.initials: T
authors.lastname: Wolff
authors.forename: Suvobrata
authors.initials: S
authors.lastname: Chakravarty
authors.forename: Feng
authors.initials: F
authors.lastname: Li
mesh_terms: D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D006801:Humans; D009981:Influenza B virus / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*; D014773:Virology / Q000379:methods
chemical_list: C061290:PA protein, influenza viruses; D014764:Viral Proteins; C077346:influenza virus polymerase basic protein 1; D008156:Luciferases; D012324:RNA-Dependent RNA Polymerase