title: Analysis of poliovirus protein 3A interactions with viral and cellular proteins in infected cells.
abstract: Poliovirus proteins 3A and 3AB are small, membrane-binding proteins that play multiple roles in viral RNA replication complex formation and function. In the infected cell, these proteins associate with other viral and cellular proteins as part of a supramolecular complex whose structure and composition are unknown. We isolated viable viruses with three different epitope tags (FLAG, hemagglutinin [HA], and c-myc) inserted into the N-terminal region of protein 3A. These viruses exhibited growth properties and characteristics very similar to those of the wild-type, untagged virus. Extracts prepared from the infected cells were subjected to immunoaffinity purification of the tagged proteins by adsorption to commercial antibody-linked beads and examined after elution for cellular and other viral proteins that remained bound to 3A sequences during purification. Viral proteins 2C, 2BC, 3D, and 3CD were detected in all three immunopurified 3A samples. Among the cellular proteins previously reported to interact with 3A either directly or indirectly, neither LIS1 nor phosphoinositol-4 kinase (PI4K) were detected in any of the purified tagged 3A samples. However, the guanine nucleotide exchange factor GBF1, which is a key regulator of membrane trafficking in the cellular protein secretory pathway and which has been shown previously to bind enteroviral protein 3A and to be required for viral RNA replication, was readily recovered along with immunoaffinity-purified 3A-FLAG. Surprisingly, we failed to cocapture GBF1 with 3A-HA or 3A-myc proteins. A model for variable binding of these 3A mutant proteins to GBF1 based on amino acid sequence motifs and the resulting practical and functional consequences thereof are discussed.
authors.affiliation: Laboratory of Infections Diseases, National Institutes of Health, Bldg. 33, Room 3W10A.2, 33 North Drive, MSC 3203, Bethesda, MD 20892-3203, USA. nteterina@niaid.nih.gov
authors.forename: Natalya L
authors.initials: NL
authors.lastname: Teterina
authors.forename: Yuval
authors.initials: Y
authors.lastname: Pinto
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Weaver
authors.forename: Kenneth S
authors.initials: KS
authors.lastname: Jensen
authors.forename: Ellie
authors.initials: E
authors.lastname: Ehrenfeld
mesh_terms: D006367:HeLa Cells; D054884:Host-Pathogen Interactions; D006801:Humans; D046911:Macromolecular Substances / Q000302:isolation & purification; D017955:Poliovirus / Q000254:growth & development*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D014758:Viral Core Proteins / Q000378:metabolism*
chemical_list: C109349:3A protein, Poliovirus; D046911:Macromolecular Substances; D014758:Viral Core Proteins