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.Poliovirus (PV) is a member of the human enterovirus C cluster in the Enterovirus genus of the virus family Picornaviridae. The PV genome encodes a single polyprotein that is proteolytically processed to generate a set of intermediate precursors and final cleavage products that are all required for virus replication. The N-terminal region of the polyprotein (P1) forms the viral capsid proteins, which are dispensable for viral RNA translation and replication but are needed for encapsidation and assembly of infectious particles. The remainder of the polyprotein (P2 and P3 regions) generates proteins that contribute catalytic and structural functions for viral RNA translation and replication, as well as for disruption and/or reorganization of numerous cellular processes and activities that could restrict or combat virus replication. All of the PV noncapsid proteins are essential for viral RNA replication; most manifest multiple activities during the virus replication cycle, and all at least partially localize to large, membraneassociated replication complexes that form from preexisting subcellular organelles after infection. Viral proteins 2B, 2C, and 3A contain hydrophobic trans-membrane regions or amphipathic helices, and these proteins as well as their larger precursor proteins bind membranes directly (7,12,...