title: Polyprotein context regulates the activity of poliovirus 2CATPase bound to bilayer nanodiscs.
abstract: Positive-strand RNA viruses generally replicate in large membrane-associated complexes. For poliovirus, these replication complexes are anchored to the membrane via the viral 2B, 2C, and 3A proteins. 2C is an AAA+ family ATPase that plays a key role in host cell membrane rearrangement, is a putative helicase, and is implicated in virion assembly and packaging. However, the membrane-binding characteristics of all of these viral proteins have made it difficult to elucidate their exact roles in virus replication. We show here that small lipid bilayers known as nanodiscs can be used to chaperone the in vitro expression of soluble poliovirus 2C, 2BC, and 2BC3AB polyproteins in a membrane-bound form. ATPase assays on these proteins show that the activity of the core 2C domain is stimulated ~0-fold compared to the larger 2BC3AB polyprotein, with most of this stimulation occurring upon removal of 2B. The proteins are active over a wide range of salt concentrations, exhibit slight lipid headgroup dependence, and show significant stimulation by acetate. Our data lead to a model wherein the replication complex can be assembled with a minimally active form of 2C that then becomes fully activated by proteolytic cleavage from the adjacent 2B viroporin domain.
authors.affiliation: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado, USA.
authors.forename: Courtney L
authors.initials: CL
authors.lastname: Springer
authors.forename: Harrison P
authors.initials: HP
authors.lastname: Huntoon
authors.forename: Olve B
authors.initials: OB
authors.lastname: Peersen
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D008051:Lipid Bilayers / Q000378:metabolism; D008954:Models, Biological; D017955:Poliovirus / Q000201:enzymology* / Q000502:physiology*; D011485:Protein Binding; D055503:Protein Multimerization; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D002352:Carrier Proteins; D008051:Lipid Bilayers; D017361:Viral Nonstructural Proteins; C076023:2C protein, viral