ABSTRACT
Picornaviruses have 3' polyadenylated RNA genomes, but the mechanisms by which these genomes are polyadenylated during viral replication remain obscure. Based on prior studies, we proposed a model wherein the poliovirus RNA-dependent RNA polymerase (3D(pol)) uses a reiterative transcription mechanism while replicating the poly(A) and poly(U) portions of viral RNA templates. To further test this model, we examined whether mutations in 3D(pol) influenced the polyadenylation of virion RNA. We identified nine alanine substitution mutations in 3D(pol) that resulted in shorter or longer 3' poly(A) tails in virion RNA. These mutations could disrupt structural features of 3D(pol) required for the recruitment of a cellular poly(A) polymerase; however, the structural orientation of these residues suggests a direct role of 3D(pol) in the polyadenylation of RNA genomes. Reaction mixtures containing purified 3D(pol) and a template RNA with a defined poly(U) sequence provided data consistent with a template-dependent reiterative transcription mechanism for polyadenylation. The phylogenetically conserved structural features of 3D(pol) involved in the polyadenylation of virion RNA include a thumb domain alpha helix that is positioned in the minor groove of the double-stranded RNA product and lysine and arginine residues that interact with the phosphates of both the RNA template and product strands.
Subject(s)
Poliovirus/enzymology , Polyadenylation , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/metabolism , Amino Acid Substitution , DNA Mutational Analysis , HeLa Cells , Humans , Models, Molecular , Mutagenesis, Site-Directed , Poliovirus/genetics , Protein Conformation , RNA-Dependent RNA Polymerase/geneticsABSTRACT
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.
