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.P icornaviruses, like many other positive-strand RNA viruses, have RNA genomes with variable-length 3= poly(A) tails (1). The poly(A) tails of picornaviruses are important for viability (2), with the length of the poly(A) tails influencing the magnitudes of both viral mRNA translation and RNA replication (3, 4). RNA sequences and structures within the 3= nontranslated region are reported to influence both the length of poly(A) tails in picornaviral RNA (5) and the efficiency of virus replication (6, 7). Viral RNA-dependent RNA polymerases (3D pol s) are predicted to catalyze the polyadenylation of picornaviral RNA in a template-dependent manner during viral RNA replication (8); however, the mechanisms by which RNA genomes are polyadenylated during viral RNA replication remain obscure. In particular, there is little understanding of the mechanisms regulating the length of poly(A) tails synthesized during RNA replication. The RNA-dependent RNA polymerases of negative-strand RNA viruses reiteratively transcribe a small poly(U) sequence within intergenic regions of the viral RNA genome to produce long poly(A) tails on viral mRNAs (9). In a similar manner, the poliovirus RNA-dependent RNA polymerase can reiteratively transcribe poly(A) and poly(U) sequences during viral RNA replication, producing poly(A) and poly(U) sequences longer than those in the respective template RNAs (8).Viral RNA-dependent RNA polymerases are well studied at the structural level (10-12), yet there have been no reports describing the structural features of viral polymerases involved in the polyadenylation of viral RNA. The RNA-dependent RNA polymerase of picornaviruses a...

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 ϳ60-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. The picornaviruses are a family of human and mammalian pathogens that include hepatitis A virus, the rhinoviruses, foot-and-mouth disease virus (FMDV), coxsackievirus, and poliovirus. Poliovirus remains a health threat in many parts of the world, where it is being kept in check by extensive immunization efforts aimed at its eventual eradication (1). Poliovirus has a 7.5-kb single-stranded, positive-sense RNA genome that encodes a single ϳ250-kDa polypeptide. Upon translation, this viral polyprotein undergoes a series of proteolytic cleavages to generate a total of 11 different proteins plus several important functional intermediates (Fig. 1A). The four proteins in the P1 region (VP1 to VP4) are the structural proteins that make up the virion capsid, while the remaining seven nonstructural proteins in the P2 and P3 regions (2A-2C and 3A-3D, respectively) are involved in viral replication biochemistry (2). Processing of the polyprotein plays an important role in mediating protein function as several viral precursor proteins have different functional roles and biochemical activities than the fully processed proteins. For example, the cleavage of poliovirus 3CD pro generates and activates 3D pol , the RNAdependent RNA polymerase that carries out replication of the viral RNA (3-5). Similarly, the 2C and precursor 2BC proteins act in concert to trigger a massive rearrangement of intracellular membranes that results in the formation of extensive "membranous web" structures (6-8), on the surfaces of which these proteins form the viral replication centers that are the sites of RNA synthesis and virion assembly.The viral 2B, 2C, and 3A proteins all contain membrane-binding regions. Both 2BC and 2C are able to induce membrane rearrangement in living cells (9-11); however, coexpres...