Structural Features of a Picornavirus Polymerase Involved in the Polyadenylation of Viral RNA

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.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...

Poliovirus Polymerase Leu420 Facilitates RNA Recombination and Ribavirin Resistance

“…Poliovirus type 1 (Mahoney) and cDNA clones thereof were used for this study (30)(31)(32). A full-length cDNA clone was used to produce infectious poliovirus RNA (Fig.…”

“…1, ⌬Capsid-⌬IRES). 3D pol substitution mutations (G64S, K126A, R128A, K133A, K276A, N409A, D412A, S416A, L419A, and L420A) were engineered into full-length infectious poliovirus and ⌬Capsid cDNA clones as previously described (30).…”

“…Viral RNAs were produced by T7 transcription of MluI-linearized cDNA clones (Ampliscribe T7 high-yield transcription kit; Cellscript Inc.). Cells were transfected as previously described (30,31). HeLa, L929, and 3T3 cells were plated in 35-mm 6-well dishes ϳ24 h before transfection, with ϳ10 6 cells per well.…”

“…Following transfection, 2 ml of culture medium (Dulbecco modified Eagle medium containing 100 units of penicillin and 100 g per ml of streptomycin, 10% fetal bovine serum, and 10 mM MgCl 2 ) was added to each well, and the cells were incubated at 37°C in 5% CO 2 . Virus was harvested at 72 h posttransfection, recovered after three rounds of freezing and thawing, cleared of cellular debris by centrifugation at 3,000 rpm, and quantified by plaque assay (30). The mean titers from triplicates were plotted, with standard deviations (error bars).…”

“…Our investigation of RNA recombination is underpinned by the atomic structure of 3D pol elongation complexes (28,29) and a series of genetically stable alanine substitution mutations in 3D pol (30). We hypothesized that phylogenetically conserved features of 3D pol may exist to mediate RNA recombination.…”