Poly(A) at the 3′ End of Positive-Strand RNA and VPg-Linked Poly(U) at the 5′ End of Negative-Strand RNA Are Reciprocal Templates during Replication of Poliovirus RNA

Abstract

A 3 poly(A) tail is a common feature of picornavirus RNA genomes and the RNA genomes of many other positive-strand RNA viruses. We examined the manner in which the homopolymeric poly(A) and poly(U) portions of poliovirus (PV) positive-and negative-strand RNAs were used as reciprocal templates during RNA replication. Poly(A) sequences at the 3 end of viral positive-strand RNA were transcribed into VPg-linked poly(U) products at the 5 end of negative-strand RNA during PV RNA replication. Subsequently, VPg-linked poly(U) sequences at the 5 ends of negative-strand RNA templates were transcribed into poly(A) sequences at the 3 ends of positive-strand RNAs. The homopolymeric poly(A) and poly(U) portions of PV RNA products of replication were heterogeneous in length and frequently longer than the corresponding homopolymeric sequences of the respective viral RNA templates. The data support a model of PV RNA replication wherein reiterative transcription of homopolymeric templates ensures the synthesis of long 3 poly(A) tails on progeny RNA genomes.Many positive-strand RNA viruses (e.g., members of the Picornavirales, Nidovirales, Togaviridae, Caliciviridae, and Astroviridae) have long poly(A) sequences at the 3Ј termini of their RNA genomes (16,25), yet the mechanisms by which 3Ј poly(A) sequences are derived during viral replication are unclear. Picornavirus RNA genomes, including that of poliovirus (PV), have a covalently linked 5Ј-terminal protein called VPg (viral protein, genome linked), a 5Ј untranslated region (UTR), a single large open reading frame, a 3Ј UTR, and a poly(A) tail of variable length (ϳ20 to 150 adenosine residues) (1). RNA polymerases encoded by viruses in the order Picornavirales utilize viral proteins (and their nucleotidylylated intermediates) to prime the initiation of RNA replication at the 3Ј termini of viral RNA templates (28,29,31,34). In this mechanism, the viral protein VPg becomes covalently linked to the 5Ј ends of both positive-and negative-strand RNAs during viral RNA replication (30, 32). PV, which is commonly studied to elucidate mechanisms of picornavirus replication, is viable when the 3Ј UTR of the genome is deleted (12, 44); however, the 3Ј poly(A) tail is essential for RNA replication (33, 39). The length of the 3Ј poly(A) tail required for virus viability and for efficient negative-strand RNA synthesis has been examined in some detail (35,45). PV RNAs with 3Ј poly(A) tails less than 9 bases long support less than 1% of wild-type negative-strand RNA synthesis, whereas poly(A) tails Ն20 bases long support wild-type levels of negative-strand RNA synthesis (35).In this investigation, we programmed PV RNAs with defined 3Ј 84-, 51-, and 32-base poly(A) sequences [designated poly(A) (84) , poly(A) (51) , and poly(A) (32) , respectively] into cell-free reactions that faithfully reconstitute all of the metabolic steps of viral mRNA translation (11,22,23) and viral RNA replication (5, 7, 27). A significant advantage of this experimental system is the ability to study one cycle of seq...

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

“…We found that alanine substitution mutations within phylogenetically conserved regions of 3D pol resulted in populations of virion RNA with altered poly(A) tail lengths, consistent with the predicted reiterative transcription mechanisms of viral RNA replication (8). Data from reaction mixtures containing purified 3D pol confirmed that 3D pol mutations influenced the amounts of reiterative transcription on a defined poly(U) template.…”

“…Poliovirus type 1 (Mahoney) was used for this study. Poliovirus cDNA clones with genetically encoded poly(A) tails 32, 51, and 84 bases in length were previously described (8). 3D pol muta-tions were introduced into a subgenomic cDNA clone with a poly (A) tail of 84 bases [pRNA2 poly A (84) ] by site-directed mutagenesis (QuikChange XL-II site-directed mutagenesis kit; Stratagene, Inc.) and verified by sequencing the 3CD genes (nucleotides 5438 to 7440).…”

“…Poliovirus and EMCV virion RNAs were 3= end labeled in reaction mixtures containing T4 single-stranded RNA ligase (New England BioLabs) and [ 32 P]pCp (MP Biomedicals, Solon, OH). Poliovirus RNA transcripts with genetically encoded poly(A) tails 32, 51, and 84 bases in length were radiolabeled in T7 transcription reaction mixtures containing [␣-32 P]ATP as previously described (8). Radiolabeled RNAs were digested with RNase T1 and fractionated by electrophoresis in 7 M urea-18% polyacrylamide gels in TBE buffer (89 mM Tris base, 89 mM boric acid, pH 8.3, 2 mM EDTA) for 6 h at 25 W (8).…”

“…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).…”

“…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.…”