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.

FIG. 1.

RNase T₁ oligonucleotides in PV positive- and negative-strand RNAs. (A) RNase T₁ oligonucleotides in PV A₍₈₄₎ positive-strand RNA. The largest RNase T₁ oligonucleotides within PV A₍₈₄₎ positive-strand RNA are illustrated. The G residue at the 3′ end of each oligonucleotide is numbered according to its location in the PV A₍₈₄₎ replicon RNA used in the experiments (as described in Materials and Methods). RNase T₁ digestion would liberate the 3′ poly(A) sequences from the heteropolymeric portion of PV RNA. (B) RNase T₁ oligonucleotides in PV A₍₈₄₎ negative-strand RNA. The four largest RNase T₁ oligonucleotides within PV A₍₈₄₎ negative-strand RNA, including their sequences, sizes, and positions relative to the 5′ end of negative-strand RNA, are illustrated.

FIG. 2.

VPg-linked poly(U) at the 5′ end of negative-strand RNA. (A) PV RF RNA fractionated by 1% agarose gel electrophoresis. PIRCs containing PV A₍₈₄₎ RNA templates were incubated in reaction mixtures containing 1 mM ATP, 250 μM GTP, 10 μM UTP, endogenous CTP from cytoplasmic extracts, and [α-³²P]UTP as described in “PV RNA replication” in Materials and Methods. Guanidine HCl (2 mM) and 3′-dCTP (200 μM) were present in specific RNA replication reaction mixtures as indicated. Radiolabeled products of the reactions were separated by 1% agarose gel electrophoresis and detected by phosphorimaging. The mobilities of PV RF RNA and VPg-linked poly(U) 3′-dCMP are indicated. (B) RNase T₁ oligonucleotides in PV RNAs. PV A₍₈₄₎ RNA templates were synthesized by T7 RNA transcription in reaction mixtures containing either [α-³²P]UTP (lane 1) or [α-³²P]ATP (lanes 2 and 3) as described in Materials and Methods, digested with RNase T₁ (lanes 1 and 3) or untreated (lane 2), and separated by electrophoresis in 7 M urea-20% polyacrylamide (see “RNase T₁ digestion” in Materials and Methods). [α-³²P]UTP-radiolabeled products of PV A₍₈₄₎ RNA replication (lanes 4 to 9) were digested with RNase T₁ and separated by electrophoresis in 7 M urea-20% polyacrylamide (see “PV RNA replication” and “RNase T₁ digestion” in Materials and Methods). PV A₍₈₄₎ RNA products were obtained from RNA replication reaction mixtures with (lanes 6 and 9) or without (lanes 4, 5, 7, and 8) 2 mM guanidine. RNase T₁ oligonucleotides were treated with proteinase K (lanes 5 and 8) or untreated (lanes 1 to 4, 6, 7, and 9). The mobilities of specific T₁ oligonucleotides and VPg-linked poly(U) are indicated.

FIG. 3.

VPg-linked poly(U) products from wild-type and CRE-independent negative-strand RNA synthesis and the influence of UTP concentrations on the length of poly(U) sequences. (A) PV RF RNA fractionated by 1% agarose gel electrophoresis. PIRCs containing PV A₍₈₄₎ RNA templates (lanes 1 to 3) or PV A₍₈₄₎ KO CRE RNA templates (lanes 4 to 6) were incubated in reaction mixtures containing 1 mM ATP, 250 μM GTP, and 250 μM CTP, either 10 μM UTP (lanes 1, 3, 4, 6, and 7) or 100 μM UTP (lanes 2 and 5), and [α-³²P]UTP, with (lanes 3 and 6) or without 2 mM guanidine HCl, as described in “PV RNA replication” in Materials and Methods. Reaction products soluble in 2 M LiCl were separated by 1% agarose gel electrophoresis and detected by phosphorimaging. The mobility of PV RF RNA is indicated. (B) RNase T₁ oligonucleotides in PV RNAs. PV A₍₈₄₎ RNA templates (lane 1) and PV A₍₈₄₎ KO CRE RNA templates (lane 2) were synthesized by T7 RNA transcription in reaction mixtures containing [α-³²P]ATP (see Materials and Methods), digested with RNase T₁, and separated by electrophoresis in 7 M urea-20% polyacrylamide (see “RNase T₁ digestion” in Materials and Methods) (lanes 1 and 2). [α-³²P]UTP-radiolabeled products of PV A₍₈₄₎ RNA replication (lanes 3 to 5) or PV A₍₈₄₎ KO CRE RNA replication (lanes 6 to 8) from reaction mixtures corresponding to lanes 1 to 6 of panel A were digested with RNase T₁ and separated by electrophoresis in 7 M urea-18% polyacrylamide (see “PV RNA replication” and “RNase T₁ digestion” in Materials and Methods). The mobilities of specific T₁ oligonucleotides and VPg-linked poly(U) are indicated. A 120-base-long RNA, corresponding to the 5′ 120 bases of PV RNA, was used as a size marker in the urea-polyacrylamide gels. (C) Size distributions of poly(A) sequences of PV A₍₈₄₎ RNA templates and the corresponding VPg-linked poly(U) products of RNA replication. Amounts of RNase T₁ oligonucleotides from PV A₍₈₄₎ RNA templates (green line), VPg-linked poly(U) products from RNA replication reaction mixtures containing 10 μM UTP (red line), and VPg-linked poly(U) products from RNA replication reaction mixtures containing 100 μM UTP (blue line) are indicated. The molar amounts of VPg-linked poly(U) products were calculated based on the corresponding molar amounts of RNase T₁ oligonucleotides from the heteropolymeric portion of each RNA product. PI, phosphorimaging.

FIG. 4.

VPg-linked poly(U) products synthesized from PV A₍₈₄₎ and PV A₍₈₄₎ A79C RNA templates. (A) Diagram of PV A₍₈₄₎ and PV A₍₈₄₎ A79C RNA templates and potential products of negative-strand RNA synthesis. ORF, open reading frame. (B) PV RF RNA fractionated by 1% agarose gel electrophoresis. PIRCs containing PV A₍₈₄₎ RNA templates (lanes 1 to 3) or PV A₍₈₄₎ A79C RNA templates (lanes 4 to 6) were incubated in reaction mixtures containing 1 mM ATP, 250 μM GTP, 250 μM CTP, 10 μM UTP, 2 mM guanidine HCl (lanes 3 and 6), and [α-³²P]UTP as described in “PV RNA replication” in Materials and Methods. Reaction products soluble in 2 M LiCl were separated by 1% agarose gel electrophoresis and detected by phosphorimaging. The mobility of PV RF RNA is indicated. (C) RNase T₁ oligonucleotides in PV RNAs. PV A₍₈₄₎ and PV A₍₈₄₎ A79C RNA templates were synthesized by T7 RNA transcription in reaction mixtures containing [α-³²P]ATP (see Materials and Methods), digested with RNase T₁, and separated by electrophoresis in 7 M urea-18% polyacrylamide (see “RNase T₁ digestion” in Materials and Methods) (lanes 1 and 2, respectively). [α-³²P]UTP-radiolabeled products of PV A₍₈₄₎ RNA replication (lanes 3 to 5) or PV A₍₈₄₎ A79C RNA replication (lanes 6 to 8) were digested with RNase T₁, treated with proteinase K (lanes 4 and 7), and separated by electrophoresis in 7 M urea-18% polyacrylamide (see “PV RNA replication” and “RNase T₁ digestion” in Materials and Methods). The mobilities of specific T₁ oligonucleotides and VPg-linked poly(U) products are indicated. The mobility of a 120-base-long RNA is noted to the left of the urea-polyacrylamide gel.

FIG. 5.

VPg-linked poly(U) products from PV A₍₈₄₎, A₍₅₁₎, and A₍₃₂₎ RNA templates. (A) PV RF RNA fractionated by 1% agarose gel electrophoresis. PIRCs containing PV A₍₈₄₎ (lanes 1 and 2), PV A₍₅₁₎ (lane 3), or PV A₍₃₂₎ (lane 4) RNA templates were incubated in reaction mixtures containing 1 mM ATP, 250 μM GTP, 250 μM CTP, 10 μM UTP, 2 mM guanidine HCl (lane 2), and [α-³²P]UTP as described in “PV RNA replication” in Materials and Methods. Reaction products soluble in 2 M LiCl were separated by 1% agarose gel electrophoresis and detected by phosphorimaging. The mobility of PV RF RNA is indicated. (B) [α-³²P]ATP-labeled PV A₍₈₄₎ (lane 1), PV A₍₅₁₎ (lane 2), or PV A₍₃₂₎ (lane 3) RNA templates and [α-³²P]UTP-labeled negative-strand RNA products of PV A₍₈₄₎ (lanes 4 to 6), PV A₍₅₁₎ (lanes 7 and 8), or PV A₍₃₂₎ (lanes 9 and 10) RNA replication were digested with RNase T₁, untreated (lanes 1 to 4, 6, 7, and 9) or treated with proteinase K (lanes 5, 8, and 10), and separated by electrophoresis in 7 M urea-18% polyacrylamide (see Materials and Methods). The mobilities of specific T₁ oligonucleotides and VPg-linked poly(U) products are indicated. The mobility of a 120-base-long RNA is noted to the left of the urea-polyacrylamide gel. (C) Size distributions of poly(U) products of RNA replication. Amounts of RNase T₁ oligonucleotides from PV A₍₈₄₎ (red), PV A₍₅₁₎ (green), and PV A₍₃₂₎ (blue) RNA templates were determined by phosphorimager analysis of data from lanes 5, 8, and 10 of panel B. Arbitrary PI units are plotted versus the relative mobilities of products in the gel. WT, wild-type. (D) Size distributions of T₁ oligonucleotides from PV A₍₈₄₎ RNA templates (green) and T₁ oligonucleotides from the corresponding VPg-linked negative-strand RNA products (blue). Data from lanes 1 and 5 of panel B were subjected to phosphorimager analyses. Arbitrary PI units are plotted versus the relative mobilities of products in the gel. (E) Size distributions of T₁ oligonucleotides from PV A₍₅₁₎ RNA templates (green) and T₁ oligonucleotides from the corresponding VPg-linked negative-strand RNA products (blue). Data from lanes 2 and 8 of panel B were subjected to phosphorimager analyses. Arbitrary PI units were plotted versus the relative mobilities of products in the gel. (F) Size distributions of T₁ oligonucleotides from PV A₍₃₂₎ RNA templates (green) and T₁ oligonucleotides from the corresponding VPg-linked negative-strand RNA products (blue). Data from lanes 3 and 10 of panel B were subjected to phosphorimager analyses. Arbitrary PI units are plotted versus the relative mobilities of products in the gel.

FIG. 6.

3′ poly(A) products of PV RNA replication. (A) PV RNAs fractionated by 1% agarose gel electrophoresis. PIRCs containing rPV A₍₈₄₎ (lanes 1 and 2) or rPV A₍₃₂₎ (lanes 3 and 4) RNA templates were incubated in reaction mixtures containing 1 mM ATP, 250 μM GTP, 250 μM CTP, 100 μM UTP, 2 mM guanidine HCl (lanes 2 and 4), and [α-³²P]ATP as described in “PV RNA replication” in Materials and Methods. Products of the reactions were separated by 1% agarose gel electrophoresis and detected by phosphorimaging. The mobilities of PV RI, RF, and positive-strand RNAs are indicated. (B) [α-³²P]ATP-labeled rPV A₍₈₄₎ (lane 1) or rPV A₍₃₂₎ (lane 2) RNA templates and [α-³²P]ATP-labeled products of rPV A₍₈₄₎ (lanes 3 and 4) or rPV A₍₃₂₎ (lanes 5 and 6) RNA replication were digested with RNase T₁, separated by electrophoresis in 7 M urea-18% polyacrylamide, and detected by phosphorimaging (see Materials and Methods). The mobilities of specific T₁ oligonucleotides and poly(A) sequences are indicated. The mobility of a 120-base-long RNA is noted to the left of the urea-polyacrylamide gel. (C) Size distributions of T₁ oligonucleotides from rPV A₍₈₄₎ RNA templates (green) and T₁ oligonucleotides from the corresponding products of RNA replication (red). Data from lanes 1 and 3 of panel B were subjected to phosphorimager analyses. Arbitrary PI units are plotted versus the relative mobilities of products in the gel. The molar amounts of 3′ poly(A) products of RNA replication were calculated based on the corresponding molar amounts of RNase T₁ oligonucleotides from the heteropolymeric portion of each positive-strand RNA product. Asterisks indicate the mobilities of heteropolymeric 37-, 36-, and 31-mers from the body of positive-strand RNA. The mobilities of rPV A₍₈₄₎ template and product poly(A) sequences are further annotated in the graph. (D) Size distributions of T₁ oligonucleotides from rPV A₍₃₂₎ RNA templates (green) and T₁ oligonucleotides from the corresponding products of RNA replication (red). Data from lanes 2 and 5 of panel B were subjected to phosphorimager analyses. Arbitrary PI units are plotted versus the relative mobilities of products in the gel. The molar amounts of 3′ poly(A) products of RNA replication were calculated based on the corresponding molar amounts of RNase T₁ oligonucleotides from the heteropolymeric portion of each positive-strand RNA product. Asterisks indicate the mobility of heteropolymeric 37-, 36-, and 31-mers from the body of positive-strand RNA. The mobilities of rPV A₍₃₂₎ template and product poly(A) sequences are further annotated in the graph.

FIG. 7.

rPV A₍₃₂₎ template sequences, poly(U) products, and corresponding poly(A) products. Size distributions of T₁ oligonucleotides from [α-³²P]ATP-labeled rPV A₍₃₂₎ RNA templates (from Fig. 4B, lane 2) (A), [α-³²P]ATP end-labeled rPV A₍₃₂₎ RNA templates (from Fig. 4B, lane 6) (B), [α-³²P]UTP-labeled PV A₍₃₂₎ negative-strand RNA products (from Fig. 5B, lane 10) (C), and [α-³²P]ATP-labeled rPV A₍₃₂₎ RNA products (from Fig. 4B, lane 5) (D) are shown. PI units are plotted versus the relative mobilities of products in the gels.

FIG. 8.

Poly(A) tails of PV RNAs recovered from HeLa cells. (A) Diagram of rPV A₍₃₂₎ RNA and mutant derivatives of rPV A₍₃₂₎ RNA containing G substitutions at poly(A) positions 10, 15, 20, and 25. (B) Sizes and sequences of poly(A) tails of T7 transcripts. (C) Sizes and sequences of poly(A) tails of PV RNA recovered from HeLa cells.

FIG. 9.

Reciprocal nature of poly(A) and poly(U) templates during PV RNA replication. Poly(A) sequences at the 3′ end of PV positive-strand RNA and poly(U) sequences at the 5′ end of negative-strand RNA function as reciprocal templates during PV RNA replication. (A) VPg-linked poly(U) synthesis. (B) 3′ poly(A) synthesis.