Altered interactions between stem-loop IV within the 5' noncoding region of coxsackievirus RNA and poly(rC) binding protein 2: effects on IRES-mediated translation and viral infectivity

Abstract

Coxsackievirus B3 (CVB3) is a causative agent of viral myocarditis, meningitis, pancreatitis, and encephalitis. Much of what is known about the coxsackievirus intracellular replication cycle is based on the information already known from a well-studied and closely related virus, poliovirus. Like that of poliovirus, the 5' noncoding region (5' NCR) of CVB3 genomic RNA contains secondary structures that function in both viral RNA replication and cap-independent translation initiation. For poliovirus IRES-mediated translation, the interaction of the cellular protein PCBP2 with a major secondary structure element (stem-loop IV) is required for gene expression. Previously, the complete secondary structure of the coxsackievirus 5' NCR was determined by chemical structure probing and overall, many of the RNA secondary structures bear significant similarity to those of poliovirus; however, the functions of the coxsackievirus IRES stem-loop structures have not been determined. Here we report that a CVB3 RNA secondary structure, stem-loop IV, folds similarly to poliovirus stem-loop IV and like its enterovirus counterpart, coxsackievirus stem-loop IV interacts with PCBP2. We used RNase foot-printing to identify RNA sequences protected following PCBP2 binding to coxsackievirus stem-loop IV. When nucleotide substitutions were separately engineered at two sites in coxsackievirus stem-loop IV to reduce PCBP2 binding, inhibition of IRES-mediated translation was observed. Both of these nucleotide substitutions were engineered into full-length CVB3 RNA and upon transfection into HeLa cells, the specific infectivities of both constructs were reduced and the recovered viruses displayed small-plaque phenotypes and slower growth kinetics compared to wild type virus.

Figure 1. Secondary structure determination of coxsackievirus stem-loop IV RNA

A. Enzymatic structure probing of coxsackievirus stem-loop IV RNA. Radio-labeled coxsackievirus stem-loop IV RNA (20–40 ng) was digested with RNase A (lanes 2–5), T₁ (lanes 6–13) and V₁ (lanes 14–17). The RNA was digested for 1–3 minutes at 25°C with RNase dilutions of 1:1 and 1:10 from stock. Urea (1 mM) was added to the T₁ digestion to obtain a G-ladder (lanes 8–9 and 12–13). The single-strand specific RNases used were RNase A, which cleaves after C and U residues, and RNase T₁, which cleaves after G residues. RNase V₁ was used to identify base-paired nucleotides. Undigested, full-length end-labeled coxsackievirus stem-loop IV RNAs are seen at the top of the gel. This shows “one-hit kinetics” of RNase digestion and ensures that the observed cleavage products are from completely formed RNA structures. The bands in the RNase-digested lanes correspond to specific nucleotides cleaved by the RNases. B. M-fold predicted secondary structure of coxsackievirus stem-loop IV RNA. Nucleotides in bold were identified through RNase structure probing. The structure shown represents nucleotides 235 to 430 for coxsackievirus B3. The poly(C)-loop, poly(Py)-bulge, and GNRA tetra-loop are indicated by dashed-line boxes.

Figure 1. Secondary structure determination of coxsackievirus stem-loop IV RNA

A. Enzymatic structure probing of coxsackievirus stem-loop IV RNA. Radio-labeled coxsackievirus stem-loop IV RNA (20–40 ng) was digested with RNase A (lanes 2–5), T₁ (lanes 6–13) and V₁ (lanes 14–17). The RNA was digested for 1–3 minutes at 25°C with RNase dilutions of 1:1 and 1:10 from stock. Urea (1 mM) was added to the T₁ digestion to obtain a G-ladder (lanes 8–9 and 12–13). The single-strand specific RNases used were RNase A, which cleaves after C and U residues, and RNase T₁, which cleaves after G residues. RNase V₁ was used to identify base-paired nucleotides. Undigested, full-length end-labeled coxsackievirus stem-loop IV RNAs are seen at the top of the gel. This shows “one-hit kinetics” of RNase digestion and ensures that the observed cleavage products are from completely formed RNA structures. The bands in the RNase-digested lanes correspond to specific nucleotides cleaved by the RNases. B. M-fold predicted secondary structure of coxsackievirus stem-loop IV RNA. Nucleotides in bold were identified through RNase structure probing. The structure shown represents nucleotides 235 to 430 for coxsackievirus B3. The poly(C)-loop, poly(Py)-bulge, and GNRA tetra-loop are indicated by dashed-line boxes.

Figure 2. Mobility shift assays of coxsackievirus stem-loop IV RNA with PCBP2 and PCBP1

A. Electrophoretic mobility shift assays of poliovirus and coxsackievirus stem-loop IV RNA with recombinant PCBP2 and PCBP1. In vitro transcribed, ³²P-labeled stem-loop IV RNA at a final concentration of 0.1 nM was incubated with increasing amounts of purified recombinant PCBP2 for 10 min at 30°C, and the reaction was then subjected to native polyacrylamide gel electrophoresis. Lanes 1, 4, 7, and 11 are the RNA alone. Poliovirus stem-loop IV RNA (lanes 2–3) and coxsackievirus stem-loop IV RNA (lanes 5–6 and 8–10) can form ribonucleoprotein (RNP) complexes with PCBP2. The RNP complex is denoted by the arrow. PCBP1 (lanes 12–13) is unable to interact with coxsackievirus stem-loop IV RNA. B. Competition electrophoretic mobility shift assay of poliovirus stem-loop IV RNA and PCBP2 using unlabeled coxsackievirus stem-loop IV RNA. In vitro transcribed ³²P-labeled poliovirus stem-loop IV RNA (1 nM) was incubated with PCBP2 (50 nM) in the presence of increasing amounts of unlabeled poliovirus (lanes 3–6) or coxsackievirus stem-loop IV RNAs (lanes 7–10) for 10 min at 30°C. The RNP complexes were then resolved on a native polyacrylamide gel. RNP complexes are denoted by the arrow. C. Graph of relative binding affinities of PCBP2 for poliovirus and coxsackievirus stem-loop IV RNA. The relative affinities of PCBP2 for the stem-loop IV RNAs were determined by measuring the band intensity of the bound RNA divided by the sum of the free RNA and bound RNA. It was observed that 15 nM unlabeled poliovirus stem-loop IV RNA was enough to dissociate half of the labeled poliovirus stem-loop IV-PCBP2 RNP complex, while it required 35 nM unlabeled coxsackievirus stem-loop IV RNA to obtain the same level of competition.

Figure 2. Mobility shift assays of coxsackievirus stem-loop IV RNA with PCBP2 and PCBP1

A. Electrophoretic mobility shift assays of poliovirus and coxsackievirus stem-loop IV RNA with recombinant PCBP2 and PCBP1. In vitro transcribed, ³²P-labeled stem-loop IV RNA at a final concentration of 0.1 nM was incubated with increasing amounts of purified recombinant PCBP2 for 10 min at 30°C, and the reaction was then subjected to native polyacrylamide gel electrophoresis. Lanes 1, 4, 7, and 11 are the RNA alone. Poliovirus stem-loop IV RNA (lanes 2–3) and coxsackievirus stem-loop IV RNA (lanes 5–6 and 8–10) can form ribonucleoprotein (RNP) complexes with PCBP2. The RNP complex is denoted by the arrow. PCBP1 (lanes 12–13) is unable to interact with coxsackievirus stem-loop IV RNA. B. Competition electrophoretic mobility shift assay of poliovirus stem-loop IV RNA and PCBP2 using unlabeled coxsackievirus stem-loop IV RNA. In vitro transcribed ³²P-labeled poliovirus stem-loop IV RNA (1 nM) was incubated with PCBP2 (50 nM) in the presence of increasing amounts of unlabeled poliovirus (lanes 3–6) or coxsackievirus stem-loop IV RNAs (lanes 7–10) for 10 min at 30°C. The RNP complexes were then resolved on a native polyacrylamide gel. RNP complexes are denoted by the arrow. C. Graph of relative binding affinities of PCBP2 for poliovirus and coxsackievirus stem-loop IV RNA. The relative affinities of PCBP2 for the stem-loop IV RNAs were determined by measuring the band intensity of the bound RNA divided by the sum of the free RNA and bound RNA. It was observed that 15 nM unlabeled poliovirus stem-loop IV RNA was enough to dissociate half of the labeled poliovirus stem-loop IV-PCBP2 RNP complex, while it required 35 nM unlabeled coxsackievirus stem-loop IV RNA to obtain the same level of competition.

Figure 3. RNase foot-printing assay of coxsackievirus stem-loop IV RNA and PCBP2

A.PCBP2 protection of multiple regions of coxsackievirus stem-loop IV RNA. Radio-labeled coxsackievirus stem-loop IV RNA (20–40 ng) was incubated with 5 μM of PCBP2, denoted by +, at 30°C for 10 minutes and subjected to RNase A (lanes 4–7), T₁ (lanes 8–13) and V₁ (lanes 14–17) digestion. The RNAs were digested for 1–3 minutes at 25°C with RNase dilution of 1:1 and 1:10 from stock. Urea (1 mM) was added to the T₁ digestion to obtain a G-ladder (lanes 10 and 13). Boxes indicate nucleotides within coxsackievirus stem-loop IV RNA that were protected by PCBP2 from RNase cleavage. B. Secondary structure showing regions of sequence in coxsackievirus stem-loop IV RNA protected by PCBP2 from RNase digestion (denoted by boxes). PCBP2 protects the poly(C)-loop, the stem leading to the internal bulge, and the helix region adjacent to the poly(Py)-bulge.

Figure 3. RNase foot-printing assay of coxsackievirus stem-loop IV RNA and PCBP2

A.PCBP2 protection of multiple regions of coxsackievirus stem-loop IV RNA. Radio-labeled coxsackievirus stem-loop IV RNA (20–40 ng) was incubated with 5 μM of PCBP2, denoted by +, at 30°C for 10 minutes and subjected to RNase A (lanes 4–7), T₁ (lanes 8–13) and V₁ (lanes 14–17) digestion. The RNAs were digested for 1–3 minutes at 25°C with RNase dilution of 1:1 and 1:10 from stock. Urea (1 mM) was added to the T₁ digestion to obtain a G-ladder (lanes 10 and 13). Boxes indicate nucleotides within coxsackievirus stem-loop IV RNA that were protected by PCBP2 from RNase cleavage. B. Secondary structure showing regions of sequence in coxsackievirus stem-loop IV RNA protected by PCBP2 from RNase digestion (denoted by boxes). PCBP2 protects the poly(C)-loop, the stem leading to the internal bulge, and the helix region adjacent to the poly(Py)-bulge.

Figure 4. Mobility shift assay of poly(G) substituted coxsackievirus stem-loop IV RNA with PCBP2

A.M-fold predicted secondary structure of wild type, poly(G)-loop and poly(G)- bulge substituted coxsackievirus stem-loop IV RNAs. Wild type sequences corresponding to the poly(C)-loop or poly(C)-bulge are boxed with solid lines while mutated sequences are boxed with dashed lines. B. In vitro transcribed ³²P-labeled wild type (lanes 1–5), poly(G)-bulge (lanes 6–10), and poly(G)-loop (lanes 11–15) coxsackievirus stem-loop IV RNAs (1 nM) were incubated with increasing amounts of purified recombinant PCBP2 (250–750 nM) for 10 min at 30^°C. The reactions were then resolved by native polyacrylamide gel electrophoresis. Lanes 1, 6, and 11 are the RNA alone. RNP complexes and free probe RNAs are denoted by arrows. Competition mobility shift assays were carried out with 100 nM unlabeled poliovirus stem-loop IV RNA (lanes 5, 10, and 15).

Figure 4. Mobility shift assay of poly(G) substituted coxsackievirus stem-loop IV RNA with PCBP2

A.M-fold predicted secondary structure of wild type, poly(G)-loop and poly(G)- bulge substituted coxsackievirus stem-loop IV RNAs. Wild type sequences corresponding to the poly(C)-loop or poly(C)-bulge are boxed with solid lines while mutated sequences are boxed with dashed lines. B. In vitro transcribed ³²P-labeled wild type (lanes 1–5), poly(G)-bulge (lanes 6–10), and poly(G)-loop (lanes 11–15) coxsackievirus stem-loop IV RNAs (1 nM) were incubated with increasing amounts of purified recombinant PCBP2 (250–750 nM) for 10 min at 30^°C. The reactions were then resolved by native polyacrylamide gel electrophoresis. Lanes 1, 6, and 11 are the RNA alone. RNP complexes and free probe RNAs are denoted by arrows. Competition mobility shift assays were carried out with 100 nM unlabeled poliovirus stem-loop IV RNA (lanes 5, 10, and 15).

Figure 5. Luciferase assay of coxsackievirus IRES with poly(G) substitutions in stem-loop IV

A.Luciferase reporter RNAs (50 fmol) containing the poliovirus 5′ NCR, coxsackievirus 5′ NCR, poly(G)-loop coxsackievirus 5′NCR, or poly(G)-bulge coxsackievirus 5′ NCR were incubated in HeLa S10 cytoplasmic extracts for 2.5 h at 30°C and then assayed for luciferase. The relative light unit (RLU) values are averages from three separate experiments.

Figure 6. Growth properties of coxsackievirus mutants with poly(G) substitutions in stem-loop IV RNA

A.Plaque size morphology. In vitro transcribed full-length wild type, poly(G)-loop, or poly(G)-bulge mutated stem-loop IV coxsackievirus RNAs were transfected into HeLa cell monolayers. Readily visible plaques formed at 3 days post-transfection. Wild type coxsackievirus RNA produced large plaques, while the poly(G) mutated stem-loop coxsackievirus RNAs produced smaller plaques. B. Single cycle growth analysis of coxsackievirus mutants with poly(rG) nucleotide substitutions in stem-loop IV. HeLa cell monolayers were infected with wild type -◆-, poly(G)-loop -■-, or poly(G)-bulge -▲-mutated coxsackieviruses at a multiplicity of infection (MOI) of 10. The infected monolayers were harvested at the indicated times after infection and the virus titers were determined by plaque assay on HeLa cells as described in Material and Methods. The virus titers are reported as the log₁₀ PFU (plaque forming units) per milliliter (ml).