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. 2003 Jan;9(1):124–137. doi: 10.1261/rna.2950603

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FIGURE 1. — Poliovirus CRE predicted structure and modification. (A) Predicted structure of the native poliovirus type 3 CRE. The designation and location of the stem regions (based on Goodfellow et al. 2000) are indicated together with modifications made to these regions. Nucleotides in bold italics in Stem 3 represent defined mutations, the substitution of two or more of which render the CRE inactive (Goodfellow et al. 2000). The G nucleotide in bold italics in Inv1 (inversion of the Stem 1 region) represents a substitution made to avoid introducing a termination codon to the sequence. The conserved functional A¹A²A³CA motif in the terminal loop of the CRE is highlighted in bold. (B) Schematic diagram of virus genomes and subgenomic replicons used in this study. The noncoding regions are indicated as thin solid horizontal lines. Coding regions are indicated as wide open (unmodified poliovirus sequence) or filled (modified by replacement or mutagenesis) boxes. The region encoding chloramphenicol acetyl transferase expressed by subgenomic replicons is indicated as a solid black filled box labeled CAT. The disrupted native CRE sequence (located within the region encoding the virus 2C protein) is shown with an X. Subgenomic replicon-based cassette vectors (pT7/Rep3/SL3c and pT7/Rep3c) were constructed by the introduction of unique restriction sites flanking a ‘‘stuffer region’’ (indicated by the character ∼), which introduces a frameshift to the encoded polyprotein (see D). Relevant naturally present restriction sites used to introduce sequences into the 5′ NCR are indicated. The panel on the right-hand side defines the role the particular construct was used for in these studies, and whether there is a functional CRE in the native location within the region of the polyprotein encoding 2C. (C) The cassette region of pT7/Rep3c and pT7/Rep3/SL3c. The sequence through the engineered cassette region is shown, together with relevant translation products (indicated using single-letter amino acid codes). Without modification the cassette region is translated in the open reading frame (ORF) highlighted ‘‘0’’, which terminates 19 residues after the carboxyl terminus of the CAT protein. Digestion with Bss HII and Sma I allows replacement of the removed cassette stuffer region (shown in bold text) with complementary oligonucleotides that include the CRE sequence of interest. The oligonucleotides were designed to restore the polyprotein open reading frame to the +1 frame as shown. The YG dipeptide cleaved by the virus 2A^pro protease is shown in double height type, below the filled blocks indicating the short spacer region that flanks the inserted sequence and the start of the 2A^pro protease open reading frame.

FIGURE 1. — Poliovirus CRE predicted structure and modification. (A) Predicted structure of the native poliovirus type 3 CRE. The designation and location of the stem regions (based on Goodfellow et al. 2000) are indicated together with modifications made to these regions. Nucleotides in bold italics in Stem 3 represent defined mutations, the substitution of two or more of which render the CRE inactive (Goodfellow et al. 2000). The G nucleotide in bold italics in Inv1 (inversion of the Stem 1 region) represents a substitution made to avoid introducing a termination codon to the sequence. The conserved functional A¹A²A³CA motif in the terminal loop of the CRE is highlighted in bold. (B) Schematic diagram of virus genomes and subgenomic replicons used in this study. The noncoding regions are indicated as thin solid horizontal lines. Coding regions are indicated as wide open (unmodified poliovirus sequence) or filled (modified by replacement or mutagenesis) boxes. The region encoding chloramphenicol acetyl transferase expressed by subgenomic replicons is indicated as a solid black filled box labeled CAT. The disrupted native CRE sequence (located within the region encoding the virus 2C protein) is shown with an X. Subgenomic replicon-based cassette vectors (pT7/Rep3/SL3c and pT7/Rep3c) were constructed by the introduction of unique restriction sites flanking a ‘‘stuffer region’’ (indicated by the character ∼), which introduces a frameshift to the encoded polyprotein (see D). Relevant naturally present restriction sites used to introduce sequences into the 5′ NCR are indicated. The panel on the right-hand side defines the role the particular construct was used for in these studies, and whether there is a functional CRE in the native location within the region of the polyprotein encoding 2C. (C) The cassette region of pT7/Rep3c and pT7/Rep3/SL3c. The sequence through the engineered cassette region is shown, together with relevant translation products (indicated using single-letter amino acid codes). Without modification the cassette region is translated in the open reading frame (ORF) highlighted ‘‘0’’, which terminates 19 residues after the carboxyl terminus of the CAT protein. Digestion with Bss HII and Sma I allows replacement of the removed cassette stuffer region (shown in bold text) with complementary oligonucleotides that include the CRE sequence of interest. The oligonucleotides were designed to restore the polyprotein open reading frame to the +1 frame as shown. The YG dipeptide cleaved by the virus 2A^pro protease is shown in double height type, below the filled blocks indicating the short spacer region that flanks the inserted sequence and the start of the 2A^pro protease open reading frame.