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Chapter 18 : The Aphtho- and Cardiovirus “Primary” 2A/2B Polyprotein “Cleavage”

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Abstract:

In the case of the aphtho- and cardioviruses, the primary cleavage in the region of the polyprotein was known to be different, occurring at the C terminus of 2A. Precursor forms spanning the 2A/2B junction are not observed in aphtho- or cardiovirus polyprotein processing. Deletions downstream of 2A did not appear to affect cleavage. Experiments analyzing the endogenous processing properties of recombinant aphthovirus indicated that the cleavage activity could be a property of the 2A oligopeptidic region alone. Consistent with this notion, studies on the endogenous processing properties of domains of the cardiovirus Theiler’s murine encephalomyelitis virus (TMEV) polyprotein localized the 2A/2B cleavage activity within the 2AB region. With artificial reporter polyprotein systems the 2A/2B cleavage activity of both EMCV and TMEV was subsequently mapped to the C-terminal 18 aa of their 2A proteins—these cardiovirus sequences being as efficient as the FMDV 2A in mediating cleavage. The molar excess of the translation product N terminal of 2A over that C-terminal of 2A is a product of inserting the 2A sequence into our artificial polyprotein systems. In summary, the authors and others have shown the aphtho- and cardiovirus 2A/2B cleavage is mediated by an oligopeptidic region, representing either the whole (aphthoviruses) or part (cardioviruses) of the 2A region.

Citation: Ryan M, Luke G, Hughes L, Cowton V, Ten Dam E, Li X, Donnelly M, Mehrotra A, Gani D. 2002. The Aphtho- and Cardiovirus “Primary” 2A/2B Polyprotein “Cleavage”, p 213-223. In Semler B, Wimmer E (ed), Molecular Biology of Picornavirus. ASM Press, Washington, DC. doi: 10.1128/9781555817916.ch18

Key Concept Ranking

Mobile Genetic Elements
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Theiler's Murine Encephalomyelitis
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Figures

Image of FIGURE 1
FIGURE 1

Picornavirus polyproteins. (A) The polyprotein organizations of entero-, rhino-, cardio-, and aphthoviruses are shown together (boxed areas) with the sites of primary polyprotein cleavage. (B) The sequences of the C-terminal region of cardioviruses and the 2A region of aphthoviruses are shown together with 2A-like sequences from other virus and cellular sequences.

Citation: Ryan M, Luke G, Hughes L, Cowton V, Ten Dam E, Li X, Donnelly M, Mehrotra A, Gani D. 2002. The Aphtho- and Cardiovirus “Primary” 2A/2B Polyprotein “Cleavage”, p 213-223. In Semler B, Wimmer E (ed), Molecular Biology of Picornavirus. ASM Press, Washington, DC. doi: 10.1128/9781555817916.ch18
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Image of FIGURE 2
FIGURE 2

Translational analyses. Artificial reporter polyproteins (boxed areas) used to program in vitro translation systems are shown together with translation profiles obtained from rabbit reticulocyte lysates. (A) The FMDV region was either N-terminally extended by the incorporation of FMDV 1D sequences ot by stepwise deletion. (B) Cleavage activities (%) are shown. (C) Site-directed mutants of FMDV 2A were constructed and the cleavage activities analyzed with in vitro translation systems.

Citation: Ryan M, Luke G, Hughes L, Cowton V, Ten Dam E, Li X, Donnelly M, Mehrotra A, Gani D. 2002. The Aphtho- and Cardiovirus “Primary” 2A/2B Polyprotein “Cleavage”, p 213-223. In Semler B, Wimmer E (ed), Molecular Biology of Picornavirus. ASM Press, Washington, DC. doi: 10.1128/9781555817916.ch18
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Image of FIGURE 3
FIGURE 3

2A-like sequences. (A) Insect virus polyproteins are shown together with the location of the 2A-like sequence (shaded rectangle), picornavirus protein 2C-like domain (open circles), proteinase domain (open squares), and polymerase domains (open diamonds). (B) The position of the type C rotavirus 2A-like sequence (shaded rectangle) is shown. (C) The sequence and position of the 2A-like sequences from spp. repeated sequences are shown (shaded rectangle).

Citation: Ryan M, Luke G, Hughes L, Cowton V, Ten Dam E, Li X, Donnelly M, Mehrotra A, Gani D. 2002. The Aphtho- and Cardiovirus “Primary” 2A/2B Polyprotein “Cleavage”, p 213-223. In Semler B, Wimmer E (ed), Molecular Biology of Picornavirus. ASM Press, Washington, DC. doi: 10.1128/9781555817916.ch18
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Image of FIGURE 4
FIGURE 4

Translational model of 2A-mediated “cleavage.” Step (i): The synthesis of the 2A peptide sequence is completed with the 2A-peptidyl-tRNA complex in the ribosomal A site. Step (ii): This complex is translocated from the A to P site by eEF2. Step (iii): Prolyl-tRNA is bound to the A site. Step (iv): Cleavage of the peptide-tRNA ester linkage occurs. Step (v): The nascent peptide is released from the ribosome. Step (vi): Prolyl-tRNA is translocated from the A to P site, the next aminoacyl-tRNA is bound to the A site, and translation of the downstream product continues.

Citation: Ryan M, Luke G, Hughes L, Cowton V, Ten Dam E, Li X, Donnelly M, Mehrotra A, Gani D. 2002. The Aphtho- and Cardiovirus “Primary” 2A/2B Polyprotein “Cleavage”, p 213-223. In Semler B, Wimmer E (ed), Molecular Biology of Picornavirus. ASM Press, Washington, DC. doi: 10.1128/9781555817916.ch18
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