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mRNA maturation in giant viruses: variation on a theme.

Priet S, Lartigue A, Debart F, Claverie JM, Abergel C - Nucleic Acids Res. (2015)

Bottom Line: Unexpectedly, the vPAPs are homodimeric and uniquely self-processive.The vPAP backbone structures exhibit a symmetrical architecture with two subdomains sharing a nucleotidyltransferase topology, suggesting that vPAPs originate from an ancestral duplication.A Poxvirus processivity factor homologue encoded by Megavirus chilensis displays a conserved 5'-GpppA 2'O methyltransferase activity but is also able to internally methylate the mRNAs' polyA tails.

View Article: PubMed Central - PubMed

Affiliation: Architecture et Fonction des Macromolécules Biologiques, CNRS UMR 7257, Aix-Marseille Université, 163 Avenue de Luminy, Case 932, 13288 Marseille cedex 9, France.

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Molecular determinant of mimiviruses PAP dimerization and processivity. (A) Mg561 dimer symmetry. Rotational symmetry axis is shown in gray. Figure 1 color code is applied to one monomer and the second monomer is in light gray. (B) Buried surface area of the Mg561 dimer. The dimer interface buries a surface area (yellow) of about 3200 Å2, which corresponds to 11.5% of each monomer's solvent accessible surface area (about 27 500 Å2). The first monomer is in cyan, the second one is in magenta. Cation locations are marked as green balls. (C) Gel filtration analysis of ΔD1 H6c-Mg561 and ΔD1 H6c-R341 mutants. Overlay of UV traces of the full-length H6c-Mg561 (dark blue curve) and ΔD1 H6c-Mg561 (light blue curve) proteins (Left panel) and of the full-length H6c-R341 (dark green curve) and ΔD1 H6c-R341 (light green curve) proteins (Right panel). Full-length H6c-Mg561 and H6c-R341 eluted as a single peak corresponding to a dimeric state: apparent molecular masses were 116.8 and 176.7 kDa, respectively, versus the calculated ones, 125.7 and 136.8 kDa. At least 90% of the ΔD1 H6c-Mg561 and ΔD1 H6c-R341 protein eluted as monomers: apparent molecular masses were 57.8 and 71.1 kDa versus the calculated 57.7 and 63.4 kDa. GE Healthcare markers were used for calibration. (D) PolyA polymerase assay of H6c-Mg561, ΔD1 H6c-Mg561, H6c-R341 and ΔD1 H6c-R341 on a 20-mers RNA primer in the presence of Mg2+ or Mn2+ as catalytic ions. Time course is in min.
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Figure 4: Molecular determinant of mimiviruses PAP dimerization and processivity. (A) Mg561 dimer symmetry. Rotational symmetry axis is shown in gray. Figure 1 color code is applied to one monomer and the second monomer is in light gray. (B) Buried surface area of the Mg561 dimer. The dimer interface buries a surface area (yellow) of about 3200 Å2, which corresponds to 11.5% of each monomer's solvent accessible surface area (about 27 500 Å2). The first monomer is in cyan, the second one is in magenta. Cation locations are marked as green balls. (C) Gel filtration analysis of ΔD1 H6c-Mg561 and ΔD1 H6c-R341 mutants. Overlay of UV traces of the full-length H6c-Mg561 (dark blue curve) and ΔD1 H6c-Mg561 (light blue curve) proteins (Left panel) and of the full-length H6c-R341 (dark green curve) and ΔD1 H6c-R341 (light green curve) proteins (Right panel). Full-length H6c-Mg561 and H6c-R341 eluted as a single peak corresponding to a dimeric state: apparent molecular masses were 116.8 and 176.7 kDa, respectively, versus the calculated ones, 125.7 and 136.8 kDa. At least 90% of the ΔD1 H6c-Mg561 and ΔD1 H6c-R341 protein eluted as monomers: apparent molecular masses were 57.8 and 71.1 kDa versus the calculated 57.7 and 63.4 kDa. GE Healthcare markers were used for calibration. (D) PolyA polymerase assay of H6c-Mg561, ΔD1 H6c-Mg561, H6c-R341 and ΔD1 H6c-R341 on a 20-mers RNA primer in the presence of Mg2+ or Mn2+ as catalytic ions. Time course is in min.

Mentions: As observed in solution (see later), the Mg561 crystal structure reveals a very stable homodimer with an almost perfect two-fold symmetry axis (179°) and a 3200 Å2 buried surface area upon dimerization (Figures 3 and 4). The dimer structure highlights a large tunnel at the interface between the two monomers. The N-terminal domain (D1) encompasses an extended strand followed by a long helix (α1) bent at the P28 residue (Figures 1 and 3). In each monomer, this helix creates a domain swap, and makes contact with the D4 loop located between α13 and β16 and the α7 helix of the D2 domains of the other monomer to form the dimer structure. In the VP55 structure (PDB: 2GA9, (26)), the equivalent helix (α8, Figure 1B) is making contacts with the D4 (α18 and β16) and the D2 (α15) domains of the same molecule. In Mg561, next to the α1 helix, another proline residue (P44) induces a change in orientation of the main chain, making the first helix of the domain D2 (red) almost perpendicular to the α1 helix. We investigated if VP55 could form a related dimer at the high protein concentration found in crystals by scrutinizing the crystallographic structure of the unliganded monomeric VP55 containing two monomers per asymmetric unit (PDB: 3OWG, (51)). Even using the symmetry related molecules, no occurrences of such a dimer was visible. Reciprocally, none of the symmetry related molecules in the Mg561 and the R341 structure reproduce the dimer found in the VP55 structure. To further characterize the molecular determinants of the dimerization of Mg561 and the role of the dimerization on the polyadenylation activity, we made deletion of the Mg561 (ΔD1 Mg561) and R341 (ΔD1 R341) N-terminal domains. Gel filtration experiments revealed that the mutants were monomeric and although they still co-purified with RNA, they were inactive (Figure 4), suggesting that the dimeric state and the N-terminal domain were required for the polyadenylation activity.


mRNA maturation in giant viruses: variation on a theme.

Priet S, Lartigue A, Debart F, Claverie JM, Abergel C - Nucleic Acids Res. (2015)

Molecular determinant of mimiviruses PAP dimerization and processivity. (A) Mg561 dimer symmetry. Rotational symmetry axis is shown in gray. Figure 1 color code is applied to one monomer and the second monomer is in light gray. (B) Buried surface area of the Mg561 dimer. The dimer interface buries a surface area (yellow) of about 3200 Å2, which corresponds to 11.5% of each monomer's solvent accessible surface area (about 27 500 Å2). The first monomer is in cyan, the second one is in magenta. Cation locations are marked as green balls. (C) Gel filtration analysis of ΔD1 H6c-Mg561 and ΔD1 H6c-R341 mutants. Overlay of UV traces of the full-length H6c-Mg561 (dark blue curve) and ΔD1 H6c-Mg561 (light blue curve) proteins (Left panel) and of the full-length H6c-R341 (dark green curve) and ΔD1 H6c-R341 (light green curve) proteins (Right panel). Full-length H6c-Mg561 and H6c-R341 eluted as a single peak corresponding to a dimeric state: apparent molecular masses were 116.8 and 176.7 kDa, respectively, versus the calculated ones, 125.7 and 136.8 kDa. At least 90% of the ΔD1 H6c-Mg561 and ΔD1 H6c-R341 protein eluted as monomers: apparent molecular masses were 57.8 and 71.1 kDa versus the calculated 57.7 and 63.4 kDa. GE Healthcare markers were used for calibration. (D) PolyA polymerase assay of H6c-Mg561, ΔD1 H6c-Mg561, H6c-R341 and ΔD1 H6c-R341 on a 20-mers RNA primer in the presence of Mg2+ or Mn2+ as catalytic ions. Time course is in min.
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Figure 4: Molecular determinant of mimiviruses PAP dimerization and processivity. (A) Mg561 dimer symmetry. Rotational symmetry axis is shown in gray. Figure 1 color code is applied to one monomer and the second monomer is in light gray. (B) Buried surface area of the Mg561 dimer. The dimer interface buries a surface area (yellow) of about 3200 Å2, which corresponds to 11.5% of each monomer's solvent accessible surface area (about 27 500 Å2). The first monomer is in cyan, the second one is in magenta. Cation locations are marked as green balls. (C) Gel filtration analysis of ΔD1 H6c-Mg561 and ΔD1 H6c-R341 mutants. Overlay of UV traces of the full-length H6c-Mg561 (dark blue curve) and ΔD1 H6c-Mg561 (light blue curve) proteins (Left panel) and of the full-length H6c-R341 (dark green curve) and ΔD1 H6c-R341 (light green curve) proteins (Right panel). Full-length H6c-Mg561 and H6c-R341 eluted as a single peak corresponding to a dimeric state: apparent molecular masses were 116.8 and 176.7 kDa, respectively, versus the calculated ones, 125.7 and 136.8 kDa. At least 90% of the ΔD1 H6c-Mg561 and ΔD1 H6c-R341 protein eluted as monomers: apparent molecular masses were 57.8 and 71.1 kDa versus the calculated 57.7 and 63.4 kDa. GE Healthcare markers were used for calibration. (D) PolyA polymerase assay of H6c-Mg561, ΔD1 H6c-Mg561, H6c-R341 and ΔD1 H6c-R341 on a 20-mers RNA primer in the presence of Mg2+ or Mn2+ as catalytic ions. Time course is in min.
Mentions: As observed in solution (see later), the Mg561 crystal structure reveals a very stable homodimer with an almost perfect two-fold symmetry axis (179°) and a 3200 Å2 buried surface area upon dimerization (Figures 3 and 4). The dimer structure highlights a large tunnel at the interface between the two monomers. The N-terminal domain (D1) encompasses an extended strand followed by a long helix (α1) bent at the P28 residue (Figures 1 and 3). In each monomer, this helix creates a domain swap, and makes contact with the D4 loop located between α13 and β16 and the α7 helix of the D2 domains of the other monomer to form the dimer structure. In the VP55 structure (PDB: 2GA9, (26)), the equivalent helix (α8, Figure 1B) is making contacts with the D4 (α18 and β16) and the D2 (α15) domains of the same molecule. In Mg561, next to the α1 helix, another proline residue (P44) induces a change in orientation of the main chain, making the first helix of the domain D2 (red) almost perpendicular to the α1 helix. We investigated if VP55 could form a related dimer at the high protein concentration found in crystals by scrutinizing the crystallographic structure of the unliganded monomeric VP55 containing two monomers per asymmetric unit (PDB: 3OWG, (51)). Even using the symmetry related molecules, no occurrences of such a dimer was visible. Reciprocally, none of the symmetry related molecules in the Mg561 and the R341 structure reproduce the dimer found in the VP55 structure. To further characterize the molecular determinants of the dimerization of Mg561 and the role of the dimerization on the polyadenylation activity, we made deletion of the Mg561 (ΔD1 Mg561) and R341 (ΔD1 R341) N-terminal domains. Gel filtration experiments revealed that the mutants were monomeric and although they still co-purified with RNA, they were inactive (Figure 4), suggesting that the dimeric state and the N-terminal domain were required for the polyadenylation activity.

Bottom Line: Unexpectedly, the vPAPs are homodimeric and uniquely self-processive.The vPAP backbone structures exhibit a symmetrical architecture with two subdomains sharing a nucleotidyltransferase topology, suggesting that vPAPs originate from an ancestral duplication.A Poxvirus processivity factor homologue encoded by Megavirus chilensis displays a conserved 5'-GpppA 2'O methyltransferase activity but is also able to internally methylate the mRNAs' polyA tails.

View Article: PubMed Central - PubMed

Affiliation: Architecture et Fonction des Macromolécules Biologiques, CNRS UMR 7257, Aix-Marseille Université, 163 Avenue de Luminy, Case 932, 13288 Marseille cedex 9, France.

Show MeSH
Related in: MedlinePlus