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An amphipathic alpha-helix controls multiple roles of brome mosaic virus protein 1a in RNA replication complex assembly and function.

Liu L, Westler WM, den Boon JA, Wang X, Diaz A, Steinberg HA, Ahlquist P - PLoS Pathog. (2009)

Bottom Line: Here we identify in BMV 1a an amphipathic alpha-helix, helix A, and use NMR analysis to define its structure and propensity to insert in hydrophobic membrane-mimicking micelles.We show that helix A is essential for efficient 1a-ER membrane association and normal perinuclear ER localization, and that deletion or mutation of helix A abolishes RNA replication.The results provide new insights into the pathways of RNA replication complex assembly and show that helix A is critical for assembly and function of the viral RNA replication complex, including its central role in targeting replication components and controlling modes of 1a action.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Virology, University of Wisconsin-Madison, Madison, Wisconsin, USA.

ABSTRACT
Brome mosaic virus (BMV) protein 1a has multiple key roles in viral RNA replication. 1a localizes to perinuclear endoplasmic reticulum (ER) membranes as a peripheral membrane protein, induces ER membrane invaginations in which RNA replication complexes form, and recruits and stabilizes BMV 2a polymerase (2a(Pol)) and RNA replication templates at these sites to establish active replication complexes. During replication, 1a provides RNA capping, NTPase and possibly RNA helicase functions. Here we identify in BMV 1a an amphipathic alpha-helix, helix A, and use NMR analysis to define its structure and propensity to insert in hydrophobic membrane-mimicking micelles. We show that helix A is essential for efficient 1a-ER membrane association and normal perinuclear ER localization, and that deletion or mutation of helix A abolishes RNA replication. Strikingly, mutations in helix A give rise to two dramatically opposite 1a function phenotypes, implying that helix A acts as a molecular switch regulating the intricate balance between separable 1a functions. One class of helix A deletions and amino acid substitutions markedly inhibits 1a-membrane association and abolishes ER membrane invagination, viral RNA template recruitment, and replication, but doubles the 1a-mediated increase in 2a(Pol) accumulation. The second class of helix A mutations not only maintains efficient 1a-membrane association but also amplifies the number of 1a-induced membrane invaginations 5- to 8-fold and enhances viral RNA template recruitment, while failing to stimulate 2a(Pol) accumulation. The results provide new insights into the pathways of RNA replication complex assembly and show that helix A is critical for assembly and function of the viral RNA replication complex, including its central role in targeting replication components and controlling modes of 1a action.

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NMR structure of helix A on SDS micelles.(A) 1H{15N}-HSQC spectrum of BMV 1a helix A bound to 100 mM SDS micelles. Peaks arise from the amide moieties in the peptide. The assignments of peaks to particular amides are shown. Boxed peaks arise from the side chain amide protons of the Asn-399 and Gln-402 side chains and are unassigned. (B) Ensemble of 20 structures (backbone atoms only) determined for the peptide bound to an SDS micelle. The coloring represents the secondary structure as predicted on the basis of Ca and Cb chemical shifts [45]. White, helix; Gray, coil. (C) NMR-based three-dimensional structure of helix A in SDS micelles from four different viewpoints as indicated. (D) Artist's rendering of the topology of helix A at the interface between polar headgroups and fatty acid chains in a lipid bilayer, based on DSA contacts and other results discussed in the text. The “front” projection of helix A from panel C is shown. For comparison, the image of one of the glycerophospholipids is shown enhanced at the top left.
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ppat-1000351-g003: NMR structure of helix A on SDS micelles.(A) 1H{15N}-HSQC spectrum of BMV 1a helix A bound to 100 mM SDS micelles. Peaks arise from the amide moieties in the peptide. The assignments of peaks to particular amides are shown. Boxed peaks arise from the side chain amide protons of the Asn-399 and Gln-402 side chains and are unassigned. (B) Ensemble of 20 structures (backbone atoms only) determined for the peptide bound to an SDS micelle. The coloring represents the secondary structure as predicted on the basis of Ca and Cb chemical shifts [45]. White, helix; Gray, coil. (C) NMR-based three-dimensional structure of helix A in SDS micelles from four different viewpoints as indicated. (D) Artist's rendering of the topology of helix A at the interface between polar headgroups and fatty acid chains in a lipid bilayer, based on DSA contacts and other results discussed in the text. The “front” projection of helix A from panel C is shown. For comparison, the image of one of the glycerophospholipids is shown enhanced at the top left.

Mentions: A helical wheel projection of the 18 aa helix A core region shows that it has the potential to form an amphipathic α-helical cylinder with one side (the right side in Fig. 2) having a cluster of hydrophobic, non-polar residues including three leucines (L396, L400, L407) and two nearby positive-charged lysines (K403, K406), and the other (left) side of the helix mostly hydrophilic and polar residues (Fig. 2, see also marked aa in Fig. 1A). To test these predictions, we used NMR to resolve the structure of an 18 aa peptide with the core sequence (aa 392–409) of helix A. NMR spectra of this peptide dissolved in water did not reveal a long term stable structure. However, upon including 100 mM SDS to provide lipid bilayer-mimicking micelles [25], the peptide showed NMR spectral changes consistent with a stable conformation (Fig. 3A). Based solely on 13C chemical shifts, NMR showed that aa 397–406 in the peptide had a >80% probability to be in a helical structure (Fig. 3B). To elucidate this further, the three dimensional structure of the peptide was calculated based on NOE distance constraints arising from spatial contact of hydrogen atoms observed to be closer than ∼5×. Additional dihedral angle constraints were derived from chemical shifts using the TALOS program [26]. The resulting structure (Fig. 3C) shows an α-helical conformation for aa 397–406, indicating that an amphipathic helix formed upon binding to the lipid membrane-mimicking SDS micelle. The constraints and overall quality of the structure are shown in Table 1.


An amphipathic alpha-helix controls multiple roles of brome mosaic virus protein 1a in RNA replication complex assembly and function.

Liu L, Westler WM, den Boon JA, Wang X, Diaz A, Steinberg HA, Ahlquist P - PLoS Pathog. (2009)

NMR structure of helix A on SDS micelles.(A) 1H{15N}-HSQC spectrum of BMV 1a helix A bound to 100 mM SDS micelles. Peaks arise from the amide moieties in the peptide. The assignments of peaks to particular amides are shown. Boxed peaks arise from the side chain amide protons of the Asn-399 and Gln-402 side chains and are unassigned. (B) Ensemble of 20 structures (backbone atoms only) determined for the peptide bound to an SDS micelle. The coloring represents the secondary structure as predicted on the basis of Ca and Cb chemical shifts [45]. White, helix; Gray, coil. (C) NMR-based three-dimensional structure of helix A in SDS micelles from four different viewpoints as indicated. (D) Artist's rendering of the topology of helix A at the interface between polar headgroups and fatty acid chains in a lipid bilayer, based on DSA contacts and other results discussed in the text. The “front” projection of helix A from panel C is shown. For comparison, the image of one of the glycerophospholipids is shown enhanced at the top left.
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Related In: Results  -  Collection

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ppat-1000351-g003: NMR structure of helix A on SDS micelles.(A) 1H{15N}-HSQC spectrum of BMV 1a helix A bound to 100 mM SDS micelles. Peaks arise from the amide moieties in the peptide. The assignments of peaks to particular amides are shown. Boxed peaks arise from the side chain amide protons of the Asn-399 and Gln-402 side chains and are unassigned. (B) Ensemble of 20 structures (backbone atoms only) determined for the peptide bound to an SDS micelle. The coloring represents the secondary structure as predicted on the basis of Ca and Cb chemical shifts [45]. White, helix; Gray, coil. (C) NMR-based three-dimensional structure of helix A in SDS micelles from four different viewpoints as indicated. (D) Artist's rendering of the topology of helix A at the interface between polar headgroups and fatty acid chains in a lipid bilayer, based on DSA contacts and other results discussed in the text. The “front” projection of helix A from panel C is shown. For comparison, the image of one of the glycerophospholipids is shown enhanced at the top left.
Mentions: A helical wheel projection of the 18 aa helix A core region shows that it has the potential to form an amphipathic α-helical cylinder with one side (the right side in Fig. 2) having a cluster of hydrophobic, non-polar residues including three leucines (L396, L400, L407) and two nearby positive-charged lysines (K403, K406), and the other (left) side of the helix mostly hydrophilic and polar residues (Fig. 2, see also marked aa in Fig. 1A). To test these predictions, we used NMR to resolve the structure of an 18 aa peptide with the core sequence (aa 392–409) of helix A. NMR spectra of this peptide dissolved in water did not reveal a long term stable structure. However, upon including 100 mM SDS to provide lipid bilayer-mimicking micelles [25], the peptide showed NMR spectral changes consistent with a stable conformation (Fig. 3A). Based solely on 13C chemical shifts, NMR showed that aa 397–406 in the peptide had a >80% probability to be in a helical structure (Fig. 3B). To elucidate this further, the three dimensional structure of the peptide was calculated based on NOE distance constraints arising from spatial contact of hydrogen atoms observed to be closer than ∼5×. Additional dihedral angle constraints were derived from chemical shifts using the TALOS program [26]. The resulting structure (Fig. 3C) shows an α-helical conformation for aa 397–406, indicating that an amphipathic helix formed upon binding to the lipid membrane-mimicking SDS micelle. The constraints and overall quality of the structure are shown in Table 1.

Bottom Line: Here we identify in BMV 1a an amphipathic alpha-helix, helix A, and use NMR analysis to define its structure and propensity to insert in hydrophobic membrane-mimicking micelles.We show that helix A is essential for efficient 1a-ER membrane association and normal perinuclear ER localization, and that deletion or mutation of helix A abolishes RNA replication.The results provide new insights into the pathways of RNA replication complex assembly and show that helix A is critical for assembly and function of the viral RNA replication complex, including its central role in targeting replication components and controlling modes of 1a action.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Virology, University of Wisconsin-Madison, Madison, Wisconsin, USA.

ABSTRACT
Brome mosaic virus (BMV) protein 1a has multiple key roles in viral RNA replication. 1a localizes to perinuclear endoplasmic reticulum (ER) membranes as a peripheral membrane protein, induces ER membrane invaginations in which RNA replication complexes form, and recruits and stabilizes BMV 2a polymerase (2a(Pol)) and RNA replication templates at these sites to establish active replication complexes. During replication, 1a provides RNA capping, NTPase and possibly RNA helicase functions. Here we identify in BMV 1a an amphipathic alpha-helix, helix A, and use NMR analysis to define its structure and propensity to insert in hydrophobic membrane-mimicking micelles. We show that helix A is essential for efficient 1a-ER membrane association and normal perinuclear ER localization, and that deletion or mutation of helix A abolishes RNA replication. Strikingly, mutations in helix A give rise to two dramatically opposite 1a function phenotypes, implying that helix A acts as a molecular switch regulating the intricate balance between separable 1a functions. One class of helix A deletions and amino acid substitutions markedly inhibits 1a-membrane association and abolishes ER membrane invagination, viral RNA template recruitment, and replication, but doubles the 1a-mediated increase in 2a(Pol) accumulation. The second class of helix A mutations not only maintains efficient 1a-membrane association but also amplifies the number of 1a-induced membrane invaginations 5- to 8-fold and enhances viral RNA template recruitment, while failing to stimulate 2a(Pol) accumulation. The results provide new insights into the pathways of RNA replication complex assembly and show that helix A is critical for assembly and function of the viral RNA replication complex, including its central role in targeting replication components and controlling modes of 1a action.

Show MeSH
Related in: MedlinePlus