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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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Membrane flotation analysis of helix A-GFP fusion protein and its mutant derivatives.Representative western blots using anti GFP antiserum are shown. Histograms on the right show average flotation efficiencies based on three independent experiments, calculated as in Figure 1.
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ppat-1000351-g004: Membrane flotation analysis of helix A-GFP fusion protein and its mutant derivatives.Representative western blots using anti GFP antiserum are shown. Histograms on the right show average flotation efficiencies based on three independent experiments, calculated as in Figure 1.

Mentions: Since the structure and DSA results implied that L396, L400, and L407 were positioned in the face of helix A most deeply immersed into the bilayer-mimicking micelle (Fig. 3C, bottom view, and 3D), we tested the importance of these three leucines for helix A-mediated membrane association. We introduced L to A mutations in the 18H-GFP fusion protein expression plasmid and tested their effects on membrane flotation efficiency. As shown in Fig. 4, the wt18H-GFP again had 30–35% flotation efficiency, while single L to A mutations reduced this to ∼7–15%. Of the three leucines, mutating the more N-proximal L396 and L400 more severely reduced membrane association than mutating L407, which paralleled the stronger micelle contact of the N-terminal half of the peptide (Fig. 3C and Table 2). These results might also explain in part the tolerance for an isoleucine at the 407-equivalent position in other bromovirus replicase proteins (Fig. 1A). A fusion protein with a combination of all three L to A mutations had near background level flotation, implying a complete loss of function of helix A in targeting cytosolic GFP to membranes. In contrast, K to E mutations reversing the charge of lysines 403 and 406 (the only basic residues in the 18 aa helix A core) showed K403E to only marginally decrease the flotation efficiency of 18H-GFP, while K406E had no significant effect (Fig. 4), consistent with the NMR observation that these amino acids have weak and no lipid contact, respectively (Fig. 3C). A double K to R mutation designed to retain the positive charge at these amino acid positions did not affect membrane association at all (Fig. 4), suggesting that K403 might contribute to membrane association via its positive charge, perhaps by neutralizing negatively charged lipid head groups. Overall, as mutations that change the leucine-rich non-polar face of the helix have more detrimental effects on membrane association than other amino acid substitutions, the results were consistent with the NMR-based structure of helix A and show that amphipathic helix A has a key role in membrane targeting.


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)

Membrane flotation analysis of helix A-GFP fusion protein and its mutant derivatives.Representative western blots using anti GFP antiserum are shown. Histograms on the right show average flotation efficiencies based on three independent experiments, calculated as in Figure 1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654722&req=5

ppat-1000351-g004: Membrane flotation analysis of helix A-GFP fusion protein and its mutant derivatives.Representative western blots using anti GFP antiserum are shown. Histograms on the right show average flotation efficiencies based on three independent experiments, calculated as in Figure 1.
Mentions: Since the structure and DSA results implied that L396, L400, and L407 were positioned in the face of helix A most deeply immersed into the bilayer-mimicking micelle (Fig. 3C, bottom view, and 3D), we tested the importance of these three leucines for helix A-mediated membrane association. We introduced L to A mutations in the 18H-GFP fusion protein expression plasmid and tested their effects on membrane flotation efficiency. As shown in Fig. 4, the wt18H-GFP again had 30–35% flotation efficiency, while single L to A mutations reduced this to ∼7–15%. Of the three leucines, mutating the more N-proximal L396 and L400 more severely reduced membrane association than mutating L407, which paralleled the stronger micelle contact of the N-terminal half of the peptide (Fig. 3C and Table 2). These results might also explain in part the tolerance for an isoleucine at the 407-equivalent position in other bromovirus replicase proteins (Fig. 1A). A fusion protein with a combination of all three L to A mutations had near background level flotation, implying a complete loss of function of helix A in targeting cytosolic GFP to membranes. In contrast, K to E mutations reversing the charge of lysines 403 and 406 (the only basic residues in the 18 aa helix A core) showed K403E to only marginally decrease the flotation efficiency of 18H-GFP, while K406E had no significant effect (Fig. 4), consistent with the NMR observation that these amino acids have weak and no lipid contact, respectively (Fig. 3C). A double K to R mutation designed to retain the positive charge at these amino acid positions did not affect membrane association at all (Fig. 4), suggesting that K403 might contribute to membrane association via its positive charge, perhaps by neutralizing negatively charged lipid head groups. Overall, as mutations that change the leucine-rich non-polar face of the helix have more detrimental effects on membrane association than other amino acid substitutions, the results were consistent with the NMR-based structure of helix A and show that amphipathic helix A has a key role in membrane targeting.

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