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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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Helix A and specific non-polar (L) and polar residues (K) in it are required for efficient 1a membrane binding and ER targeting.(A) Distribution of wt 1a or 1a helix A mutants in membrane flotation gradients. Representative western blots using anti-1a antiserum are shown on the left, the histogram on the right shows average flotation efficiencies based on three independent experiments, calculated as in Figure 1. The levels of commonly observed less-than-full-length 1a-derived degradation products (whose separation from 1a depends on the particular gel composition and run time used) were not included in the calculations. (B) Fluorescence microscopy images of cells expressing wt 1a or 1a helix mutants and Sec63-GFP as an ER marker.
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ppat-1000351-g005: Helix A and specific non-polar (L) and polar residues (K) in it are required for efficient 1a membrane binding and ER targeting.(A) Distribution of wt 1a or 1a helix A mutants in membrane flotation gradients. Representative western blots using anti-1a antiserum are shown on the left, the histogram on the right shows average flotation efficiencies based on three independent experiments, calculated as in Figure 1. The levels of commonly observed less-than-full-length 1a-derived degradation products (whose separation from 1a depends on the particular gel composition and run time used) were not included in the calculations. (B) Fluorescence microscopy images of cells expressing wt 1a or 1a helix mutants and Sec63-GFP as an ER marker.

Mentions: To extend the results from helix A-GFP fusion proteins, the contribution of helix A to membrane association of full-length 1a was assessed using biochemical and cell imaging approaches. By membrane flotation gradient analyses, the flotation efficiency of wt 1a was ∼ 96% (Fig. 5A), confirming 1a's previously established high affinity for membranes [24]. Deleting the 35 aa or 18 aa helices reduced 1a-membrane association by over two-fold (Fig. 5A). The three L to A mutations, either as single mutations or as a triple combination, similarly reduced the flotation efficiency of full length 1a to ∼45%. Single alanine insertions immediately downstream from L396 and L400 reduced flotation efficiency to levels similar to full helix A deletions (Fig. 5A), confirming the importance of correct spacing to maintain the amphipathic characteristics of helix A. The importance of the charged lysines at positions 403 and 406 at the hydrophilic face of helix A was assessed using alanine or arginine substitutions. Single position substitution mutants and double mutants K403/406A and K403/406R maintained full flotation efficiency (Fig. 5A, single mutations not shown). 1a mutants K403E, K406E, and double mutant K403/406E retained intermediate flotation efficiencies showing that although the positive charge at these positions is not required, reversing it to a negative charge destabilizes membrane association (Fig. 5A). The K403/406E single and double mutations showed a somewhat greater inhibition of membrane association in the context of full length 1a (∼63% for the double mutant in Fig. 5) than in the context of the 18 aa helix fused to GFP (∼77%, Fig. 4), suggesting the possibility that residues outside of the 18 aa helix core might cooperatively influence membrane association.


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)

Helix A and specific non-polar (L) and polar residues (K) in it are required for efficient 1a membrane binding and ER targeting.(A) Distribution of wt 1a or 1a helix A mutants in membrane flotation gradients. Representative western blots using anti-1a antiserum are shown on the left, the histogram on the right shows average flotation efficiencies based on three independent experiments, calculated as in Figure 1. The levels of commonly observed less-than-full-length 1a-derived degradation products (whose separation from 1a depends on the particular gel composition and run time used) were not included in the calculations. (B) Fluorescence microscopy images of cells expressing wt 1a or 1a helix mutants and Sec63-GFP as an ER marker.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654722&req=5

ppat-1000351-g005: Helix A and specific non-polar (L) and polar residues (K) in it are required for efficient 1a membrane binding and ER targeting.(A) Distribution of wt 1a or 1a helix A mutants in membrane flotation gradients. Representative western blots using anti-1a antiserum are shown on the left, the histogram on the right shows average flotation efficiencies based on three independent experiments, calculated as in Figure 1. The levels of commonly observed less-than-full-length 1a-derived degradation products (whose separation from 1a depends on the particular gel composition and run time used) were not included in the calculations. (B) Fluorescence microscopy images of cells expressing wt 1a or 1a helix mutants and Sec63-GFP as an ER marker.
Mentions: To extend the results from helix A-GFP fusion proteins, the contribution of helix A to membrane association of full-length 1a was assessed using biochemical and cell imaging approaches. By membrane flotation gradient analyses, the flotation efficiency of wt 1a was ∼ 96% (Fig. 5A), confirming 1a's previously established high affinity for membranes [24]. Deleting the 35 aa or 18 aa helices reduced 1a-membrane association by over two-fold (Fig. 5A). The three L to A mutations, either as single mutations or as a triple combination, similarly reduced the flotation efficiency of full length 1a to ∼45%. Single alanine insertions immediately downstream from L396 and L400 reduced flotation efficiency to levels similar to full helix A deletions (Fig. 5A), confirming the importance of correct spacing to maintain the amphipathic characteristics of helix A. The importance of the charged lysines at positions 403 and 406 at the hydrophilic face of helix A was assessed using alanine or arginine substitutions. Single position substitution mutants and double mutants K403/406A and K403/406R maintained full flotation efficiency (Fig. 5A, single mutations not shown). 1a mutants K403E, K406E, and double mutant K403/406E retained intermediate flotation efficiencies showing that although the positive charge at these positions is not required, reversing it to a negative charge destabilizes membrane association (Fig. 5A). The K403/406E single and double mutations showed a somewhat greater inhibition of membrane association in the context of full length 1a (∼63% for the double mutant in Fig. 5) than in the context of the 18 aa helix fused to GFP (∼77%, Fig. 4), suggesting the possibility that residues outside of the 18 aa helix core might cooperatively influence membrane association.

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