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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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Sequential order of BMV RNA replication complex assembly steps.The order of replication complex assembly steps shown is as inferred from the effects of Class I and Class II mutations in helix A on complex assembly and other data (see main text for further details). The black arrows show the inferred progression of replication complex assembly for wt 1a. Consistent with the effects of Class I and II mutant phenotypes on membrane interaction and 2aPol recruitment (see below), and with the ability of 1a to recruit nascent 2aPol from translating, cytoplasmic polysomes [19], 1a and 2aPol interact in the cytoplasm prior to membrane association. For Class II mutants, subsequent 1a–membrane association and 1a-induced membrane rearrangement is correlated with inhibition of 1a-2aPol interaction. The effects of mutations in the C-proximal 1a NTPase/helicase domain imply that 1a-mediated recruitment of viral RNA templates to the membrane-associated, protected state required for replication occurs after 1a-induced membrane rearrangement [21], as shown. Red and green arrows show the opposite shifts in assembly equilibrium induced by Class I and Class II mutations. Class I 1a mutants have lost helix A–mediated ER membrane association and all capability to invaginate or otherwise modify membranes, but retain efficient interaction with 2aPol. In contrast, Class II 1a mutants retain efficient ER membrane association and show greatly increased levels of membrane invagination and RNA template recruitment, but have decreased interaction with 2aPol.
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ppat-1000351-g012: Sequential order of BMV RNA replication complex assembly steps.The order of replication complex assembly steps shown is as inferred from the effects of Class I and Class II mutations in helix A on complex assembly and other data (see main text for further details). The black arrows show the inferred progression of replication complex assembly for wt 1a. Consistent with the effects of Class I and II mutant phenotypes on membrane interaction and 2aPol recruitment (see below), and with the ability of 1a to recruit nascent 2aPol from translating, cytoplasmic polysomes [19], 1a and 2aPol interact in the cytoplasm prior to membrane association. For Class II mutants, subsequent 1a–membrane association and 1a-induced membrane rearrangement is correlated with inhibition of 1a-2aPol interaction. The effects of mutations in the C-proximal 1a NTPase/helicase domain imply that 1a-mediated recruitment of viral RNA templates to the membrane-associated, protected state required for replication occurs after 1a-induced membrane rearrangement [21], as shown. Red and green arrows show the opposite shifts in assembly equilibrium induced by Class I and Class II mutations. Class I 1a mutants have lost helix A–mediated ER membrane association and all capability to invaginate or otherwise modify membranes, but retain efficient interaction with 2aPol. In contrast, Class II 1a mutants retain efficient ER membrane association and show greatly increased levels of membrane invagination and RNA template recruitment, but have decreased interaction with 2aPol.

Mentions: The Class I and II 1a mutant phenotypes reveal significant insights into the pathways by which BMV RNA replication complexes assemble (Fig. 12). Immunogold electron microscopy and stoichiometric calculations of the various viral components in wt BMV replication complexes indicate that each spherule replication complex contains ∼200–400 BMV 1a molecules [13]. Calculations of spherule surface area and the predicted size of the 1a protein, 1a's strong affinity for the cytoplasmic face of the ER membrane [24], 1a self-interaction [29], and other results all imply that 1a forms an inner shell inside the spherules, explaining the formation and maintenance of these high-energy membrane deformations [12],[13]. Similar conclusions, based on electron microscope tomography and multiple other approaches, recently emerged for the role of transmembrane viral replication protein A in spherule RNA replication complexes formed by flock house nodavirus on mitochondrial membranes [23]. The observation that the Class II cluster of helix A mutations alters the size of the induced membrane spherules (Table 3) suggests that this part of helix A affects 1a-membrane and/or 1a-1a self-interactions that determine the diameter of the inner protein shell. Such altered interactions, together with the ∼3-fold reduced volume of Class II spherules, explain how Class II 1a mutants produce significantly more spherules than wt 1a (Table 3) from a similar or only slightly increased number of 1a proteins (Fig. 3C).


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)

Sequential order of BMV RNA replication complex assembly steps.The order of replication complex assembly steps shown is as inferred from the effects of Class I and Class II mutations in helix A on complex assembly and other data (see main text for further details). The black arrows show the inferred progression of replication complex assembly for wt 1a. Consistent with the effects of Class I and II mutant phenotypes on membrane interaction and 2aPol recruitment (see below), and with the ability of 1a to recruit nascent 2aPol from translating, cytoplasmic polysomes [19], 1a and 2aPol interact in the cytoplasm prior to membrane association. For Class II mutants, subsequent 1a–membrane association and 1a-induced membrane rearrangement is correlated with inhibition of 1a-2aPol interaction. The effects of mutations in the C-proximal 1a NTPase/helicase domain imply that 1a-mediated recruitment of viral RNA templates to the membrane-associated, protected state required for replication occurs after 1a-induced membrane rearrangement [21], as shown. Red and green arrows show the opposite shifts in assembly equilibrium induced by Class I and Class II mutations. Class I 1a mutants have lost helix A–mediated ER membrane association and all capability to invaginate or otherwise modify membranes, but retain efficient interaction with 2aPol. In contrast, Class II 1a mutants retain efficient ER membrane association and show greatly increased levels of membrane invagination and RNA template recruitment, but have decreased interaction with 2aPol.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000351-g012: Sequential order of BMV RNA replication complex assembly steps.The order of replication complex assembly steps shown is as inferred from the effects of Class I and Class II mutations in helix A on complex assembly and other data (see main text for further details). The black arrows show the inferred progression of replication complex assembly for wt 1a. Consistent with the effects of Class I and II mutant phenotypes on membrane interaction and 2aPol recruitment (see below), and with the ability of 1a to recruit nascent 2aPol from translating, cytoplasmic polysomes [19], 1a and 2aPol interact in the cytoplasm prior to membrane association. For Class II mutants, subsequent 1a–membrane association and 1a-induced membrane rearrangement is correlated with inhibition of 1a-2aPol interaction. The effects of mutations in the C-proximal 1a NTPase/helicase domain imply that 1a-mediated recruitment of viral RNA templates to the membrane-associated, protected state required for replication occurs after 1a-induced membrane rearrangement [21], as shown. Red and green arrows show the opposite shifts in assembly equilibrium induced by Class I and Class II mutations. Class I 1a mutants have lost helix A–mediated ER membrane association and all capability to invaginate or otherwise modify membranes, but retain efficient interaction with 2aPol. In contrast, Class II 1a mutants retain efficient ER membrane association and show greatly increased levels of membrane invagination and RNA template recruitment, but have decreased interaction with 2aPol.
Mentions: The Class I and II 1a mutant phenotypes reveal significant insights into the pathways by which BMV RNA replication complexes assemble (Fig. 12). Immunogold electron microscopy and stoichiometric calculations of the various viral components in wt BMV replication complexes indicate that each spherule replication complex contains ∼200–400 BMV 1a molecules [13]. Calculations of spherule surface area and the predicted size of the 1a protein, 1a's strong affinity for the cytoplasmic face of the ER membrane [24], 1a self-interaction [29], and other results all imply that 1a forms an inner shell inside the spherules, explaining the formation and maintenance of these high-energy membrane deformations [12],[13]. Similar conclusions, based on electron microscope tomography and multiple other approaches, recently emerged for the role of transmembrane viral replication protein A in spherule RNA replication complexes formed by flock house nodavirus on mitochondrial membranes [23]. The observation that the Class II cluster of helix A mutations alters the size of the induced membrane spherules (Table 3) suggests that this part of helix A affects 1a-membrane and/or 1a-1a self-interactions that determine the diameter of the inner protein shell. Such altered interactions, together with the ∼3-fold reduced volume of Class II spherules, explain how Class II 1a mutants produce significantly more spherules than wt 1a (Table 3) from a similar or only slightly increased number of 1a proteins (Fig. 3C).

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