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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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1a helix A mutations abolish BMV RNA replication in yeast cells and barley.(A) Analysis of positive and minus strand RNA3 and RNA4 from yeast cells expressing 2aPol, RNA3 and either wt 1a or Class I or Class II 1a helix mutants. (B) Viral RNA levels in barley leaves 9 days post-inoculation with in vitro transcribed wt RNA1 or helix A mutant RNA1 and RNA2 and RNA3. 18S rRNA was measured as a loading control in (A) and (B).
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ppat-1000351-g010: 1a helix A mutations abolish BMV RNA replication in yeast cells and barley.(A) Analysis of positive and minus strand RNA3 and RNA4 from yeast cells expressing 2aPol, RNA3 and either wt 1a or Class I or Class II 1a helix mutants. (B) Viral RNA levels in barley leaves 9 days post-inoculation with in vitro transcribed wt RNA1 or helix A mutant RNA1 and RNA2 and RNA3. 18S rRNA was measured as a loading control in (A) and (B).

Mentions: Yeast cells expressing wt 1a, 2aPol and RNA3 supported efficient viral RNA replication (Fig. 10A). In contrast, in cells expressing Class I 1a mutants, 2aPol and RNA3, only weak RNA3 signals were detected, similar to the levels of RNA3 derived entirely from plasmid-based transcription in cells lacking 1a (Fig. 10A). In cells expressing Class II 1a mutants, 2aPol and RNA3, positive-strand RNA3 accumulated to levels intermediate between those in cells with and without wt 1a (Fig. 10A), consistent with the ability of class II 1a mutants to mediate RNA3 recruitment to a membrane-protected state (Fig. 9). However, positive-strand RNA4 and negative-strand strand RNA3, which are only synthesized as products of viral RNA replication, were undetectable in cells expressing any of the Class I 1a mutants, and reached only 5–10% of wt levels in cells expressing most Class II 1a mutants (Fig. 10A). The only exception was Class II mutant 1aT397A, which weakly stimulated 2aPol accumulation (Fig. 8C) and retained ∼25% of wt 1a replication levels (Fig. 10A). Thus, BMV RNA replication was severely inhibited by the helix A mutations in both classes.


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)

1a helix A mutations abolish BMV RNA replication in yeast cells and barley.(A) Analysis of positive and minus strand RNA3 and RNA4 from yeast cells expressing 2aPol, RNA3 and either wt 1a or Class I or Class II 1a helix mutants. (B) Viral RNA levels in barley leaves 9 days post-inoculation with in vitro transcribed wt RNA1 or helix A mutant RNA1 and RNA2 and RNA3. 18S rRNA was measured as a loading control in (A) and (B).
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Related In: Results  -  Collection

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

ppat-1000351-g010: 1a helix A mutations abolish BMV RNA replication in yeast cells and barley.(A) Analysis of positive and minus strand RNA3 and RNA4 from yeast cells expressing 2aPol, RNA3 and either wt 1a or Class I or Class II 1a helix mutants. (B) Viral RNA levels in barley leaves 9 days post-inoculation with in vitro transcribed wt RNA1 or helix A mutant RNA1 and RNA2 and RNA3. 18S rRNA was measured as a loading control in (A) and (B).
Mentions: Yeast cells expressing wt 1a, 2aPol and RNA3 supported efficient viral RNA replication (Fig. 10A). In contrast, in cells expressing Class I 1a mutants, 2aPol and RNA3, only weak RNA3 signals were detected, similar to the levels of RNA3 derived entirely from plasmid-based transcription in cells lacking 1a (Fig. 10A). In cells expressing Class II 1a mutants, 2aPol and RNA3, positive-strand RNA3 accumulated to levels intermediate between those in cells with and without wt 1a (Fig. 10A), consistent with the ability of class II 1a mutants to mediate RNA3 recruitment to a membrane-protected state (Fig. 9). However, positive-strand RNA4 and negative-strand strand RNA3, which are only synthesized as products of viral RNA replication, were undetectable in cells expressing any of the Class I 1a mutants, and reached only 5–10% of wt levels in cells expressing most Class II 1a mutants (Fig. 10A). The only exception was Class II mutant 1aT397A, which weakly stimulated 2aPol accumulation (Fig. 8C) and retained ∼25% of wt 1a replication levels (Fig. 10A). Thus, BMV RNA replication was severely inhibited by the helix A mutations in both classes.

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