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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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Class I and Class II mutations in 1a helix A have opposite effects on recruiting genomic RNA3 to a membrane-associated, nuclease-resistant state.(A) RNA3 levels in yeast cells expressing RNA3 either alone, with wt 1a, or with 1a bearing Class I or Class II mutations in helix A. 18S rRNA was measured as a loading control. (B) Distribution of RNA3 in membrane flotation gradients in yeast cells expressing either RNA3 alone or with wt 1a or Class I or Class II 1a helix A mutants. (C) Distribution of RNA3 in supernatant (S) and pellet (P) fractions of lysates incubated with or without micrococcal nuclease from yeast cells expressing RNA3 alone or with wt 1a or Class I or Class II 1a helix A mutants.
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ppat-1000351-g009: Class I and Class II mutations in 1a helix A have opposite effects on recruiting genomic RNA3 to a membrane-associated, nuclease-resistant state.(A) RNA3 levels in yeast cells expressing RNA3 either alone, with wt 1a, or with 1a bearing Class I or Class II mutations in helix A. 18S rRNA was measured as a loading control. (B) Distribution of RNA3 in membrane flotation gradients in yeast cells expressing either RNA3 alone or with wt 1a or Class I or Class II 1a helix A mutants. (C) Distribution of RNA3 in supernatant (S) and pellet (P) fractions of lysates incubated with or without micrococcal nuclease from yeast cells expressing RNA3 alone or with wt 1a or Class I or Class II 1a helix A mutants.

Mentions: In yeast cells, the half-life of RNA3 increases from 5–10 min in the absence of 1a to more than 3 hours in the presence of 1a, which is reflected in a marked increase in RNA3 accumulation [22]. Accordingly, as shown in Fig. 9A, lanes 1 and 2, GAL1 promoter-driven wt 1a increased RNA3 accumulation ∼20-fold. Strikingly, the effects of the Class I and Class II mutations on 1a stimulation of RNA3 accumulation were opposite to each other and opposite to the effects of each mutant on 2aPol. Co-expressing class II 1a mutants stimulated RNA3 accumulation ∼40-fold, or double the stimulation by wt 1a (Fig. 9A), in parallel with the increased frequency of spherule formation by these mutants (Table 3). In contrast, Class I 1a mutants showed no ability to stimulate RNA3 accumulation, so that RNA3 levels in cells expressing Class I 1a mutants were similar to those in cells lacking 1a (Fig. 9A).


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)

Class I and Class II mutations in 1a helix A have opposite effects on recruiting genomic RNA3 to a membrane-associated, nuclease-resistant state.(A) RNA3 levels in yeast cells expressing RNA3 either alone, with wt 1a, or with 1a bearing Class I or Class II mutations in helix A. 18S rRNA was measured as a loading control. (B) Distribution of RNA3 in membrane flotation gradients in yeast cells expressing either RNA3 alone or with wt 1a or Class I or Class II 1a helix A mutants. (C) Distribution of RNA3 in supernatant (S) and pellet (P) fractions of lysates incubated with or without micrococcal nuclease from yeast cells expressing RNA3 alone or with wt 1a or Class I or Class II 1a helix A mutants.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654722&req=5

ppat-1000351-g009: Class I and Class II mutations in 1a helix A have opposite effects on recruiting genomic RNA3 to a membrane-associated, nuclease-resistant state.(A) RNA3 levels in yeast cells expressing RNA3 either alone, with wt 1a, or with 1a bearing Class I or Class II mutations in helix A. 18S rRNA was measured as a loading control. (B) Distribution of RNA3 in membrane flotation gradients in yeast cells expressing either RNA3 alone or with wt 1a or Class I or Class II 1a helix A mutants. (C) Distribution of RNA3 in supernatant (S) and pellet (P) fractions of lysates incubated with or without micrococcal nuclease from yeast cells expressing RNA3 alone or with wt 1a or Class I or Class II 1a helix A mutants.
Mentions: In yeast cells, the half-life of RNA3 increases from 5–10 min in the absence of 1a to more than 3 hours in the presence of 1a, which is reflected in a marked increase in RNA3 accumulation [22]. Accordingly, as shown in Fig. 9A, lanes 1 and 2, GAL1 promoter-driven wt 1a increased RNA3 accumulation ∼20-fold. Strikingly, the effects of the Class I and Class II mutations on 1a stimulation of RNA3 accumulation were opposite to each other and opposite to the effects of each mutant on 2aPol. Co-expressing class II 1a mutants stimulated RNA3 accumulation ∼40-fold, or double the stimulation by wt 1a (Fig. 9A), in parallel with the increased frequency of spherule formation by these mutants (Table 3). In contrast, Class I 1a mutants showed no ability to stimulate RNA3 accumulation, so that RNA3 levels in cells expressing Class I 1a mutants were similar to those in cells lacking 1a (Fig. 9A).

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