Membrane synthesis, specific lipid requirements, and localized lipid composition changes associated with a positive-strand RNA virus RNA replication protein - PubMed (original) (raw)

Membrane synthesis, specific lipid requirements, and localized lipid composition changes associated with a positive-strand RNA virus RNA replication protein

Wai-Ming Lee et al. J Virol. 2003 Dec.

Abstract

Multifunctional RNA replication protein 1a of brome mosaic virus (BMV), a positive-strand RNA virus, localizes to the cytoplasmic face of endoplasmic reticulum (ER) membranes and induces ER lumenal spherules in which viral RNA synthesis occurs. We previously showed that BMV RNA replication in yeast is severely inhibited prior to negative-strand RNA synthesis by a single-amino-acid substitution in the ole1w allele of yeast Delta9 fatty acid (FA) desaturase, which converts saturated FAs (SFAs) to unsaturated FAs (UFAs). Here we further define the relationships between 1a, membrane lipid composition, and RNA synthesis. We show that 1a expression increases total membrane lipids in wild-type (wt) yeast by 25 to 33%, consistent with recent results indicating that the numerous 1a-induced spherules are enveloped by invaginations of the outer ER membrane. 1a did not alter total membrane lipid composition in wt or ole1w yeast, but the ole1w mutation selectively depleted 18-carbon, monounsaturated (18:1) FA chains and increased 16:0 SFA chains, reducing the UFA-to-SFA ratio from approximately 2.5 to approximately 1.5. Thus, ole1w inhibition of RNA replication was correlated with decreased levels of UFA, membrane fluidity, and plasticity. The ole1w mutation did not alter 1a-induced membrane synthesis, 1a localization to the perinuclear ER, or colocalization of BMV 2a polymerase, nor did it block spherule formation. Moreover, BMV RNA replication templates were still recovered from cell lysates in a 1a-induced, 1a- and membrane-associated, and nuclease-resistant but detergent-susceptible state consistent with spherules. However, unlike nearby ER membranes, the membranes surrounding spherules in ole1w cells were not distinctively stained with osmium tetroxide, which interacts specifically with UFA double bonds. Thus, in ole1w cells, spherule-associated membranes were locally depleted in UFAs. This localized UFA depletion helps to explain why BMV RNA replication is more sensitive than cell growth to reduced UFA levels. The results imply that 1a preferentially interacts with one or more types of membrane lipids.

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Figures

FIG. 1.

FIG. 1.

Effect of BMV 1a expression and the yeast ole1w mutation on total accumulation and composition of yeast FAs. wt yeast and mutant yeast were grown in defined, galactose-containing medium to mid-log phase. Total FA was extracted, converted to methyl esters, and separated by chain length and degree of saturation by using gas-liquid chromatography. The molar amount of 16:0, 16:1, 18:0, and 18:1 FA was measured by using a flame ionization detector and was normalized to the OD600 of each yeast culture. (A) Relative levels of 16:0, 16:1, 18:0, and 18:1 FA in each sample, as a molar percentage of total FA. Yeast genotype (wt or ole1w) and the presence or absence of 1a (− or +) are indicated at the bottom for each sample. Total FA is the sum of 16:0, 16:1, 18:0, and 18:1 in each sample. (B) Ratio of total UFA (16:1 + 18:1) to SFA (16:0 + 18:0). (C) Total FA per cell, shown as a percentage of the total FA per cell of wt yeast lacking 1a. Each histogram shows the averages and standard deviations of four experiments.

FIG. 2.

FIG. 2.

ole1w yeast supports normal localization of BMV 1a protein to ER membranes. wt yeast and ole1w mutant yeast expressing 1a and a fusion protein linking GFP to sec63p, an integral ER membrane protein, were fixed with formaldehyde, spheroplasted, permeabilized with Triton X-100, and immunostained with rabbit anti-1a antiserum followed by secondary antibodies conjugated to Texas red. sec63p-GFP was visualized by its intrinsic fluorescence. The intracellular distributions of sec63p-GFP (top row, green) and 1a (middle row, red) were sequentially imaged in the same 0.5-μm optical section by confocal microscopy and were digitally superimposed for further comparison (bottom row). Representative images are shown. (A) wt yeast. (B) ole1w yeast. Shown are 1a and sec63p-GFP distributions representative of >95% of the cells. (C) ole1w yeast. Shown are 1a and sec63p-GFP distributions seen in a small percentage of the cells.

FIG. 3.

FIG. 3.

ole1w yeast supports normal, 1a-induced localization of BMV 2a protein to ER membranes. wt yeast (A) and ole1w yeast (B) expressing BMV 1a and a 2a-GFP fusion protein were processed as described in the Fig. 2 legend. 1a (top row, red) was visualized by indirect immunofluorescence, 2a-GFP (middle row, green) was visualized by intrinsic fluorescence, and the images were digitally superimposed for futher comparison (bottom row). Representative images are shown.

FIG. 4.

FIG. 4.

ole1w yeast supports normal, 1a-induced transfer of BMV RNA3 to a membrane-associated, nuclease-resistant state. (A) Membrane association of RNA3. wt yeast and ole1w yeast expressing 1a and RNA3 were spheroplasted and lysed osmotically, and the total lysate (T) was fractionated by centrifugation (5 min at 20,000 × g) into a membrane-depleted supernatant (S) and a membrane-enriched pellet (P). RNA3 levels in each fraction were analyzed by Northern blotting. (B) Nuclease resistance of RNA3. Northern blot analysis of RNA3 in the pellet fraction (P) from panel A after no additional treatment (lanes 1 and 4), incubation with 0.01 U of micrococcal nuclease (MNase)/μl for 15 min at 30°C (lanes 2 and 5), or incubation with 0.5% NP-40 for 15 min at 0°C followed by micrococcal nuclease (lanes 3 and 6).

FIG. 5.

FIG. 5.

Yeast ole1w mutation preferentially alters the membranes bounding 1a-induced spherules. Representative electron micrographs of wt yeast (panels A and C) and ole1w mutant yeast (panels B and D) expressing 1a are shown. Panels C and D show enlarged portions of panels A and B, respectively, to reveal further details of the spherule membranes. Labels represent nucleoplasm (Nuc), nuclear envelope (NE), cytoplasm (Cyto), and 1a-induced spherules (Sph). Please see Results for further comments.

FIG. 6.

FIG. 6.

BMV 1a protein is localized to perinuclear spherule cores in both wt and ole1w yeast. Representative electron micrographs of immunogold localization of 1a in wt yeast (A) and ole1w mutant yeast (B and C) expressing 1a are shown. Labels indicate nucleoplasm (Nuc), nuclear envelope (NE), and cytoplasm (Cyto). To preserve 1a antigenicity for immunogold staining, yeast expressing 1a was fixed by using aldehydes without OsO4, causing membranes to appear as white, electron-lucent strips, rather than as osmium-stained black lines in Fig. 5A and C. 1a was visualized by immunostaining thin sections with rabbit anti-1a antiserum and secondary antibodies conjugated to 12-nm gold particles.

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