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

Multifunctional RNA replication protein la 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 la, membrane lipid composition, and RNA synthesis. We show that la expression increases total membrane lipids in wild-type (wt) yeast by 25 to 33%, consistent with recent results indicating that the numerous la-induced spherules are enveloped by invaginations of the outer ER membrane. la 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 similar to2.5 to similar to1.5. Thus, ole1w inhibition of RNA replication was correlated with decreased levels of UFA, membrane fluidity, and plasticity. The ole1w mutation did not alter la-induced membrane synthesis, la 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 la-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.