Intersection of the Multivesicular Body Pathway and Lipid Homeostasis in RNA Replication by a Positive-Strand RNA Virus

Abstract

Like many positive-strand RNA viruses, brome mosaic virus (BMV) RNA replication occurs in membrane-invaginated vesicular compartments. BMV RNA replication compartments show parallels with membrane-enveloped, budding retrovirus virions, whose release depends on the cellular multivesicular body (MVB) sorting pathway. BMV RNA replication compartments are not released from their parent membranes, but might depend on MVB functions for membrane invagination. Prior results show that BMV RNA replication is severely inhibited by deletion of the crucial MVB gene DOA4 or BRO1. We report here that involvement of DOA4 and BRO1 in BMV RNA replication is not dependent on the MVB pathway's membrane-shaping functions but rather is due to their roles in recycling ubiquitin from MVB cargos. We show that deleting DOA4 or BRO1 inhibits the ubiquitination- and proteasome-dependent activation of homologous transcription factors Mga2p and Spt23p, which regulate many lipid metabolism genes, including the fatty acid desaturase gene OLE1, which is essential for BMV RNA replication. However, Mga2p processing and BMV RNA replication are restored by supplementing free ubiquitin, which is depleted in doa4Δ and bro1Δ cells. The results identify Mga2p and Spt23p processing and lipid regulation as sensitive targets of ubiquitin depletion and correctly predict multiple effects of modulating additional host genes RFU1, UBP6, and UFD3. Our results also show that BMV RNA replication depends on additional Mga2p-regulated genes likely involved in lipid metabolism beyond OLE1. Among other points, these findings show the potential for blocking viral RNA replication by modulating lipid synthesis at multiple levels.