Abstract
Positive-strand RNA viruses genome replication invariably is associated with vesicles or other rearranged cellular membranes. Brome mosaic virus (BMV) RNA replication occurs on perinuclear endoplasmic reticulum (ER) membranes in ~70 nm vesicular invaginations (spherules). BMV RNA replication vesicles show multiple parallels with membrane-enveloped, budding retrovirus virions, whose envelopment and release depend on the host ESCRT (endosomal sorting complexes required for transport) membrane-remodeling machinery. We now find that deleting components of the ESCRT pathway results in at least two distinct BMV phenotypes. One group of genes regulate RNA replication and the frequency of viral replication complex formation, but had no effect on spherule size, while a second group of genes regulate RNA replication in a way or ways independent of spherule formation. In particular, deleting SNF7 inhibits BMV RNA replication > 25-fold and abolishes detectable BMV spherule formation, even though the BMV RNA replication proteins accumulate and localize normally on perinuclear ER membranes. Moreover, BMV ESCRT recruitment and spherule assembly depend on different sets of protein-protein interactions from those used by multivesicular body vesicles, HIV-1 virion budding, or tomato bushy stunt virus (TBSV) spherule formation. These and other data demonstrate that BMV requires cellular ESCRT components for proper formation and function of its vesicular RNA replication compartments. The results highlight growing but diverse interactions of ESCRT factors with many viruses and viral processes, and potential value of the ESCRT pathway as a target for broad-spectrum antiviral resistance.
Highlights
A universal feature of positive-strand RNA ((+)RNA) viruses is that they multiply their RNA on intracellular membranes, usually in association with vesiculation or other membrane rearrangements [1,2,3]
We show that knocking out several components of the cellular Endosomal Complex Required for Transport (ESCRT) machinery resulted in parallel defects in brome mosaic virus (BMV) RNA replication and replication compartment formation, whereas other ESCRT components affected RNA replication independently of replication compartment formation
BMV’s interaction with the ESCRT machinery appears to be distinct from that reported for other viruses and from the ESCRT requirements for forming vesicles in cellular multivesicular bodies
Summary
A universal feature of positive-strand RNA ((+)RNA) viruses is that they multiply their RNA on intracellular membranes, usually in association with vesiculation or other membrane rearrangements [1,2,3]. The techniques of yeast genetics and molecular biology have greatly facilitated investigation of BMV replication and host-virus interactions [5, 16, 17]. In both yeast and plant cells, BMV RNA replication depends on the viral 1a and 2apol proteins and specific cis-acting RNA signals [18], generates a considerable excess of positive- to negative-strand RNA [15], and efficiently directs subgenomic mRNA synthesis [15]. Replication factor 1a localizes to the outer perinuclear ER membranes and induces 50–75 nm vesicular invaginations or spherules that, in the presence of 2apol and a replicable RNA template, serve as compartments or mini-organelles for RNA replication [22]. Similar spherular invaginations of the perinuclear ER membrane are prominent features of natural plant infections by BMV and its close relatives CCMV and BBMV [23, 24], and equivalent invaginations of other membranes are associated with genome replication in plant cells by other Bromoviridae [25], Tymoviridae [26, 27], Tombusviridae [28] and other plant viruses, and in animal cells by human- and animal-infecting alphaviruses [29,30,31]
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