Morphogenesis of Endoplasmic Reticulum Membrane-Invaginated Vesicles during Beet Black Scorch Virus Infection: Role of Auxiliary Replication Protein and New Implications of Three-Dimensional Architecture

Abstract

All well-characterized positive-strand RNA viruses[(؉)RNA viruses] induce the formation of host membrane-bound viral replication complexes (VRCs), yet the underlying mechanism and machinery for VRC formation remain elusive. We report here the biogenesis and topology of the Beet black scorch virus (BBSV) replication complex. Distinct cytopathological changes typical of endoplasmic reticulum (ER) aggregation and vesiculation were observed in BBSV-infected Nicotiana benthamiana cells. Immunogold labeling of the auxiliary replication protein p23 and double-stranded RNA (dsRNA) revealed that the ER-derived membranous spherules provide the site for BBSV replication. Further studies indicated that p23 plays a crucial role in mediating the ER rearrangement. Three-dimensional electron tomographic analysis revealed the formation of multiple ER-originated vesicle packets. Each vesicle packet enclosed a few to hundreds of independent spherules that were invaginations of the ER membranes into the lumen. Strikingly, these vesicle packets were connected to each other via tubules, a rearrangement event that is rare among other virus-induced membrane reorganizations. Fibrillar contents within the spherules were also reconstructed by electron tomography, which showed diverse structures. Our results provide the first, to our knowledge, three-dimensional ultrastructural analysis of membrane-bound VRCs of a plant (؉)RNA virus and should help to achieve a better mechanistic understanding of the organization and microenvironment of plant (؉)RNA virus replication complexes. IMPORTANCE Assembly of virus replication complexes

Biogenesis and architecture of arterivirus replication organelles

“…Using immuno electron microscopy (IEM) to detect BrUTP incorporated into viral RNA, the interior of these spherules was shown to contain newly-synthesized viral RNA that is thought to be exported to the cytosol through a neck-like channel of ∼10 nm in diameter, which could be clearly visualized in the 3D reconstruction. Since this first study, the membrane modifications induced by, for example, coronaviruses (Knoops et al, 2008;Maier et al, 2013a), arteriviruses (Knoops et al, 2012), flaviviruses (Gillespie et al, 2010;Junjhon et al, 2014;Miorin et al, 2013;Offerdahl et al, 2012;Welsch et al, 2009), hepaciviruses (Romero-Brey and Bartenschlager, 2014), togaviruses (Fontana et al, 2010), picornaviruses (Belov and van Kuppeveld, 2012;Limpens et al, 2011) and tombusviruses (Cao et al, 2015) have been characterized using ET.…”

“…The morphotype of virus-induced invaginations seems to extend to plant +RNA viruses (reviewed in Laliberte and Sanfacon, 2010; for an ET characterization see Cao et al, 2015). The striking similarities in the morphology of these invaginations across widely different virus groups strongly suggest a highly conserved mechanism behind their formation.…”

Killing two birds with one stone: How do Plant Viruses Break Down Plant Defenses and Manipulate Cellular Processes to Replicate Themselves?

“…By using ER membranes, viruses induce the formation of replication vesicles (RV) providing physical support to a coordinated translation and replication of RNA, also, to keep close cellular components necessary to efficient viral replication, such as tRNAs and nucleotides and proteins, for example. Additionally, RVs formed in the ER membranes are a safety environment acting as a shield protecting viral RNA from host immune defense (i.e., nucleases) ensuring a minimal or even no exposure of viral genome to cellular receptors that can initiate defense mechanisms (Cao et al 2015;Romero-Brey et al 2016;Verchot 2016a).…”

“…The most studied models of virus-induced membrane modification in ER are those analyzed during plant infection caused by viruses from Bromoviridae and Tombusviridae families (Restrepo-Hartwig et al 1996;Cao et al 2015). Regarding plant viruses belonging to the Bromoviridae family, Brome Mosaic Virus (BMV) and Cowpea chlorotic mottle virus (CCMV) are good models to understand how these viruses can cause morphological alterations in ER membranes.…”

“…During replication process, BMV replicates its RNA inside of a 60 nm spherule produced in ER-membranes, which is very close to the nucleus, whereas CCMV causes spherules in the ER lumen in perinuclear and peripheral portions (Kim 1977;Restrepo-Hartwig et al 1996). On the other hand, the Beat black scorch virus (BBSV) belonging to the Tombusviridae family induces ER aggregation and vesiculation (Cao et al 2015). Besides Bromoviruses and Tombusviruses, Potyviruses and Potexviruses also stimulate ER-membranes rearrangements leading to vesicle production with two goals (1) improve and protect virus replication process and (2) use these vesicles for intracellular movement (Grangeon et al 2012;Hashimoto et al 2015).…”