Assembly-hub function of ER-localized SNARE proteins in biogenesis of tombusvirus replication compartment

Abstract

Positive-strand RNA viruses assemble numerous membrane-bound viral replicase complexes within large replication compartments to support their replication in infected cells. Yet the detailed mechanism of how given subcellular compartments are subverted by viruses is incompletely understood. Although, Tomato bushy stunt virus (TBSV) uses peroxisomal membranes for replication, in this paper, we show evidence that the ER-resident SNARE (soluble NSF attachment protein receptor) proteins play critical roles in the formation of active replicase complexes in yeast model host and in plants. Depletion of the syntaxin 18-like Ufe1 and Use1, which are components of the ER SNARE complex in the ERAS (ER arrival site) subdomain, in yeast resulted in greatly reduced tombusvirus accumulation. Over-expression of a dominant-negative mutant of either the yeast Ufe1 or the orthologous plant Syp81 syntaxin greatly interferes with tombusvirus replication in yeast and plants, thus further supporting the role of this host protein in tombusvirus replication. Moreover, tombusvirus RNA replication was low in cell-free extracts from yeast with repressed Ufe1 or Use1 expression. We also present evidence for the mislocalization of the tombusviral p33 replication protein to the ER membrane in Ufe1p-depleted yeast cells. The viral p33 replication protein interacts with both Ufe1p and Use1p and co-opts them into the TBSV replication compartment in yeast and plant cells. The co-opted Ufe1 affects the virus-driven membrane contact site formation, sterol-enrichment at replication sites, recruitment of several pro-viral host factors and subversion of the Rab5-positive PE-rich endosomes needed for robust TBSV replication. In summary, we demonstrate a critical role for Ufe1 and Use1 SNARE proteins in TBSV replication and propose that the pro-viral functions of Ufe1 and Use1 are to serve as assembly hubs for the formation of the extensive TBSV replication compartments in cells. Altogether, these findings point clearly at the ERAS subdomain of ER as a critical site for the biogenesis of the TBSV replication compartment.

Perception of double‐stranded RNA in plant antiviral immunity

“…Tombusvirus replication, for instance, induces a replication compartment consisting of aggregated peroxysomes, endoplasmic reticulum and early endosomal proteins and membranes (Nagy, 2016). The tombusvirus p33 replication protein co-opts host factors involved in membrane fusion for virus replication (Sasvari et al, 2018). Also the Turnip mosaic virus (TuMV) 6K2 protein has recently been shown to interact with and relocate a host protein involved in ER fusion for efficient replication and movement (Movahed et al, 2019).…”

Susceptibility Genes to Plant Viruses

“…Through interactions with 1a, membrane-shaping reticulon proteins (RHP) are critical to the formation of BMV replication compartments [ 55 ]. Similarly, through interaction with the p33 replication protein, ER-resident SNARE protein encoded by SYP81 is essential in the formation of TBSV replication compartments [ 59 ]. RAB5 encodes a GTPase protein that interacts with p33 and redistributes phosphatidylethanolamine to the replication compartments to favor TBSV replication [ 58 ].…”

“…Based on the model that viral factors interact and may form complexes with cellular factors, yeast two-hybrid assays or immunoprecipitation of viral factors followed by mass spectrometry have led to the identification of cellular factors needed for translation [ 50 ], replication complex formation [ 59 ], viral RNA replication [ 37 ], movement [ 91 , 92 ], gene silencing suppression [ 100 ], and others [ 109 ] ( Table 1 ).…”

ScreeningLegionellaeffectors for antiviral effects reveals Rab1 GTPase as a proviral factor coopted for tombusvirus replication

“…TBSV usurps the ERAS (ER arrival site) subdomain in the ER as an assembly hub for the biogenesis of the TBSV replication compartment (34). The ERAS consists of the ER-resident SNARE proteins, namely the syntaxin 18-like Ufe1, Use1, and Sec20, and the Dsl1 tethering complex (35).…”

“…Through binding to Ufe1 and Use1 SNARE proteins (34), the p33 replication protein might utilize the SNARE and tethering proteins to capture Rab1 and COPII vesicles. This was tested by depleting Ufe1 or Use1 levels through repressing GAL1 promoter-based expression of either Ufe1 or Use1 mRNAs in GALS::UFE1 and GAL1::USE1 yeast cells, respectively (34). Confocal microscopy revealed the lack of colocalization of Rab1 with p33 replication protein in yeasts with depleted levels of either Ufe1 or Use1 (Fig.…”