Sterol Binding by the Tombusviral Replication Proteins Is Essential for Replication in Yeast and Plants

Xu, Kai; Nagy, Peter D.

doi:10.1128/jvi.01984-16

Cited by 33 publications

(32 citation statements)

References 45 publications

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“…The major role of the TBSV-induced vMCSs is the enrichment of various sterols at the site of replication to facilitate tombusvirus replication [ 43 , 45 ]. Accordingly, expression of Ufe1ΔTM in yeast replicating TBSV repRNA reduced the enrichment of sterols at internal locations ( Fig 8E ), which have been shown to represent replication compartments [ 43 ].…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2018

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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.

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Section: Resultsmentioning

confidence: 99%

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

et al. 2018

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“…Similar to the process that occurs during BMV infection, tombusvirus-induced membrane deformation requires lipid synthesis to support the huge proliferation of ER and peroxisome membranes (Sharma et al, 2011 ; Barajas et al, 2014c ). Furthermore, sterols and phospholipids are enriched in the cellular locations at which TBSV RNA replication occurs in both plant cells and yeast (Sharma et al, 2010 ; Xu and Nagy, 2015 , 2017 ). Sterols are essential membrane components that determine the curvature and fluidity of membranes (Lorizate and Kräusslich, 2011 ).…”

Section: Biogenesis Of Viral Replication Compartmentsmentioning

confidence: 99%

“…Sterols are essential membrane components that determine the curvature and fluidity of membranes (Lorizate and Kräusslich, 2011 ). The replication proteins p33 and p92 bind directly to sterols in vitro (Xu and Nagy, 2017 ). In addition, TBSV p33 interacts with oxysterol-binding ORP and VAMP-associated proteins (VAP), which mediate the redistribution of sterols to viral replication sites and facilitate the bending of membranes (Barajas et al, 2014b ).…”

Section: Biogenesis Of Viral Replication Compartmentsmentioning

confidence: 99%

Three-Dimensional Architecture and Biogenesis of Membrane Structures Associated with Plant Virus Replication

et al. 2018

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Positive-sense (+) RNA viruses represent the most abundant group of viruses and are dependent on the host cell machinery to replicate. One remarkable feature that occurs after (+) RNA virus entry into cells is the remodeling of host endomembranes, leading to the formation of viral replication factories. Recently, rapid progress in three-dimensional (3D) imaging technologies, such as electron tomography (ET) and focused ion beam-scanning electron microscopy (FIB-SEM), has enabled researchers to visualize the novel membrane structures induced by viruses at high resolution. These 3D imaging technologies provide new mechanistic insights into the viral infection cycle. In this review, we summarize the latest reports on the cellular remodeling that occurs during plant virus infection; in particular, we focus on studies that provide 3D architectural information on viral replication factories. We also outline the mechanisms underlying the formation of these membranous structures and discuss possible future research directions.

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“…It is well known that organelle-specific lipids contribute to the unique identity of cellular compartments, enabling the sorting of proteins during membrane trafficking, and acting as receptors for the recruitment of specific enzymes and signaling molecules ( van Meer et al, 2008 ; Klose et al, 2013 ). It is thus very likely that protein/lipid interactions contribute predominantly to the specificity of replication proteins/VRCs targeting ( Lampio et al, 2000 ; Xu and Nagy, 2017 ; Altan-Bonnet, 2017 ), although stabilization by specific host factors cannot be excluded at this stage.…”

Section: Discussionmentioning

confidence: 99%

Identification and Molecular Characterization of the Chloroplast Targeting Domain of Turnip yellow mosaic virus Replication Proteins

Moriceau¹,

Jomat²,

Bressanelli³

et al. 2017

Front. Plant Sci.

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Turnip yellow mosaic virus (TYMV) is a positive-strand RNA virus infecting plants. The TYMV 140K replication protein is a key organizer of viral replication complex (VRC) assembly, being responsible for recruitment of the viral polymerase and for targeting the VRCs to the chloroplast envelope where viral replication takes place. However, the structural requirements determining the subcellular localization and membrane association of this essential viral protein have not yet been defined. In this study, we investigated determinants for the in vivo chloroplast targeting of the TYMV 140K replication protein. Subcellular localization studies of deletion mutants identified a 41-residue internal sequence as the chloroplast targeting domain (CTD) of TYMV 140K; this sequence is sufficient to target GFP to the chloroplast envelope. The CTD appears to be located in the C-terminal extension of the methyltransferase domain—a region shared by 140K and its mature cleavage product 98K, which behaves as an integral membrane protein during infection. We predicted the CTD to fold into two amphipathic α-helices—a folding that was confirmed in vitro by circular dichroism spectroscopy analyses of a synthetic peptide. The importance for subcellular localization of the integrity of these amphipathic helices, and the function of 140K/98K, was demonstrated by performing amino acid substitutions that affected chloroplast targeting, membrane association and viral replication. These results establish a short internal α-helical peptide as an unusual signal for targeting proteins to the chloroplast envelope membrane, and provide new insights into membrane targeting of viral replication proteins—a universal feature of positive-strand RNA viruses.

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Sterol Binding by the Tombusviral Replication Proteins Is Essential for Replication in Yeast and Plants

Cited by 33 publications

References 45 publications

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

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

Three-Dimensional Architecture and Biogenesis of Membrane Structures Associated with Plant Virus Replication

Identification and Molecular Characterization of the Chloroplast Targeting Domain of Turnip yellow mosaic virus Replication Proteins

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