Screening<i>Legionella</i>effectors for antiviral effects reveals Rab1 GTPase as a proviral factor coopted for tombusvirus replication

Inaba, Jun-ichi; Xu, Kai; Kovalev, Nikolay; Ramanathan, Harish N.; Roy, Craig R.; Lindenbach, Brett D.; Nagy, Peter D.

doi:10.1073/pnas.1911108116

Cited by 27 publications

(23 citation statements)

References 46 publications

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“…257 out of a total of 2930 proteins of the L. pneumophila strain Philadelphia-1 (Proteome ID: UP000000609) contain the FxxxLxxxK sequence motif. Of these, 46 proteins, including SidH and VpdB, belong to the 280 known Legionella effectors 35 , indicating that this sequence is enriched in the effector proteins (16.4% of effectors vs. 8.0% of non-effectors). However, the FxxxLxxxK motif is not present in the C-terminal regions of SetA and PieA, which can also bind to LvgA.…”

Section: Discussionmentioning

confidence: 99%

Structural basis for effector protein recognition by the Dot/Icm Type IVB coupling protein complex

et al. 2020

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The Legionella pneumophila Dot/Icm type IVB secretion system (T4BSS) is extremely versatile, translocating~300 effector proteins into host cells. This specialized secretion system employs the Dot/Icm type IVB coupling protein (T4CP) complex, which includes IcmS, IcmW and LvgA, that are known to selectively assist the export of a subclass of effectors. Herein, the crystal structure of a four-subunit T4CP subcomplex bound to the effector protein VpdB reveals an interaction between LvgA and a linear motif in the C-terminus of VpdB. The same binding interface of LvgA also interacts with the C-terminal region of three additional effectors, SidH, SetA and PieA. Mutational analyses identified a FxxxLxxxK binding motif that is shared by VpdB and SidH, but not by SetA and PieA, showing that LvgA recognizes more than one type of binding motif. Together, this work provides a structural basis for how the Dot/Icm T4CP complex recognizes effectors, and highlights the multiple substrate-binding specificities of its adaptor subunit.

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Section: Discussionmentioning

confidence: 99%

Structural basis for effector protein recognition by the Dot/Icm Type IVB coupling protein complex

et al. 2020

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“…The following yeast expression plasmids have been previously described: pGAD-LEU-Cup1-His92 ( 102 The following plant expression plasmids have been previously described: pGD-p33-RFP, pGD-p33-GFP, pGD-p33-BFP, and pGD-RFP-SKL (26, 30, 67) and pEarleyGate100-DrrA (35).…”

Section: Methodsmentioning

confidence: 99%

“…TBSV replication triggers characteristic alterations in cells, including stabilization of the actin network, recruitment of endosomal and COPII vesicles into the viral replication compartments, induction of subcellular membrane proliferation, aggregation of peroxisomes, and exploitation of the ATP-generating glycolytic and fermentation pathways (26,(30)(31)(32)(33)(34)(35)(36). Another feature of TBSV replication is the formation/stabilization of membrane contact sites (MCSs) between the endoplasmic reticulum (ER) and peroxisomes, which are required for sterol enrichment within the replication compartment to support efficient virus replication (5,19,(37)(38)(39).…”

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confidence: 99%

Co-opted Cellular Sac1 Lipid Phosphatase and PI(4)P Phosphoinositide Are Key Host Factors during the Biogenesis of the Tombusvirus Replication Compartment

Sasvari

Lin

Inaba

et al. 2020

J Virol

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Positive-strand RNA [(+)RNA] viruses assemble numerous membrane-bound viral replicase complexes (VRCs) with the help of viral replication proteins and co-opted host proteins within large viral replication compartments in the cytosol of infected cells. In this study, we found that deletion or depletion of Sac1 phosphatidylinositol 4-phosphate [PI(4)P] phosphatase reduced tomato bushy stunt virus (TBSV) replication in yeast (Saccharomyces cerevisiae) and plants. We demonstrate a critical role for Sac1 in TBSV replicase assembly in a cell-free replicase reconstitution assay. The effect of Sac1 seems to be direct, based on its interaction with the TBSV p33 replication protein, its copurification with the tombusvirus replicase, and its presence in the virus-induced membrane contact sites and within the TBSV replication compartment. The proviral functions of Sac1 include manipulation of lipid composition, sterol enrichment within the VRCs, and recruitment of additional host factors into VRCs. Depletion of Sac1 inhibited the recruitment of Rab5 GTPase-positive endosomes and enrichment of phosphatidylethanolamine in the viral replication compartment. We propose that Sac1 might be a component of the assembly hub for VRCs, likely in collaboration with the co-opted the syntaxin18-like Ufe1 SNARE protein within the TBSV replication compartments. This work also led to demonstration of the enrichment of PI(4)P phosphoinositide within the replication compartment. Reduction in the PI(4)P level due to chemical inhibition in plant protoplasts; depletion of two PI(4)P kinases, Stt4p and Pik1p; or sequestration of free PI(4)P via expression of a PI(4)P-binding protein in yeast strongly inhibited TBSV replication. Altogether, Sac1 and PI(4)P play important proviral roles during TBSV replication. IMPORTANCE Replication of positive-strand RNA viruses depends on recruitment of host components into viral replication compartments or organelles. Using TBSV, we uncovered the critical roles of Sac1 PI(4)P phosphatase and its substrate, PI(4)P phosphoinositide, in promoting viral replication. Both Sac1 and PI(4)P are recruited to the site of viral replication to facilitate the assembly of the viral replicase complexes, which perform viral RNA replication. We found that Sac1 affects the recruitment of other host factors and enrichment of phosphatidylethanolamine and sterol lipids within the subverted host membranes to promote optimal viral replication. In summary, this work demonstrates the novel functions of Sac1 and PI(4)P in TBSV replication in the model host yeast and in plants.

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“…Why do tombusviruses need the help of these co-opted tethering proteins? The tombusvirus replication proteins interact with numerous cellular proteins with pro-viral as well as antiviral functions [45,[90][91][92]. Therefore, tombusvirus replication proteins might be too promiscuous and not be suitable to build vMCSs with targeted organelles without the contribution of the specialized tethering host factors.…”

Section: Plos Pathogensmentioning

confidence: 99%

Dynamic interplay between the co-opted Fis1 mitochondrial fission protein and membrane contact site proteins in supporting tombusvirus replication

et al. 2021

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Plus-stranded RNA viruses have limited coding capacity and have to co-opt numerous pro-viral host factors to support their replication. Many of the co-opted host factors support the biogenesis of the viral replication compartments and the formation of viral replicase complexes on subverted subcellular membrane surfaces. Tomato bushy stunt virus (TBSV) exploits peroxisomal membranes, whereas the closely-related carnation Italian ringspot virus (CIRV) hijacks the outer membranes of mitochondria. How these organellar membranes can be recruited into pro-viral roles is not completely understood. Here, we show that the highly conserved Fis1 mitochondrial fission protein is co-opted by both TBSV and CIRV via direct interactions with the p33/p36 replication proteins. Deletion of FIS1 in yeast or knockdown of the homologous Fis1 in plants inhibits tombusvirus replication. Instead of the canonical function in mitochondrial fission and peroxisome division, the tethering function of Fis1 is exploited by tombusviruses to facilitate the subversion of membrane contact site (MCS) proteins and peroxisomal/mitochondrial membranes for the biogenesis of the replication compartment. We propose that the dynamic interactions of Fis1 with MCS proteins, such as the ER resident VAP tethering proteins, Sac1 PI4P phosphatase and the cytosolic OSBP-like oxysterol-binding proteins, promote the formation and facilitate the stabilization of virus-induced vMCSs, which enrich sterols within the replication compartment. We show that this novel function of Fis1 is exploited by tombusviruses to build nuclease-insensitive viral replication compartment.

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

Cited by 27 publications

References 46 publications

Structural basis for effector protein recognition by the Dot/Icm Type IVB coupling protein complex

Structural basis for effector protein recognition by the Dot/Icm Type IVB coupling protein complex

Co-opted Cellular Sac1 Lipid Phosphatase and PI(4)P Phosphoinositide Are Key Host Factors during the Biogenesis of the Tombusvirus Replication Compartment

Dynamic interplay between the co-opted Fis1 mitochondrial fission protein and membrane contact site proteins in supporting tombusvirus replication

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