Key Interplay between the Co-Opted Sorting Nexin-BAR Proteins and PI3P Phosphoinositide in the Formation of the Tombusvirus Replicase

Authors

Zhike Feng, University of KentuckyFollow
Nikolay Kovalev, University of KentuckyFollow
Peter D. Nagy, University of KentuckyFollow

Abstract

Positive-strand RNA viruses replicate in host cells by forming large viral replication organelles, which harbor numerous membrane-bound viral replicase complexes (VRCs). In spite of its essential role in viral replication, the biogenesis of the VRCs is not fully understood. The authors identified critical roles of cellular membrane-shaping proteins and PI(3)P (phosphatidylinositol 3-phosphate) phosphoinositide, a minor lipid with key functions in endosomal vesicle trafficking and autophagosome biogenesis, in VRC formation for tomato bushy stunt virus (TBSV). The authors show that TBSV co-opts the endosomal SNX-BAR (sorting nexin with Bin/Amphiphysin/Rvs- BAR domain) proteins, which bind to PI(3)P and have membrane-reshaping function during retromer tubular vesicle formation, directly into the VRCs to boost progeny viral RNA synthesis. We find that the viral replication protein-guided recruitment and pro-viral function of the SNX-BAR proteins depends on enrichment of PI(3)P at the site of viral replication. Depletion of SNX-BAR proteins or PI(3)P renders the viral double-stranded (ds)RNA replication intermediate RNAi-sensitive within the VRCs in the surrogate host yeast and in planta and ribonuclease-sensitive in cell-free replicase reconstitution assays in yeast cell extracts or giant unilamellar vesicles (GUVs). Based on our results, we propose that PI(3)P and the co-opted SNX-BAR proteins are coordinately exploited by tombusviruses to promote VRC formation and to play structural roles and stabilize the VRCs during viral replication. Altogether, the interplay between the co-opted SNX-BAR membrane-shaping proteins, PI(3)P and the viral replication proteins leads to stable VRCs, which provide the essential protection of the viral RNAs against the host antiviral responses.

Document Type

Article

Publication Date

12-28-2020

Notes/Citation Information

Published in PLOS Pathogens, v. 16, issue 12, e1009120.

© 2020 Feng et al.

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Digital Object Identifier (DOI)

https://doi.org/10.1371/journal.ppat.1009120

Funding Information

This work was supported by the National Science Foundation (MCB-1122039 and IOS-1922895) and a USDA hatch grant (KY012042) to PDN. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Related Content

Descriptions of the figures that are available for download as the additional files listed at the end of this record:

S1 Fig. Pro-viral roles of the endosomal SNX-BAR proteins in tombusvirus replication in yeast and plant protoplasts.

(A) Top image: Northern blot analysis shows decreased CIRV (+)repRNA accumulation in vps5Δ yeast strain. Vps5p and its deletion mutants were expressed from the constitutive TEF1 promoter from a plasmid. The accumulation level of repRNA was normalized based on 18S rRNA levels (second panel). The accumulation of Strep-p36, Strep-p95 and Vps5-Flag is measured by western blotting and anti-Strep or anti-Flag antibodies. See further details in Fig 1A. (B) Top image: Northern blot analysis shows decreased TBSV (+)repRNA accumulation in vps17Δ yeast strain. Vps17p SNX-BAR protein was expressed from the TEF1 promoter from a plasmid. The accumulation level of repRNA was normalized based on 18S rRNA levels (second panel). (C-D-E) VIGS-based knock-down of both SNX1 and SNX2b mRNA levels inhibits the accumulation of TBSV, CIRV and the related TCV RNAs in N. benthamiana protoplasts. Top panel: The accumulation of TBSV, CIRV and TCV gRNA and sgRNAs was measured using Northern blot analysis of total RNA samples obtained from N. benthamiana protoplasts. Second panel: ethidium-bromide stained gels show ribosomal RNA level. Protoplasts were isolated from the upper leaves of SNX1/SNX2b-silenced N. benthamiana on the 12th day, followed by transformation of TBSV, CIRV and TCV RNA, respectively. Twenty-four hours later, total RNA was analyzed by Northern blotting. (F) Up-regulation of Snx1 and Snx2b expression in TBSV and CIRV-infected N. benthamiana leaves. The mRNA levels for the SNX-BAR proteins were estimated by semi-quantitative RT-PCR in total RNA samples obtained from TBSV or CIRV-infected versus mock-infected N. benthamiana leaves. Tubulin mRNA and ribosomal RNA were used as controls (bottom panels). (G-H) The endosomal Vps5p SNX-BAR protein is not required for nodavirus replication in yeast. Top image: Northern blot analysis shows the accumulation level of NoV and FHV (+)RNA1 and the subgenomic RNA3 in vps5Δ yeast strain. The accumulation level of repRNA was normalized based on 18S rRNA levels (second panel). Bottom panel: The flag-tagged Protein A replication protein expression was measured with western blotting using anti-Flag antibody. https://doi.org/10.1371/journal.ppat.1009120.s007

S2 Fig. The TBSV and CIRV replication proteins interact with the endosomal SNX-BAR proteins in plant cells.

(A) Interaction between TBSV p92^pol replication protein and the SNX-BAR proteins was detected by BiFC assay in planta. TBSV p92-cYFP and the nYFP-Snx2b, nYFP-Snx2a or nYFP-Snx1 proteins were co-expressed from the 35S promoter after co-agroinfiltration into N. benthamiana leaves. Bottom three panels: negative control was GST-cYFP and the SNX-BAR proteins analyzed by BiFC assay in planta. Note that the plants were infected with TBSV to induce the viral replication compartments in cells. Co-localization of RFP-SKL peroxisomal luminar marker with the BiFC signals demonstrates that the interaction between p92pol and SNX-BAR proteins occurs in the viral replication compartments. Scale bars represent 10 μm. (B) Western blots show the accumulation levels of the proteins used in the BiFC assays. Top panel for S2A Fig, bottom panels for Fig 3A. (C) Interactions between CIRV p36 replication protein and the SNX-BAR proteins were detected by BiFC assay in planta. CIRV p36-cYFP and nYFP-Snx2b, nYFP-Snx2a or nYFP-Snx1 proteins were co-expressed from the 35S promoter after co-agroinfiltration into N. benthamiana leaves. nYFP-GST was co-expressed with p36-cYFP to serve as the negative control for BiFC assay. Note that the plants were infected with CIRV to induce the viral replication compartments in cells. Scale bars represent 10 μm. https://doi.org/10.1371/journal.ppat.1009120.s008

S3 Fig. Recruitment of the endosomal SNX-BAR proteins into the viral replication compartments by tombusviral replication proteins in yeast and plant cells.

(A-B) Co-localization of TBSV p33-BFP with the GFP-tagged Snx1 and GFP-tagged Snx2b in N. benthamiana cells is detected by confocal laser microscopy. Scale bars represent 5 μm. (C) Localization pattern of Snx1-GFP in the absence of viral components in N. benthamiana cells is detected by confocal laser microscopy. (D) Co-localization of TBSV GFP-p33 and CIRV GFP-p36, respectively, with Vps5-Flag SNX-BAR protein in yeast cells replicating repRNA. Vps5-Flag was detected with anti-Flag antibody. Each experiment was repeated three times. (E) Absent of excessive co-localization of TBSV GFP-p33 with Vps5yr-AA-Flag mutant protein in yeast cells replicating repRNA. https://doi.org/10.1371/journal.ppat.1009120.s009

S4 Fig. Both PI(3)P phosphoinositide and endosomal SNX-BAR proteins are co-opted into the viral replication compartments in plant cells.

(A) Co-localization of TBSV p33-BFP with the GFP-tagged Snx1 and with RFP-2xFYVE protein in N. benthamiana leaf tissues and protoplasts is detected by confocal laser microscopy. RFP-2xFYVE protein binds PI(3)P selectively. Top two panels: Co-localization of TBSV p33-BFP with the GFP-tagged Snx1 and with RFP-2xFYVE protein in N. benthamiana leaves. Middle panel represents images in the absence of viral components. Scale bars represent 10 μm. Bottom panel shows the co-localization of TBSV p33-BFP with the GFP-tagged Snx1 and with RFP-2xFYVE protein in N. benthamiana protoplasts. Scale bars represent 20 μm. (B) Co-localization of TBSV p33-BFP with the GFP-tagged Snx2b and with RFP-2xFYVE protein in N. benthamiana leaf tissues and protoplasts is detected by confocal laser microscopy. Top panel: Co-localization of TBSV p33-BFP with the GFP-tagged Snx1 and with RFP-2xFYVE protein in N. benthamiana leaves. Second panel represents images in the absence of viral components. Scale bars represent 10 μm. Third panel shows the co-localization of TBSV p33-BFP with the GFP-tagged Snx2b and with RFP-2xFYVE protein in N. benthamiana protoplasts. Bottom panel represents images in the absence of viral components. Scale bars represent 20 μm. https://doi.org/10.1371/journal.ppat.1009120.s010

S5 Fig. PI(3)P phosphoinositide is required for recruitment of Snx1 protein into the viral replication compartments in plant cells.

(A-B) BiFC assay shows the reduced level of interaction between p33 replication protein and the Snx1 protein in N. benthamiana treated with Wortmannin, a Vps34 PI3K inhibitor, or with DMSO as a negative control. TBSV p33-cYFP and nYFP-Snx1 proteins were co-expressed from the 35S promoter after co-agroinfiltration into N. benthamiana leaves. RFP-SKL was expressed as a peroxisomal marker to identify the viral replication compartments. Scale bars represent 10 μm. The BiFC signals were quantified via Image J. Each experiment was repeated. (C) Western blot analysis shows the accumulation level of nYFP-Snx1 and nYFP-Snx2b proteins and p33-cYFP in Wortmannin or DMSO-treated N. benthamiana leaves. (D) BiFC assay shows that nYFP-Snx1rry protein was not recruited by CIRV p36-cYFP replication protein into the viral replication compartment in N. benthamiana infected with CIRV. The CIRV replication compartment was decorated with CoxIV-RFP mitochondria marker protein. Scale bars represent 10 μm. (E) Pull-down assay shows direct interaction of TBSV p33 replication protein with the shown Snx1 mutants in vitro. Top panel: Western blot analysis of the captured GST-Snx1 mutants with the MBP-affinity purified p33 (lanes 1, 3, 5 and 7). The negative control was MBP (lanes 2, 4, 6 and 8). Bottom panel: The captured MBP-p33 and MBP were detected with anti-MBP antibody. Note that equal amount of each GST fusion protein was incubated with MBP-p33 or MBP. (F) Expression of RavZ^CT-p33 fusion protein inhibits TBSV replication in yeast. Reduced repRNA accumulation by expression of RavZ^CT-p33 fusion protein in comparison with p33 replication protein in wt yeast replicating TBSV repRNA. RavZ^CT-p33 fusion protein lacking the PI(3)P-binding domain (i.e., RavZ^CT-P) loses the strong inhibitory effect on TBSV replication. Top panel shows the northern blot analysis of (+)repRNA accumulation. Middle panel: the 18S rRNA level. Accumulation of p33 and the fusion proteins was shown in bottom panel. (G) Co-expression of RavZ^CT or RavZ^CT-P with viral p33, p92 and repRNA does not affect TBSV replication in wt yeast. The accumulation of repRNA was normalized based on 18S rRNA levels (second panel). The protein level of RavZ^CT or RavZ^CT-P was shown in bottom panel. https://doi.org/10.1371/journal.ppat.1009120.s011

S6 Fig. Recruitment of retromer proteins and the endosomal SNX-BAR proteins into VROs by tombusviral replication proteins in plant cells.

(A) Co-localization of TBSV p33-BFP with the GFP-tagged retromer proteins (Vps26, Vps35 and Vps29) and RFP-tagged Snx2b in N. benthamiana cells is detected by confocal laser microscopy. The plants were infected with TBSV. Scale bars represent 10 μm. (B) Different co-localization pattern of the GFP-tagged retromer proteins (Vps26, Vps35 and Vps29) and RFP-tagged Snx2b in the absence of viral components in N. benthamiana cells is detected by confocal laser microscopy. Scale bars represent 10 μm. https://doi.org/10.1371/journal.ppat.1009120.s012

S7 Fig. Vps5 SNX-BAR protein and PI(3)P are required for TBSV replication and protection of the viral dsRNA in vitro.

(A-B) Reduced repRNA production by the tombusvirus replicase assembled in vps5Δ yeast strain. The yeast strains expressed the p33 and p92^pol replication proteins and (+)repRNA, allowing for the assembly of the viral replicase complex in yeast peroxisomal membranes. The CFEs were treated with MNase 20 min latter, followed by the inactivation of MNase after 15 min with EGTA. Non-denaturing PAGE analysis shows the ³²P-labeled TBSV repRNA products from the in vitro assay. (C-D) In vitro reconstitution of the TBSV replicase using purified recombinant p33 and p92^pol replication proteins and TBSV (+)repRNA. The CFEs were prepared from wt yeast and were pre-incubated with purified recombinant Flag-tagged Ymr1 PI(3)P phosphatase to reduce the PI(3)P level in the CFE. The MNase treatments, which lasted for 15 min, were done at three different time points as shown. Non-denaturing PAGE analysis was done as in Fig 5. (E-F) In vitro association of Vps5 or Snx1 SNX-BAR proteins with the viral RNAs. In vitro RNA gel mobility shift assay shows that GST-Vps5 or GST-Snx1 bind to the ³²P-labeled (+)repRNA and (-)repRNA, respectively. Purified GST-Vps5, GST-Snx1 or GST was added in increasing amounts (1, 3, 6, 15 and 30 pMol for GST, 1, 3 and 6 pmol for GST-Vps5, and 6, 15 and 30 pmol for GST-Snx1) to the assays. The Vps5-repRNA or Snx1-repRNA complex was analyzed on non-denaturing 5% polyacrylamide gels. Each experiment was repeated. (G) Scheme of the MS2-CP-based RNA purification assay. Flag-p33, Flag-p92 and Vps5-3xHA and the repRNA derivatives were expressed in yeast to allow the formation of RNA-protein complexes. This scheme is to explain the experiments presented in Fig 7. https://doi.org/10.1371/journal.ppat.1009120.s013

Repository Citation

Feng, Zhike; Kovalev, Nikolay; and Nagy, Peter D., "Key Interplay between the Co-Opted Sorting Nexin-BAR Proteins and PI3P Phosphoinositide in the Formation of the Tombusvirus Replicase" (2020). Plant Pathology Faculty Publications. 97.
https://uknowledge.uky.edu/plantpath_facpub/97