Resumen
Replication of positive-strand RNA viruses depends on usurped cellular membranes and co-opted host proteins. Based on pharmacological inhibition and genetic and biochemical approaches, the authors identified critical roles of the cellular Cdc48 unfoldase/segregase protein in facilitating the replication of tomato bushy stunt virus (TBSV). We show that TBSV infection induces the expression of Cdc48 in Nicotiana benthamiana plants. Cdc48 binds to the TBSV replication proteins through its N-terminal region. In vitro TBSV replicase reconstitution experiments demonstrated that Cdc48 is needed for efficient replicase assembly and activity. Surprisingly, the in vitro replication experiments also showed that excess amount of Cdc48 facilitates the disassembly of the membrane-bound viral replicase-RNA template complex. Cdc48 is also needed for the recruitment of additional host proteins. Because several human viruses, including flaviviruses, utilize Cdc48, also called VCP/p97, for replication, we suggest that Cdc48 might be a common panviral host factor for plant and animal RNA viruses.
| Idioma original | English |
|---|---|
| Páginas (desde-hasta) | 1-17 |
| Número de páginas | 17 |
| Publicación | Virology |
| Volumen | 576 |
| DOI | |
| Estado | Published - nov 2022 |
Nota bibliográfica
Publisher Copyright:© 2022 Elsevier Inc.
Financiación
We thank Dr. Judit Pogany for comments on the manuscript. The contributions by Dr. Songbai Zhang to the initial experiments are appreciated by the authors. The authors also thank Drs. Herman B. Scholthof, Texas A&M and Robert Mullen, University of Guelph, Canada for the anti-p33 primary antibody. In addition, the authors are grateful for Dr. Nihal Altan-Bonnet (NIH) and Brett Tyler (Oregon State University) for providing the GFP-2xFYVE construct. The authors thank Dr. Charles Boone (University of Toronto) for the temperature-sensitive yeast mutants. This work was supported by the National Science Foundation ( MCB-1517751 and IOS-1922895 ), USDA ( NIFA , 2020-70410-32901 ) and a USDA hatch grant (KY012042) to PDN. In the third set of experiments, we reconstituted the TBSV replicase in WT yeast CFE, followed by addition of purified GST-Cdc48 (10 pM amount) at 0, 20, 40, 60 and 80 min time-points. Then, the replication assay is stopped after 3 h incubation, followed by the addition of RNase III to destroy the unprotected dsRNAs. At the end of the assay, we measured the level of the TBSV dsRNA replication intermediate [produced by minus-strand synthesis on the (+)RNA template], which is always present in the viral replicase complex (Kovalev et al., 2014). As a control, we added purified GST into the replicase assay. The WT CFE supports the assembly of the TBSV replicase within the first 40–60 min, after which the replicase and the dsRNA become mostly inaccessible to RNase III (Kovalev et al., 2017; Kovalev et al., 2014). Accordingly, the control samples showed high level of dsRNA protection by the replicase at each time point (Fig. 9E). Surprisingly, the addition of Cdc48 rendered the TBSV dsRNA sensitive to RNase III at each time point, including the late time points (60 and 80 min) and inhibited TBSV replication (Fig. 9E). These data suggest that excess amount of Cdc48 helps destabilizing the VRCs and eliminates the VRC provided protection of the viral dsRNA from RNase III nuclease. This suggests that the segregase function of Cdc48 is capable of targeting the assembled membranous replicase in vitro.In vitro TBSV replication assay. To support in vitro TBSV replication, cell-free extracts (CFE) were prepared from untransformed R1158, TET:CDC48, BY4741, cdc48-1, cdc48-2, and cdc48-3 cdc48-4601 yeast strains as described earlier (Pogany et al., 2008; Xu and Nagy, 2015). Reaction mixture for the in vitro TBSV replication assays contained 2 μl of CFE, 0.5 μg (+) DI-72 RNA transcripts, 400 ng affinity-purified MBP-p33, 400 ng affinity-purified MBP-p92pol in 20 μl total volume. 8 pmol of purified GST-tagged Cdc48 proteins or GST protein were used in the CFE assay. The reactions were performed for 3 h at 25 oC. The RNA samples were extracted with phenol-chloroform and precipitated, and subjected to nondenaturing PAGE analysis.We thank Dr. Judit Pogany for comments on the manuscript. The contributions by Dr. Songbai Zhang to the initial experiments are appreciated by the authors. The authors also thank Drs. Herman B. Scholthof, Texas A&M and Robert Mullen, University of Guelph, Canada for the anti-p33 primary antibody. In addition, the authors are grateful for Dr. Nihal Altan-Bonnet (NIH) and Brett Tyler (Oregon State University) for providing the GFP-2xFYVE construct. The authors thank Dr. Charles Boone (University of Toronto) for the temperature-sensitive yeast mutants. This work was supported by the National Science Foundation (MCB-1517751 and IOS-1922895), USDA (NIFA, 2020-70410-32901) and a USDA hatch grant (KY012042) to PDN.
| Financiadores | Número del financiador |
|---|---|
| VRCs | |
| WT CFE | |
| National Science Foundation Arctic Social Science Program | MCB-1517751, IOS-1922895 |
| National Institutes of Health (NIH) | |
| U.S. Department of Agriculture | |
| US Department of Agriculture National Institute of Food and Agriculture, Agriculture and Food Research Initiative | 2020-70410-32901, KY012042 |
| Texas AandM University | |
| Oregon State University | |
| Center for Infectious Disease and Vaccine Research La Jolla Institute for Immunology (LJI) | CDC48, BY4741, cdc48-3 cdc48-4601, R1158 |
| Conseil Français de l'Énergie |
ASJC Scopus subject areas
- Virology
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