Impacts of p97 on Proteome Changes in Human Cells during Coronaviral Replication
Abstract
Human coronavirus (HCoV) similar to other viruses rely on host cell machinery for both replication and to spread. The p97/VCP ATPase is associated with diverse pathways that may favor HCoV replication. In this study, we assessed the role of p97 and associated host responses in human lung cell line H1299 after HCoV-229E or HCoV-OC43 infection. Inhibition of p97 function by small molecule inhibitors shows antiviral activity, particularly at early stages of the virus life cycle, during virus uncoating and viral RNA replication. Importantly, p97 activity inhibition protects human cells against HCoV-induced cytopathic effects. The p97 knockdown also inhibits viral production in infected cells. Unbiased quantitative proteomics analyses reveal that HCoV-OC43 infection resulted in proteome changes enriched in cellular senescence and DNA repair during virus replication. Further analysis of protein changes between infected cells with control and p97 shRNA identifies cell cycle pathways for both HCoV-229E and HCoV-OC43 infection. Together, our data indicate a role for the essential host protein p97 in supporting HCoV replication, suggesting that p97 is a therapeutic target to treat HCoV infection.
Additional Information
© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). Received: 15 September 2021; Accepted: 25 October 2021; Published: 29 October 2021. This research was funded by the National Institute of Neurological Disorders and Stroke, grant number R01NS102279, and the Merkin Institute for Translational Research at Caltech. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.3390/cells10112953/s1, Figure S1: Full blot images of results shown in Figure 1B; Figure S2: Cell viability of HCoV-infected H1299 cells in the presence of CB-5083; Figure S3: Full blot images of results shown in Figure 2B; Figure S4: Cell viability of HCoV-infected H1299 cells in the stage-limited inhibition assay; Figure S5: Full blot images of results shown in Figure 3B; Figure S6: Knockdown of p97 expression in H1299 cells; Figure S7: Cell viability of H1299 cells with inducible control shRNA (Ctrl shRNA) or p97 shRNA after HCoV infection; Figure S8: Full blot images of results shown in Figure 4B; Figure S9: Overlapping host responses in cells after HCoV-229E, HCoV-OC43, or SARS-CoV-2 infection; Figure S10: Proteins enriched in "Cellular responses to stress"; Figure S11: Proteins enriched "Host interactions of HIV factors"; Figure S12: Proteins enriched "Mitotic anaphases"; Table S1: TagMan probes list; Table S2: Proteomics of H1299 cells with control shRNA and p97 shRNA after HCoV-229E infection; Table S3: Proteomics of H1299 cells with control shRNA and p97 shRNA after HCoV-OC43 infection; Table S4: Reactome pathway enrichment analysis; Table S5: Multi-list Reactome pathway enrichment analysis. Author Contributions: T.-F.C. conceived and supervised the project. K.-W.C. designed and performed experiments. S.L. and F.W. assisted in sample preparation for proteomics. N.M.R.-L. provided technique support for RNA extraction and real-time PCR. S.L., F.W. and N.H. assisted in proteomics data analysis and provided suggestions. K.-W.C. wrote the manuscript with support from T.-F.C., N.M.R.-L. and N.H. edited the manuscript. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: All relevant data generated during this study are included in the article and the supplementary information. The mass spectrometry raw data are deposited to the ProteomeXchance Consortium (https://www.ebi.ac.uk/pride/ (accessed on 21 October 2021)) via the PRIDE repository with the dataset identifier PXD026216 and 10.6019/PXD026216). Additional raw data generated during the current study and relevant information are available from the corresponding authors upon request. The authors declare no conflict of interest.Attached Files
Published - cells-10-02953-v2.pdf
Supplemental Material - cells-10-02953-s001.zip
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Additional details
- PMCID
- PMC8616207
- Eprint ID
- 111925
- Resolver ID
- CaltechAUTHORS:20211117-180505059
- R01NS102279
- NIH
- Caltech Merkin Institute for Translational Research
- Created
-
2021-11-17Created from EPrint's datestamp field
- Updated
-
2023-10-05Created from EPrint's last_modified field
- Caltech groups
- COVID-19, Richard N. Merkin Institute for Translational Research, Division of Biology and Biological Engineering