Nuclear Transport of Respiratory Syncytial Virus Matrix Protein Is Regulated by Dual Phosphorylation Sites
Respiratory syncytial virus (RSV) is a major cause of respiratory infections in infants and the elderly. Although the RSV matrix (M) protein has key roles in the nucleus early in infection, and in the cytoplasm later, the molecular basis of switching between the nuclear and cytoplasmic compartments...
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| Format: | Article |
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MDPI AG
2022-07-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/14/7976 |
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| author | Reena Ghildyal Michael N. Teng Kim C. Tran John Mills Marco G. Casarotto Philip G. Bardin David A. Jans |
| author_facet | Reena Ghildyal Michael N. Teng Kim C. Tran John Mills Marco G. Casarotto Philip G. Bardin David A. Jans |
| author_sort | Reena Ghildyal |
| collection | DOAJ |
| description | Respiratory syncytial virus (RSV) is a major cause of respiratory infections in infants and the elderly. Although the RSV matrix (M) protein has key roles in the nucleus early in infection, and in the cytoplasm later, the molecular basis of switching between the nuclear and cytoplasmic compartments is not known. Here, we show that protein kinase CK2 can regulate M nucleocytoplasmic distribution, whereby inhibition of CK2 using the specific inhibitor 4,5,6,7-tetrabromobenzo-triazole (TBB) increases M nuclear accumulation in infected cells as well as when ectopically expressed in transfected cells. We use truncation/mutagenic analysis for the first time to show that serine (S) 95 and threonine (T) 205 are key CK2 sites that regulate M nuclear localization. Dual alanine (A)-substitution to prevent phosphorylation abolished TBB- enhancement of nuclear accumulation, while aspartic acid (D) substitution to mimic phosphorylation at S95 increased nuclear accumulation. D95 also induced cytoplasmic aggregate formation, implying that a negative charge at S95 may modulate M oligomerization. A95/205 substitution in recombinant RSV resulted in reduced virus production compared with wild type, with D95/205 substitution resulting in an even greater level of attenuation. Our data support a model where unphosphorylated M is imported into the nucleus, followed by phosphorylation of T205 and S95 later in infection to facilitate nuclear export and cytoplasmic retention of M, respectively, as well as oligomerization/virus budding. In the absence of widely available, efficacious treatments to protect against RSV, the results raise the possibility of antiviral strategies targeted at CK2. |
| first_indexed | 2024-03-09T10:17:02Z |
| format | Article |
| id | doaj.art-12c2b8e768624e24aed6f50d71d51a84 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T10:17:02Z |
| publishDate | 2022-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-12c2b8e768624e24aed6f50d71d51a842023-12-01T22:16:26ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-07-012314797610.3390/ijms23147976Nuclear Transport of Respiratory Syncytial Virus Matrix Protein Is Regulated by Dual Phosphorylation SitesReena Ghildyal0Michael N. Teng1Kim C. Tran2John Mills3Marco G. Casarotto4Philip G. Bardin5David A. Jans6Centre for Research in Therapeutic Solutions, Faculty of Science and Technology, University of Canberra, Canberra 2617, AustraliaDepartment of Internal Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL 33612, USADepartment of Internal Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL 33612, USAFaculty of Medicine, Monash University, Burnet Institute for Medical Research, The Alfred Hospital Department of Infectious Diseases, Melbourne 3004, AustraliaResearch School of Biology, Australian National University, Canberra 2601, AustraliaMonash Lung & Sleep and Hudson Institute, Monash University, Melbourne 3181, AustraliaNuclear Signalling Lab., Department of Biochemistry and Molecular Biology, Monash University, Melbourne 3181, AustraliaRespiratory syncytial virus (RSV) is a major cause of respiratory infections in infants and the elderly. Although the RSV matrix (M) protein has key roles in the nucleus early in infection, and in the cytoplasm later, the molecular basis of switching between the nuclear and cytoplasmic compartments is not known. Here, we show that protein kinase CK2 can regulate M nucleocytoplasmic distribution, whereby inhibition of CK2 using the specific inhibitor 4,5,6,7-tetrabromobenzo-triazole (TBB) increases M nuclear accumulation in infected cells as well as when ectopically expressed in transfected cells. We use truncation/mutagenic analysis for the first time to show that serine (S) 95 and threonine (T) 205 are key CK2 sites that regulate M nuclear localization. Dual alanine (A)-substitution to prevent phosphorylation abolished TBB- enhancement of nuclear accumulation, while aspartic acid (D) substitution to mimic phosphorylation at S95 increased nuclear accumulation. D95 also induced cytoplasmic aggregate formation, implying that a negative charge at S95 may modulate M oligomerization. A95/205 substitution in recombinant RSV resulted in reduced virus production compared with wild type, with D95/205 substitution resulting in an even greater level of attenuation. Our data support a model where unphosphorylated M is imported into the nucleus, followed by phosphorylation of T205 and S95 later in infection to facilitate nuclear export and cytoplasmic retention of M, respectively, as well as oligomerization/virus budding. In the absence of widely available, efficacious treatments to protect against RSV, the results raise the possibility of antiviral strategies targeted at CK2.https://www.mdpi.com/1422-0067/23/14/7976RSV matrix proteinCK2 phosphorylationnucleocytoplasmic shuttling |
| spellingShingle | Reena Ghildyal Michael N. Teng Kim C. Tran John Mills Marco G. Casarotto Philip G. Bardin David A. Jans Nuclear Transport of Respiratory Syncytial Virus Matrix Protein Is Regulated by Dual Phosphorylation Sites International Journal of Molecular Sciences RSV matrix protein CK2 phosphorylation nucleocytoplasmic shuttling |
| title | Nuclear Transport of Respiratory Syncytial Virus Matrix Protein Is Regulated by Dual Phosphorylation Sites |
| title_full | Nuclear Transport of Respiratory Syncytial Virus Matrix Protein Is Regulated by Dual Phosphorylation Sites |
| title_fullStr | Nuclear Transport of Respiratory Syncytial Virus Matrix Protein Is Regulated by Dual Phosphorylation Sites |
| title_full_unstemmed | Nuclear Transport of Respiratory Syncytial Virus Matrix Protein Is Regulated by Dual Phosphorylation Sites |
| title_short | Nuclear Transport of Respiratory Syncytial Virus Matrix Protein Is Regulated by Dual Phosphorylation Sites |
| title_sort | nuclear transport of respiratory syncytial virus matrix protein is regulated by dual phosphorylation sites |
| topic | RSV matrix protein CK2 phosphorylation nucleocytoplasmic shuttling |
| url | https://www.mdpi.com/1422-0067/23/14/7976 |
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