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Updated: Sep 3, 2025
Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses
Published on: April 4, 2019
Nuclear Transport of Respiratory Syncytial Virus Matrix Protein Is Regulated by Dual Phosphorylation Sites
Reena Ghildyal1, Michael N Teng2, Kim C Tran2
1Centre for Research in Therapeutic Solutions, Faculty of Science and Technology, University of Canberra, Canberra 2617, Australia.
Abstract:
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.
Insights
Protein kinase CK2 regulates the movement of respiratory syncytial virus (RSV) M protein between the nucleus and cytoplasm. Targeting CK2 may offer new antiviral strategies against RSV infections.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Respiratory syncytial virus (RSV) causes significant respiratory illness in infants and the elderly.
- The RSV matrix (M) protein shuttles between the nucleus and cytoplasm during infection, but the mechanism controlling this switch is unknown.
Purpose of the Study:
- To investigate the role of protein kinase CK2 in regulating the nucleocytoplasmic distribution of the RSV M protein.
- To identify specific phosphorylation sites on the M protein that influence its localization and function.
Main Methods:
- Utilized the CK2 inhibitor TBB to assess its effect on M protein localization in infected and transfected cells.
- Employed truncation and mutagenic analysis, including alanine and aspartic acid substitutions, to map key regulatory sites on the M protein.
- Generated recombinant RSV with specific M protein mutations to evaluate their impact on virus production.
Main Results:
- Inhibition of CK2 with TBB increased M protein accumulation in the nucleus.
- Serine 95 (S95) and Threonine 205 (T205) were identified as critical CK2 phosphorylation sites regulating M nuclear localization.
- Mutations mimicking phosphorylation at S95 led to cytoplasmic aggregation, suggesting a role in M oligomerization.
- Recombinant RSV with mutated M proteins (A95/205 and D95/205) exhibited reduced virus production compared to wild-type.
Conclusions:
- A model is proposed where M protein nuclear import is followed by T205 and S95 phosphorylation, facilitating nuclear export and cytoplasmic retention, respectively.
- Phosphorylation at S95 appears to modulate M protein oligomerization and viral budding.
- Targeting protein kinase CK2 presents a potential therapeutic strategy for developing novel antiviral treatments against RSV.
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