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Characterization of the Interaction Between SARS-CoV-2 Membrane Protein (M) and Proliferating Cell Nuclear Antigen
Érika Pereira Zambalde1, Isadora Carolina Betim Pavan2, Mariana Camargo Silva Mancini1
1Multidisciplinary Laboratory of Food and Health, School of Applied Sciences, University of Campinas (Unicamp), Limeira, Brazil.
Abstract:
SARS-CoV-2 is an emerging virus from the Coronaviridae family and is responsible for the ongoing COVID-19 pandemic. In this work, we explored the previously reported SARS-CoV-2 structural membrane protein (M) interaction with human Proliferating Cell Nuclear Antigen (PCNA). The M protein is responsible for maintaining virion shape, and PCNA is a marker of DNA damage which is essential for DNA replication and repair. We validated the M-PCNA interaction through immunoprecipitation, immunofluorescence co-localization, and PLA (Proximity Ligation Assay). In cells infected with SARS-CoV-2 or transfected with M protein, using immunofluorescence and cell fractioning, we documented a reallocation of PCNA from the nucleus to the cytoplasm and the increase of PCNA and γH2AX (another DNA damage marker) expression. We also observed an increase in PCNA and γH2AX expression in the lung of a COVID-19 patient by immunohistochemistry. In addition, the inhibition of PCNA translocation by PCNA I1 and Verdinexor led to a reduction of plaque formation in an in vitro assay. We, therefore, propose that the transport of PCNA to the cytoplasm and its association with M could be a virus strategy to manipulate cell functions and may be considered a target for COVID-19 therapy.
Insights
The SARS-CoV-2 M protein interacts with human PCNA, a DNA repair protein. This interaction causes PCNA to move to the cytoplasm, potentially offering a new target for COVID-19 therapies.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- SARS-CoV-2, the virus causing COVID-19, belongs to the Coronaviridae family.
- The viral structural membrane (M) protein plays a role in virion structure.
- Proliferating Cell Nuclear Antigen (PCNA) is crucial for DNA replication and repair and is a marker of DNA damage.
Purpose of the Study:
- To investigate the interaction between the SARS-CoV-2 M protein and human PCNA.
- To understand the functional consequences of this interaction in infected cells.
- To explore potential therapeutic strategies targeting this interaction.
Main Methods:
- Immunoprecipitation and Proximity Ligation Assay (PLA) to confirm M-PCNA interaction.
- Immunofluorescence and cell fractionation to track PCNA localization.
- Immunohistochemistry on patient lung tissue.
- In vitro plaque formation assays with PCNA translocation inhibitors.
Main Results:
- Validated the interaction between SARS-CoV-2 M protein and human PCNA.
- Observed PCNA reallocation from the nucleus to the cytoplasm in infected cells and M protein-transfected cells.
- Documented increased expression of PCNA and γH2AX (DNA damage marker) in infected cells and COVID-19 patient lungs.
- Inhibition of PCNA translocation reduced viral plaque formation in vitro.
Conclusions:
- The SARS-CoV-2 M protein interacts with PCNA, inducing its cytoplasmic translocation.
- This M-PCNA association may be a viral strategy to manipulate host cell functions.
- Targeting PCNA translocation presents a potential therapeutic avenue for COVID-19.
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