Characterization of the interaction between SARS-CoV-2 Membrane Protein and Proliferating Cell Nuclear Antigen (PCNA) as a Potential Therapeutic Target

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 responsi...

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Published in:	bioRxiv 2021-12
Main Authors:	Erika Pereira Zambalde, Mariana Camargo Silva Mancini, Orlando Bonito Scudero, Isadora Carolina Betim Pavan, Severi, Matheus Brandemarte, Ana Paula Morelli, Amorim, Mariene R, Karina Bispo Dos Santos, Pierina Lorencini Parise, Gois, Mariana Marcela, Toledo-Teixeira, Daniel A, Mauad, Thais, Paulo Hilario Nascimento Saldiva, Dolhnikoff, Marisa, Proenca-Modena, Jose Luiz, Marques-Souza, Henrique, Armando Morais Ventura, Simabuco, Fernando M
Format:	Article
Language:	eng
Subjects:	Antigens Coronaviruses COVID-19 Cytoplasm Deoxyribonucleic acid DNA DNA biosynthesis DNA damage DNA repair Immunofluorescence Immunohistochemistry Immunoprecipitation Localization M protein Membrane proteins Pandemics Proliferating cell nuclear antigen Proteins Severe acute respiratory syndrome coronavirus 2 Virions
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Summary:	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 a PLA 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 of 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. Competing Interest Statement The authors have declared no competing interest.