INTRODUCTION: Neutrophils are key players in the hyperinflammatory response during SARS-CoV-2 infection. The cytosolic proliferating cell nuclear antigen (PCNA) is a scaffolding protein highly dependent on the microenvironment status and known to interact with numerous proteins that regulate neutrophil functions. This study aimed to examine the cytosolic protein content and PCNA interactome in neutrophils from COVID-19 patients. METHODS: Proteomic analyses were performed on neutrophil cytosols from healthy donors and patients with severe or critical COVID-19. In vitro approaches were used to explore the biological significance of the COVID-19-specific PCNA interactome. RESULTS: Neutrophil cytosol analysis revealed a strong interferon (IFN) protein signature, with variations according to disease severity. Interactome analysis identified associations of PCNA with proteins involved in interferon signaling, cytoskeletal organization, and neutrophil extracellular trap (NET) formation, such as protein arginine deiminase type-4 (PADI4) and histone H3, particularly in critical patients. Functional studies of interferon signaling showed that T2AA, a PCNA scaffold inhibitor, downregulated IFN-related genes, including STAT1, MX1, IFIT1, and IFIT2 in neutrophils. Additionally, T2AA specifically inhibited the secretion of CXCL10, an IFN-dependent cytokine. PCNA was also found to interact with key effector proteins implicated in NET formation, such as histone H3, especially in critical COVID-19 cases. CONCLUSION: The analysis of the PCNA interactome has unveiled new protein partners that enhance the interferon pathway, thereby modulating immune responses and contributing to hyperinflammation in COVID-19. These findings provide valuable insights into interferon dysregulation in other immune-related conditions. INTRODUCTION: Neutrophils are key players in the hyperinflammatory response during SARS-CoV-2 infection. The cytosolic proliferating cell nuclear antigen (PCNA) is a scaffolding protein highly dependent on the microenvironment status and known to interact with numerous proteins that regulate neutrophil functions. This study aimed to examine the cytosolic protein content and PCNA interactome in neutrophils from COVID-19 patients. METHODS: Proteomic analyses were performed on neutrophil cytosols from healthy donors and patients with severe or critical COVID-19. In vitro approaches were used to explore the biological significance of the COVID-19-specific PCNA interactome. RESULTS: Neutrophil cytosol analysis revealed a strong interferon (IFN) protein signature, with variations according to disease severity. Interactome analysis identified associations of PCNA with proteins involved in interferon signaling, cytoskeletal organization, and neutrophil extracellular trap (NET) formation, such as protein arginine deiminase type-4 (PADI4) and histone H3, particularly in critical patients. Functional studies of interferon signaling showed that T2AA, a PCNA scaffold inhibitor, downregulated IFN-related genes, including STAT1, MX1, IFIT1, and IFIT2 in neutrophils. Additionally, T2AA specifically inhibited the secretion of CXCL10, an IFN-dependent cytokine. PCNA was also found to interact with key effector proteins implicated in NET formation, such as histone H3, especially in critical COVID-19 cases. CONCLUSION: The analysis of the PCNA interactome has unveiled new protein partners that enhance the interferon pathway, thereby modulating immune responses and contributing to hyperinflammation in COVID-19. These findings provide valuable insights into interferon dysregulation in other immune-related conditions.