Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, poses a significant global health threat. The virus's spike (S) protein, composed of S1 and S2 subunits, is critical for host cell entry. S1 mediates receptor recognition (primarily ACE2), while S2 facilitates membrane fusion. While previous research has identified various receptors or co-receptors for S1, the role of S2 in interacting with host factors remains largely unexplored. Neutrophil extracellular traps (NETs), released by neutrophils in response to pathogens, typically exhibit antiviral activity by capturing and killing viruses. However, elevated levels of NETs and extracellular histones have been observed in severe COVID-19 patients, raising questions about their role in SARS-CoV-2 infection. This study investigates the potential impact of NETosis, particularly the released histones, on SARS-CoV-2 infectivity.

Existing literature establishes the antiviral role of NETs against viruses like influenza, HIV, and RSV. This primarily involves the electrostatic binding of viral particles to the NET's chromatin backbone and the antiviral effects of NET components such as myeloperoxidase and α-defensins. The positively charged histones within NETs are also known to contribute to antiviral effects. However, the role of NETs in SARS-CoV-2 infection is less clear. Although some studies reported increased NETs in severe COVID-19 patients, the effect of NETs or NETosis on SARS-CoV-2 infectivity was unknown. Studies also suggest that SARS-CoV-2 may trigger NET release. This study aimed to clarify the relationship between NETosis, histone release, and SARS-CoV-2 infectivity, focusing on the possible interaction of histones with the S2 subunit of the viral spike protein.

The study employed a multifaceted approach using live SARS-CoV-2 and two pseudovirus systems (luciferase and EGFP reporters). Vero E6 cells and ACE2-expressing HEK-293T cells were used for infection assays. Neutrophils were activated with phorbol myristate acetate (PMA) to induce NETosis. The impact of PMA-activated neutrophils, NETosis inhibitors (Cl-amidine), and DNase I on SARS-CoV-2 infectivity was assessed. The role of individual histones (H2A, H2B, H3, H4) in enhancing pseudovirus infectivity was determined. Biochemical binding assays, surface plasmon resonance (SPR), and molecular simulations (ZDOCK and MD) were used to investigate the interaction between histones (particularly H3 and H4) and the SARS-CoV-2 spike protein (S1, S2, and ectodomain). Mutagenesis of the S2 subunit was performed to identify specific amino acid residues involved in histone binding. The role of sialic acid on the host cell surface was explored using neuraminidase (NA), free sialic acid (Neu5Ac), and polysialic acid (PSA). A cell-cell fusion assay evaluated the effect of histones and sialic acid on membrane fusion. Finally, a mouse model of SARS-CoV-2-induced acute respiratory distress syndrome was used to assess the in vivo effects of NETosis inhibitors, histones, and sialic acid on viral replication (sgRNA copies) and apoptosis. Confocal microscopy was used to visualize NETosis and co-localization of histones with the spike protein in lung tissue.

Contrary to expectations, the study found that PMA-activated neutrophils significantly enhanced SARS-CoV-2 infectivity compared to unactivated neutrophils. This effect was reversed by Cl-amidine, indicating a role for NETosis. Histones H3 and H4, but not H2A or H2B, significantly enhanced pseudovirus infectivity in a dose-dependent manner. Binding assays and SPR revealed that histones H3 and H4 selectively bind to the S2 subunit of the spike protein, specifically to a negatively charged domain containing D1139, E1144, E1150, and D1153. Molecular simulations supported this interaction and showed that histones can bridge between S2 and sialic acid on the host cell surface. Removal of sialic acid by neuraminidase or blocking with free sialic acid or PSA significantly reduced the enhancement of infectivity mediated by histones. Histones also enhanced cell-cell fusion mediated by the spike protein, an effect inhibited by neuraminidase. In the mouse model, treatment with Cl-amidine or Neu5Ac reduced viral sgRNA copies and apoptosis, while treatment with histones H3 or H4 increased both. Immunofluorescence imaging of lung tissue from the mouse model revealed co-localization of histones and spike protein, confirming the in vivo interaction.

These findings challenge the traditional view of NETs as solely protective against viral infections. The study demonstrates a novel mechanism where SARS-CoV-2 hijacks histones released during NETosis to enhance its infectivity. The specific interaction between histones (H3 and H4) and the negatively charged region of the S2 subunit, acting as a bridge to sialic acid on host cells, represents a previously unidentified aspect of SARS-CoV-2 entry. This mechanism differs from the previously described interaction between S1 and heparan sulfate, which occurs in a separate binding site. The enhanced membrane fusion observed in the presence of histones suggests that this bridging mechanism promotes closer proximity of the viral and host cell membranes, facilitating the fusion process. The results from the mouse model support the in vitro findings, demonstrating the in vivo relevance of this mechanism. The observed reduction in viral replication and apoptosis upon inhibiting NETosis or blocking sialic acid points to potential therapeutic targets.

This study reveals a previously unknown mechanism by which SARS-CoV-2 utilizes histones from NETosis to enhance its infectivity. The interaction between histones H3/H4, the S2 subunit of the spike protein, and sialic acid on host cells represents a novel target for antiviral therapies. Further research could focus on developing inhibitors targeting this interaction, exploring the role of specific histone modifications in this process, and investigating the broader implications of this mechanism in other viral infections. Understanding this interaction could significantly improve the treatment of COVID-19 and other viral diseases.

The study primarily used in vitro and in vivo mouse models. While these models provide valuable insights, translating these findings directly to human patients requires further investigation. The focus on histones H3 and H4 may overlook the potential contributions of other NET components. The use of recombinant histones in some experiments, although justified by the literature, may differ slightly from naturally occurring histones released during NETosis. Furthermore, the precise in vivo dynamics of the interaction between histones, the S2 subunit, and sialic acid need further elucidation.