Introduction: influenza virus causes epidemics and pandemics, with significant impact on public health. The cytoskeleton is hijacked by influenza virus to reach the replication site. Previous data assessed the role of actin- and tubulin-stabilization as restriction factors of human influenza A/NWS/33 virus (IAV) infection in rhesus monkey-kidney (LLC-MK2) cells (semi-permissive model). This study aims to investigate the involvement of cytokeratin 8 (K8) phosphorylation, a post-translational modification of intermediate filaments (IF), on IAV infection in LLC-MK2 and human type II alveolar epithelial (A549; permissive model) cells. Materials and Methods: LLC-MK2 and A549 cells were infected for 24 h with IAV at a multiplicity of infection (MOI) of 0.1 or 2 plaque-forming units (PFU)/cell. IF-depolymerization was induced with acrylamide (5 mM), while IF-phosphorylation with okadaic acid (OA, phosphatase inhibitor; 0.05 microg/ml) and 12-O-tetradecanoylphorbol-13-acetate (TPA, protein-kinase activator; 25-50 nM), applied for different periods. Immunofluorescence and Western blotting assays were carried out using anti-viral nucleoprotein (NP) (Argene/BioMérieux), anti-K8 (Thermo Fischer Scientific), and anti-phospho-K8 (Thermo Fischer Scientific) antibodies. Secondary antibodies conjugated with isothiocyanate of rodamine/fluorescein or alcaline phosphatase were used. Fifty percent tissue culture infectious dose (TCID50) assays were performed to evaluate viral yields. Results: K8 showed a higher expression in A549 vs LLC-MK2 cells, while K8 phosphorylation was low in both models. IF-depolymerization with acrylamide raised NP expression in both cells, almost unchanging the viral yields. IF-phosphorylation with OA increased NP expression in A549 but not in LLC-MK2 cells, modifying the K8 pattern mostly in A549 cells. IF-phosphorylation with TPA decreased NP expression in LLC-MK2, while increased that of A549 cells, raising K8 phosphorylation mostly in A549 cells. Viral yields were consistent with the obtained results. IAV infection strongly modulated K8 organization and increased K8 phosphorylation in A549 cells, leaving almost unmodified LLC-MK2. Discussion and Conclusions: the depolymerization of IF favours IAV infection in the examined models. The drug-mediated IF-phosphorylation promotes IAV infection in A549 cells, showing no significant effects in LLC-MK2. Accordingly, viral infection prevalently modulates K8 organization and phosphorylation in A549 cells. These results evidence the presence of different regulation mechanisms acting on IF post-translational modifications in the studied models. Understanding how specific cell functions/components regulate influenza virus replication may provide new starting points for innovative anti-viral researches.

Role of post-translational modifications of cytoskeletal intermediate filaments on influenza A/NWS/33 virus (H1N1) infection in mammalian cells: focus on cytokeratin 8 phosphorylation / DE CONTO, Flora; Conversano, Francesca; Arcangeletti, Maria Cristina; Chezzi, Carlo; Calderaro, Adriana. - (2018), pp. 51-51. ((Intervento presentato al convegno 46° Congresso Nazionale della Società Italiana di Microbiologia tenutosi a Palermo (Italia) nel 26-29 settembre 2018.

Role of post-translational modifications of cytoskeletal intermediate filaments on influenza A/NWS/33 virus (H1N1) infection in mammalian cells: focus on cytokeratin 8 phosphorylation

FLORA DE CONTO;FRANCESCA CONVERSANO;MARIA CRISTINA ARCANGELETTI;CARLO CHEZZI;ADRIANA CALDERARO
2018

Abstract

Introduction: influenza virus causes epidemics and pandemics, with significant impact on public health. The cytoskeleton is hijacked by influenza virus to reach the replication site. Previous data assessed the role of actin- and tubulin-stabilization as restriction factors of human influenza A/NWS/33 virus (IAV) infection in rhesus monkey-kidney (LLC-MK2) cells (semi-permissive model). This study aims to investigate the involvement of cytokeratin 8 (K8) phosphorylation, a post-translational modification of intermediate filaments (IF), on IAV infection in LLC-MK2 and human type II alveolar epithelial (A549; permissive model) cells. Materials and Methods: LLC-MK2 and A549 cells were infected for 24 h with IAV at a multiplicity of infection (MOI) of 0.1 or 2 plaque-forming units (PFU)/cell. IF-depolymerization was induced with acrylamide (5 mM), while IF-phosphorylation with okadaic acid (OA, phosphatase inhibitor; 0.05 microg/ml) and 12-O-tetradecanoylphorbol-13-acetate (TPA, protein-kinase activator; 25-50 nM), applied for different periods. Immunofluorescence and Western blotting assays were carried out using anti-viral nucleoprotein (NP) (Argene/BioMérieux), anti-K8 (Thermo Fischer Scientific), and anti-phospho-K8 (Thermo Fischer Scientific) antibodies. Secondary antibodies conjugated with isothiocyanate of rodamine/fluorescein or alcaline phosphatase were used. Fifty percent tissue culture infectious dose (TCID50) assays were performed to evaluate viral yields. Results: K8 showed a higher expression in A549 vs LLC-MK2 cells, while K8 phosphorylation was low in both models. IF-depolymerization with acrylamide raised NP expression in both cells, almost unchanging the viral yields. IF-phosphorylation with OA increased NP expression in A549 but not in LLC-MK2 cells, modifying the K8 pattern mostly in A549 cells. IF-phosphorylation with TPA decreased NP expression in LLC-MK2, while increased that of A549 cells, raising K8 phosphorylation mostly in A549 cells. Viral yields were consistent with the obtained results. IAV infection strongly modulated K8 organization and increased K8 phosphorylation in A549 cells, leaving almost unmodified LLC-MK2. Discussion and Conclusions: the depolymerization of IF favours IAV infection in the examined models. The drug-mediated IF-phosphorylation promotes IAV infection in A549 cells, showing no significant effects in LLC-MK2. Accordingly, viral infection prevalently modulates K8 organization and phosphorylation in A549 cells. These results evidence the presence of different regulation mechanisms acting on IF post-translational modifications in the studied models. Understanding how specific cell functions/components regulate influenza virus replication may provide new starting points for innovative anti-viral researches.
Role of post-translational modifications of cytoskeletal intermediate filaments on influenza A/NWS/33 virus (H1N1) infection in mammalian cells: focus on cytokeratin 8 phosphorylation / DE CONTO, Flora; Conversano, Francesca; Arcangeletti, Maria Cristina; Chezzi, Carlo; Calderaro, Adriana. - (2018), pp. 51-51. ((Intervento presentato al convegno 46° Congresso Nazionale della Società Italiana di Microbiologia tenutosi a Palermo (Italia) nel 26-29 settembre 2018.
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11381/2850654
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact