The microtubule (MT) cytoskeleton supports vital cell functions such as cell motility, cytoplasmic transport, and mitosis. Viruses principally exploit MTs to ensure the intracellular trafficking to sites of replication, assembly and egress. The 26S proteasomes are large multisubunit proteolytic complexes located in the nucleus and cytoplasm. The degradation of functional, misfolded, damaged and incorrectly synthesized proteins occurs in the 20S core-particle of the 26S proteasome. Previous data assessed that highly stable MTs induce restriction to human influenza A/NWS/33 (NWS) virus infection in LLC-MK2 cells. Here we investigated the relationship between 20S proteasomes and stable MTs (i.e. acetylated alpha-tubulin and MT-associated protein 4) during the infectious cycle of both NWS and avian Mallard/Italy/303394-11/03 influenza A viruses (H1N1) in semi-permissive LLC-MK2 cells. By immunofluorescence and Western blotting assays, we first evaluated the outcome of pharmacological proteasome modulation on both influenza virus growth and MT reorganization. Then, the co-localization between viral nucleoprotein (NP), 20S proteasomes and markers for stabilized MTs was investigated by confocal microscopy. Proteasome inhibition increased the levels of stable MTs in infected LLC-MK2 cells. Conversely, proteasome activation reduced the amount of acetylated alpha-tubulin in mock-infected LLC-MK2 cells. Furthermore, proteasome inhibition, contrarily to activation, weakened NP expression in infected LLC-MK2 cells. Finally, we assessed that in infected LLC-MK2 cells NP co-localizes with 20S proteasomes and the patterns of stabilized MTs are perturbed. The obtained data suggest that proteasomes are favourably hijacked by both influenza strains to counteract MT-mediated restriction mechanisms and support viral replication in LLC-MK2 cells. Further studies will be necessary to elucidate the signalling that underlies MT reorganization and its possible relationship with key antiviral-defence mechanisms.

INFLUENZA A VIRUS (H1N1)-MEDIATED RECRUITMENT OF PROTEASOMES AGAINST STABLE MICROTUBULES IN LLC-MK2 CELLS / DE CONTO, Flora; Fazzi, Alessandra; Arcangeletti, Maria Cristina; Medici, Maria Cristina; Chezzi, Carlo; Calderaro, Adriana. - STAMPA. - 12th National Congress of the Italian Society for Virology:(2014), pp. 57-58. (Intervento presentato al convegno 12th National Congress of the Italian Society for Virology A Joint Meeting with the Spanish Society for Virology (SEV) tenutosi a Orvieto nel 22-24 settembre 2014).

INFLUENZA A VIRUS (H1N1)-MEDIATED RECRUITMENT OF PROTEASOMES AGAINST STABLE MICROTUBULES IN LLC-MK2 CELLS

DE CONTO, Flora;FAZZI, Alessandra;ARCANGELETTI, Maria Cristina;MEDICI, Maria Cristina;CHEZZI, Carlo;CALDERARO, Adriana
2014-01-01

Abstract

The microtubule (MT) cytoskeleton supports vital cell functions such as cell motility, cytoplasmic transport, and mitosis. Viruses principally exploit MTs to ensure the intracellular trafficking to sites of replication, assembly and egress. The 26S proteasomes are large multisubunit proteolytic complexes located in the nucleus and cytoplasm. The degradation of functional, misfolded, damaged and incorrectly synthesized proteins occurs in the 20S core-particle of the 26S proteasome. Previous data assessed that highly stable MTs induce restriction to human influenza A/NWS/33 (NWS) virus infection in LLC-MK2 cells. Here we investigated the relationship between 20S proteasomes and stable MTs (i.e. acetylated alpha-tubulin and MT-associated protein 4) during the infectious cycle of both NWS and avian Mallard/Italy/303394-11/03 influenza A viruses (H1N1) in semi-permissive LLC-MK2 cells. By immunofluorescence and Western blotting assays, we first evaluated the outcome of pharmacological proteasome modulation on both influenza virus growth and MT reorganization. Then, the co-localization between viral nucleoprotein (NP), 20S proteasomes and markers for stabilized MTs was investigated by confocal microscopy. Proteasome inhibition increased the levels of stable MTs in infected LLC-MK2 cells. Conversely, proteasome activation reduced the amount of acetylated alpha-tubulin in mock-infected LLC-MK2 cells. Furthermore, proteasome inhibition, contrarily to activation, weakened NP expression in infected LLC-MK2 cells. Finally, we assessed that in infected LLC-MK2 cells NP co-localizes with 20S proteasomes and the patterns of stabilized MTs are perturbed. The obtained data suggest that proteasomes are favourably hijacked by both influenza strains to counteract MT-mediated restriction mechanisms and support viral replication in LLC-MK2 cells. Further studies will be necessary to elucidate the signalling that underlies MT reorganization and its possible relationship with key antiviral-defence mechanisms.
2014
INFLUENZA A VIRUS (H1N1)-MEDIATED RECRUITMENT OF PROTEASOMES AGAINST STABLE MICROTUBULES IN LLC-MK2 CELLS / DE CONTO, Flora; Fazzi, Alessandra; Arcangeletti, Maria Cristina; Medici, Maria Cristina; Chezzi, Carlo; Calderaro, Adriana. - STAMPA. - 12th National Congress of the Italian Society for Virology:(2014), pp. 57-58. (Intervento presentato al convegno 12th National Congress of the Italian Society for Virology A Joint Meeting with the Spanish Society for Virology (SEV) tenutosi a Orvieto nel 22-24 settembre 2014).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11381/2759100
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