Limits...
Highly dynamic microtubules improve the effectiveness of early stages of human influenza A/NWS/33 virus infection in LLC-MK2 cells.

De Conto F, Di Lonardo E, Arcangeletti MC, Chezzi C, Medici MC, Calderaro A - PLoS ONE (2012)

Bottom Line: Furthermore, mock-infected LLC-MK2 cells were shown to have higher levels of both acetylated alpha-tubulin and microtubule-associated protein 4 (MAP4), the latter being essential for the maintenance of normal microtubule polymer levels in interphase epithelial cells.Finally, to obtain highly dynamic microtubules in LLC-MK2 cells, we knocked down the expression of MAP4 by using a RNA-mediated RNA interference approach.The results evidenced that MAP4 silencing improves NWS growth in LLC-MK2 cells.

View Article: PubMed Central - PubMed

Affiliation: Section of Microbiology, Department of Pathology and Laboratory Medicine, University of Parma, Parma, Italy. flora.deconto@unipr.it

ABSTRACT

Background: This study aims to investigate the role of microtubule dynamics in the initiation of NWS/33 human influenza A (NWS) virus infection in MDCK and LLC-MK2 mammalian kidney cells. We previously demonstrated a host-dependent role of the actin cytoskeleton in inducing restriction during the early phases of NWS infection. Furthermore, we showed the differential infectious entry of NWS virus in the above mentioned cell models.

Methodology/principal findings: By first employing a panel of microtubule-modulators, we evidenced that microtubule-stabilization negatively interferes with NWS replication in LLC-MK2 but not in MDCK cells. Conversely, microtubule-depolymerization improves NWS growth in LLC-MK2 but not in the MDCK model. By using immunofluorescence labelling and Western blotting analyses upon NWS infection in mammalian kidney cells, it was observed that the occurrence of alpha-tubulin hyperacetylation--a post-translational modified form suggestive of stable microtubules--was significantly delayed in LLC-MK2 when compared to MDCK cells. Furthermore, mock-infected LLC-MK2 cells were shown to have higher levels of both acetylated alpha-tubulin and microtubule-associated protein 4 (MAP4), the latter being essential for the maintenance of normal microtubule polymer levels in interphase epithelial cells. Finally, to obtain highly dynamic microtubules in LLC-MK2 cells, we knocked down the expression of MAP4 by using a RNA-mediated RNA interference approach. The results evidenced that MAP4 silencing improves NWS growth in LLC-MK2 cells.

Conclusion: By evidencing the cell type-dependent regulatory role of microtubule dynamics on NWS replication in mammalian kidney cells, we demonstrated that microtubule-stabilization represents a restriction factor for the initiation of NWS infection in LLC-MK2 but not in MDCK cells.

Show MeSH

Related in: MedlinePlus

Differential expression of MAP4 in mock-infected and NWS-infected mammalian kidney cells.LLC-MK2 and MDCK cells were infected with NWS virus at a m.o.i. of 1 for 24 h or 48 h, then Western blotting detection of MAP4 was carried out. Total lysates from uninfected cells (CC) were examined in parallel. Essentially similar results were obtained in two independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3401105&req=5

pone-0041207-g007: Differential expression of MAP4 in mock-infected and NWS-infected mammalian kidney cells.LLC-MK2 and MDCK cells were infected with NWS virus at a m.o.i. of 1 for 24 h or 48 h, then Western blotting detection of MAP4 was carried out. Total lysates from uninfected cells (CC) were examined in parallel. Essentially similar results were obtained in two independent experiments.

Mentions: To further shed light on the role of MT-stabilization in modulating NWS infection, we next explored the possible contribution of MAP4, a MT-associated protein showing rescue-promoting activities during the in vitro assembly of MTs. To this aim, LLC-MK2 and MDCK cells were either uninfected or infected with NWS virus (m.o.i. = 1 p.f.u./cell) for 24 or 48 h, before Western blotting analyses. The results in Figure 7 evidenced the presence of a huge amount of MAP4 in uninfected LLC-MK2 cells, whilst only faint signals were discernible in the MDCK model. Moreover, upon NWS infection we observed a progressive decrease of MAP4 expression in LLC-MK2 cells, whilst in MDCK cells its poor amount was unchanged.


Highly dynamic microtubules improve the effectiveness of early stages of human influenza A/NWS/33 virus infection in LLC-MK2 cells.

De Conto F, Di Lonardo E, Arcangeletti MC, Chezzi C, Medici MC, Calderaro A - PLoS ONE (2012)

Differential expression of MAP4 in mock-infected and NWS-infected mammalian kidney cells.LLC-MK2 and MDCK cells were infected with NWS virus at a m.o.i. of 1 for 24 h or 48 h, then Western blotting detection of MAP4 was carried out. Total lysates from uninfected cells (CC) were examined in parallel. Essentially similar results were obtained in two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3401105&req=5

pone-0041207-g007: Differential expression of MAP4 in mock-infected and NWS-infected mammalian kidney cells.LLC-MK2 and MDCK cells were infected with NWS virus at a m.o.i. of 1 for 24 h or 48 h, then Western blotting detection of MAP4 was carried out. Total lysates from uninfected cells (CC) were examined in parallel. Essentially similar results were obtained in two independent experiments.
Mentions: To further shed light on the role of MT-stabilization in modulating NWS infection, we next explored the possible contribution of MAP4, a MT-associated protein showing rescue-promoting activities during the in vitro assembly of MTs. To this aim, LLC-MK2 and MDCK cells were either uninfected or infected with NWS virus (m.o.i. = 1 p.f.u./cell) for 24 or 48 h, before Western blotting analyses. The results in Figure 7 evidenced the presence of a huge amount of MAP4 in uninfected LLC-MK2 cells, whilst only faint signals were discernible in the MDCK model. Moreover, upon NWS infection we observed a progressive decrease of MAP4 expression in LLC-MK2 cells, whilst in MDCK cells its poor amount was unchanged.

Bottom Line: Furthermore, mock-infected LLC-MK2 cells were shown to have higher levels of both acetylated alpha-tubulin and microtubule-associated protein 4 (MAP4), the latter being essential for the maintenance of normal microtubule polymer levels in interphase epithelial cells.Finally, to obtain highly dynamic microtubules in LLC-MK2 cells, we knocked down the expression of MAP4 by using a RNA-mediated RNA interference approach.The results evidenced that MAP4 silencing improves NWS growth in LLC-MK2 cells.

View Article: PubMed Central - PubMed

Affiliation: Section of Microbiology, Department of Pathology and Laboratory Medicine, University of Parma, Parma, Italy. flora.deconto@unipr.it

ABSTRACT

Background: This study aims to investigate the role of microtubule dynamics in the initiation of NWS/33 human influenza A (NWS) virus infection in MDCK and LLC-MK2 mammalian kidney cells. We previously demonstrated a host-dependent role of the actin cytoskeleton in inducing restriction during the early phases of NWS infection. Furthermore, we showed the differential infectious entry of NWS virus in the above mentioned cell models.

Methodology/principal findings: By first employing a panel of microtubule-modulators, we evidenced that microtubule-stabilization negatively interferes with NWS replication in LLC-MK2 but not in MDCK cells. Conversely, microtubule-depolymerization improves NWS growth in LLC-MK2 but not in the MDCK model. By using immunofluorescence labelling and Western blotting analyses upon NWS infection in mammalian kidney cells, it was observed that the occurrence of alpha-tubulin hyperacetylation--a post-translational modified form suggestive of stable microtubules--was significantly delayed in LLC-MK2 when compared to MDCK cells. Furthermore, mock-infected LLC-MK2 cells were shown to have higher levels of both acetylated alpha-tubulin and microtubule-associated protein 4 (MAP4), the latter being essential for the maintenance of normal microtubule polymer levels in interphase epithelial cells. Finally, to obtain highly dynamic microtubules in LLC-MK2 cells, we knocked down the expression of MAP4 by using a RNA-mediated RNA interference approach. The results evidenced that MAP4 silencing improves NWS growth in LLC-MK2 cells.

Conclusion: By evidencing the cell type-dependent regulatory role of microtubule dynamics on NWS replication in mammalian kidney cells, we demonstrated that microtubule-stabilization represents a restriction factor for the initiation of NWS infection in LLC-MK2 but not in MDCK cells.

Show MeSH
Related in: MedlinePlus