Highly Dynamic Microtubules Improve the Effectiveness of Early Stages of Human Influenza A/NWS/33 Virus Infection in LLC-MK2 Cells

Abstract

BackgroundThis 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 FindingsBy 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.ConclusionBy 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.

Influenza A Virus Uses Intercellular Connections To Spread to Neighboring Cells

“…Similar results demonstrating the limited effect of nocodazole treatment on IAV virion production in MDCK cells are shown in Fig. 4A and have also been reported previously (44). Given the lack of requirement for microtubules and sensitivity to paclitaxel, the formation of these connections may be enhanced as a downstream signaling response to a loss of microtubules during IAV infection.…”

Friend or Foe: The Role of the Cytoskeleton in Influenza A Virus Assembly

“…The discrepancies in the role of F-actin in HA-M2 association between the two above-mentioned studies might be due to differences in the cell types used in these studies (Chinese Hamster Ovary cells in [151] versus primary human blood derived macrophages in [104]). Cell-type dependent differences for the role of the F-actin and microtubules at different stages of the IAV life cycle have also been reported before [94,98,101,179,180,181]. In addition, technical differences could account for the discrepancies; for example, the presence of other viral components in infection-based experiments [104] or attachment of fluorescent proteins to the cytoplasmic domains of HA and M2 in the FRET study [151] may affect the interaction with subcortical actin.…”

“…Cofilin-1 [89] and cortactin [90], which can promote the disassembly and assembly of F-actin, respectively, are both thought to play positive roles during IAV infection. With respect to microtubules, the disruption of microtubules has no [99] to a moderately negative [39,100] effect on virus propagation, whereas a reduction in efficiency of microtubule assembly increases virus titers in a rhesus monkey kidney cell line [101]. In addition, an increase in tubulin acetylation status, which promotes microtubule function in trafficking [102], was observed to correlate with an increase in virus titers in one study [103], but not in the other [99].…”

“…Obviously, however, studies conducted using specific inhibitors or genetic ablation should provide more definitive evidence on the role of specific cytoskeletal components in the virus life cycle. Nonetheless, some studies using these strategies show contradictory data, even as to whether the cytoskeleton of interest plays a positive role or not [39,89,94,95,96,98,99,100,101]. This could partially be attributed to the use of viral titers as readout for IAV infection.…”

Entry of influenza A virus: host factors and antiviral targets

“…With the aim of visualizing viruses along the endosomal trafficking pathway, synchronized infection was established as an important tool that allows monitoring early infection events (Matlin et al , 1981). It has been demonstrated that endosomal trafficking of the virus involves actin- and microtubule-dependent processes (Nielsen et al , 1999; Sun & Whittaker, 2007; De Conto et al , 2012). Using single virus trajectories from imaging fluorescently labelled virions, viral transport was dissected into three different stages (Lakadamyali et al , 2003).…”