FIG 6.

PHEV entry into Neuro-2a cells is dependent on the cytoskeleton. (A) Detection of colocalization between PHEV and actin. Neuro-2a cells were infected with DiD-labeled PHEV (red) at 37°C for the indicated times, fixed, and stained for actin by use of Alexa Fluor 488-phalloidin (1:500; green) for 30 min. At 10 min postinfection, multiple virus particles (red) bound to actin-rich protrusions, and 20 min later, PHEV particles were colocalized with actin filaments near the cell membrane during the early stage of PHEV infection. PHEV particles were found to localize in dot-like actin filaments at approximately 90 min postinfection, and images were captured immediately. (B) Neuro-2a cells were left untreated or pretreated with CytoD or EIPA at the indicated concentrations for 1 h and then subjected to PHEV internalization assays. The entry efficiency was examined by qRT-PCR, and the data shown are means ± SD for three independent experiments. (C) Neuro-2a cells were incubated under control conditions or pretreated with CytoD at 50 μM for 1 h and then subjected to a PHEV infection assay. In the uptake assays, Dx-AF488 was added for 30 min on ice, after which the cells were fixed and processed for antibody staining. Representative micrographs and quantitative analyses of average fluorescence intensity showed that PHEV infection and Dx-AF488 uptake were inhibited by CytoD and that the cells took on a more rounded shape with a smooth margin, while transient blebs filled with actin formed at the cell surface. (D) The number of PHEV genome equivalents was estimated by qRT-PCR after CytoD or EIPA treatment of Neuro-2a cells. (E) After suppression of PHEV protein synthesis by pretreatment with CytoD or EIPA for 1 h, cell lysates were collected and analyzed for the expression level of the major viral S protein by Western blotting. (F) Cells were treated with various concentrations of CytoD to inhibit polymerization of actin. As shown in the schematic, the drug was added either 60 min prior to a 1-h inoculation with virus or at 1 hpi and maintained until analysis at 4 hpi. (G) Neuro-2a cells were left untreated or pretreated with EIPA at 50 μM for 1 h and then subjected to PHEV infection and Dx-AF488 uptake assays. Images from a representative experiment out of three independent replications showed that EIPA marginally reduced Dx-AF488 uptake but did not obviously affect PHEV infection. Bars, 10 μm (A, C, and G). *, P < 0.05; **, P < 0.01.