Fig. 1

Actin plays a critical role in the late stages of HPIV3 infection. A–D Impact of cytoskeleton inhibitors on HPIV3 replication. HeLa cells infected with HPIV3 for 18 h were treated with Nocodazole (NCZ) or Cytochalasin D (Cyto D). At 24 h post infection (hpi), the virions released in the cell supernatant (A) and the quantity of intracellular progeny virus particles (B) were determined by TCID50 assays. Cells were collected for RT-qPCR analysis of intracellular viral RNA (C), and Western Blot (WB) analysis of intracellular viral protein (D). E Influence of cytoskeleton inhibitors on HPIV3 VLPs production. 293T cells transfected with indicated plasmids for 30 h were treated with NCZ or Cyto D for 6 h. Cell lysates and VLPs were prepared and analyzed by WB. F Impact of cytoskeleton inhibitors on vRNP distribution. HeLa cells infected with HPIV3HA-P (MOI = 0.01) for 18 h were treated with NCZ or Cyto D for 6 h. Fluorescent microscopy was employed to examine vRNP distribution using an anti-HA antibody (green), plasma membrane stained by Dil (red), and nuclei were stained with DAPI. Arrowheads indicate plasma membrane. Scale bar, 50 μm. G Progeny virions released via actin filaments. HPIV3HA-P-infected HeLa cells were immunofluorescently labeled for vRNP (green), actin (red), and tubulin (magenta). Scale bar, 50 μm. All experiments were independently repeated at least twice with reproducible results. Statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001) was determined by two-sided unpaired t test. Data are means ± SEM