GFP Labelled Human Respiratory Syncytial Virus

Human respiratory syncytial virus (RSV) is an enveloped RNA virus that is the most important viral cause of acute lower respiratory tract illness in children worldwide. A genome-wide siRNA screen in human lung epithelial A549 cells identified actin-related protein 2 (ARP2) as a host factor involved in RSV infection. ARP2 knockdown did not reduce RSV entry or significantly reduce gene expression within the first 24 hours post-infection, but reduced viral gene expression thereafter, an effect that appeared to be due to inhibition of viral spread to neighboring cells. Consistent with reduced spread, release of infectious progeny virions was reduced 10-fold in ARP2-depleted cells at 72 hours post-infection. Furthermore, the researchers found that RSV infection induced filopodia formation and increased cell motility in A549 cells and that this phenotype was dependent on ARP2. Filopodia appear to deliver RSV to nearby uninfected cells, thereby facilitating viral spread. In A549 cells, expression of RSV F protein alone by plasmid or heterologous viral vector induced filopodia, suggesting a novel role for RSV F protein, namely driving filopodia induction and viral spread.

To exclude a possible role of ARP2 in RSV entry, A549 cells were studied at early time points post infection. Flow cytometry showed that at 16 hpi, the proportion of GFP-expressing cells in siARP2- and siControl-treated cells was similar in cultures infected at an MOI of 1 or 3 (Figure 1A). To exclude possible artifacts caused by transfection, a stable ARP2 knockdown A549 cell line (ARP2/KD-A549 cells) was generated using a lentiviral vector system expressing three ARP2 small hairpin RNAs and RSV-GFP infection was analyzed by flow cytometry at 6 or 12 hpi, using GFP expression as an infection marker. It was found that the proportion of infected individuals (ARP2/KD-A549 cells) did not differ from that in non-transduced A549 cells, even at higher MOIs (e.g., MOI = 5) (Figure 1B). These results suggest that ARP2 does not function in the early stages of RSV infection.

Next, the possible role of ARP2 in RSV entry was assessed using the compound CK-666, an inhibitor of ARP2/3 complex-driven actin nucleation that acts by stabilizing the ARP2/3 complex in an inactive conformation. For comparison, EIPA (5-ethylisopropyl amiloride), a potent inhibitor of macropinosome formation that has been shown to inhibit RSV entry, was also assessed and was therefore a positive control for inhibition of entry. The study showed that the macropinocytosis inhibitor EIPA reduced the uptake of dextran by A549 cells (Figure 1C). The macropinocytosis inhibitor EIPA blocked RSV entry, but the ARP2/3 complex-driven actin nucleation inhibitor CK-666 did not (Figure 1D).

Figure 1. ARP2 knockdown does not reduce RSV entry. (A) GFP expression of RSV-GFP at 16 hpi is not affected by ARP2 knockdown. (B) GFP expression of RSV-GFP at 6 and 12 hpi is not affected in the A549 cell line with constitutive ARP2 knockdown. (C) The macropinocytosis inhibitor EIPA reduces dextran uptake in A549 cells. (D) The macropinocytosis inhibitor EIPA, but not the ARP2/3 complex-driven actin nucleation inhibitor CK-666, blocks RSV entry. (Mehedi M, et al., 2016)