PIK-24 Inhibits RSV-Induced Syncytium Formation via Direct Interaction with the p85α Subunit of PI3K

ABSTRACT

Phosphoinositide-3 kinase (PI3K) signaling regulates many cellular processes, including cell survival, differentiation, proliferation, cytoskeleton reorganization, and apoptosis. The actin cytoskeleton regulated by PI3K signaling plays an important role in plasma membrane rearrangement. Currently, it is known that respiratory syncytial virus (RSV) infection requires PI3K signaling. However, the regulatory pattern or corresponding molecular mechanism of PI3K signaling on cell-to-cell fusion during syncytium formation remains unclear. This study synthesized a novel PI3K inhibitor PIK-24 designed with PI3K as a target and used it as a molecular probe to investigate the involvement of PI3K signaling in syncytium formation during RSV infection. The results of the antiviral mechanism revealed that syncytium formation required PI3K signaling to activate RHO family GTPases Cdc42, to upregulate the inactive form of cofilin, and to increase the amount of F-actin in cells, thereby causing actin cytoskeleton reorganization and membrane fusion between adjacent cells. PIK-24 treatment significantly abolished the generation of these events by blocking the activation of PI3K signaling. Moreover, PIK-24 had an obvious binding activity with the p85α regulatory subunit of PI3K. The anti-RSV effect similar to PIK-24 was obtained after knockdown of p85α in vitro or knockout of p85α in vivo, suggesting that PIK-24 inhibited RSV infection by targeting PI3K p85α. Most importantly, PIK-24 exerted a potent anti-RSV activity, and its antiviral effect was stronger than that of the classic PI3K inhibitor LY294002, PI-103, and broad-spectrum antiviral drug ribavirin. Thus, PIK-24 has the potential to be developed into a novel anti-RSV agent targeting cellular PI3K signaling.

IMPORTANCE PI3K protein has many functions and regulates various cellular processes. As an important regulatory subunit of PI3K, p85α can regulate the activity of PI3K signaling. Therefore, it serves as the key target for virus infection. Indeed, p85α-regulated PI3K signaling facilitates various intracellular plasma membrane rearrangement events by modulating the actin cytoskeleton, which may be critical for RSV-induced syncytium formation. In this study, we show that a novel PI3K inhibitor inhibits RSV-induced PI3K signaling activation and actin cytoskeleton reorganization by targeting the p85α protein, thereby inhibiting syncytium formation and exerting a potent antiviral effect. Respiratory syncytial virus (RSV) is one of the most common respiratory pathogens, causing enormous morbidity, mortality, and economic burden. Currently, no effective antiviral drugs or vaccines exist for RSV infection. This study contributes to understanding the molecular mechanism by which PI3K signaling regulates syncytium formation and provides a leading compound for anti-RSV infection drug development.

INTRODUCTION

Respiratory syncytial virus (RSV) is one of the most common respiratory pathogens worldwide. Severe RSV infection can cause lower respiratory diseases such as pneumonia, bronchiolitis, and asthma. Infants and young children are extremely susceptible to RSV (1). In addition, adults (especially the elderly) and immunocompromised individuals are increasingly recognized as the target groups of RSV infection (2, 3). RSV infections have resulted in approximately 33 million people suffering from lower respiratory tract diseases, 3.2 million hospital admissions, and 66,000 in-hospital deaths worldwide (4). In developed countries such as the United States and Canada, the annual medical cost of children and the elderly infected with RSV is as high as 1 billion dollars (5). However, there is neither an effective vaccine nor a therapeutic drug with low side effects for RSV in the clinic. Ribavirin is the only broad-spectrum antiviral drug approved by the US Food and Drug Administration (FDA) to treat RSV infection. However, the main problem with ribavirin is its potential toxic side effects and clinical efficacy concerns (6). Palivizumab is a highly potent RSV-neutralizing monoclonal antibody (MAb) and is mainly restricted to high-risk infants. The clinical use of palivizumab is limited due to its high cost, long treatment cycle, and repeated injections (7). Therefore, the development of new and effective anti-RSV drugs is an urgent problem to be solved.

RSV is an enveloped negative-stranded RNA virus that belongs to the Pneumovirus genus of the Paramyxoviridae family. The fusion of infected cells is a sign of all paramyxoviruses (8). The fused cell mass is called “syncytium,” from which the middle name of RSV is derived. As with many paramyxoviruses, syncytium formation of RSV likely involves the fusion (F) protein distributing to the surface of the infected cells, followed by plasma membrane rearrangement and membrane fusion between adjacent cells, which provides an efficient way for progeny viruses of RSV to spread cell to cell (8–10). Important advances have been obtained regarding the role of RSV-F protein in membrane fusion and syncytium formation (11, 12). In addition to RSV-F protein, several host factors such as calcium, profilin, and RhoA have also been found to be involved in RSV-induced syncytium formation (13–15), and recent studies have shown that host cells expressing higher levels of matrix metalloproteinase 9 (MMP9) form greater numbers of syncytia response to RSV infection (16). Therefore, host cell molecules and signaling pathways are indispensable for syncytium formation.

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases, which comprise three types based on substrate specificity, activating signals, and subcellular distribution (17). The most relevant and widely studied class for viral targeting is the class I PI3Ks. Class I PI3Ks is a heterodimer composed of a regulatory (p85α/p55α/p50α, p85β, p55γ, p101, or p84) and a catalytic (p110α, p110β, p110γ, or p110δ) subunit (18). As an important regulatory subunit of PI3K protein, p85α regulates the activity of PI3K protein (19) and serves as the key target for virus infection of host cells. Under the stimulation of pathogens, cytokines, or chemokines, PI3K can be activated by phosphorylation of p85α and subsequently transforms membrane-bound phosphatidylinositol 4,5-bisphosphate (PIP2) into phosphatidylinositol 3,4,5-trisphosphate (PIP3). The serine/threonine kinase Akt is a vital downstream intermediate in the PI3K-dependent signaling pathway and directly or indirectly regulates a variety of molecules to carry out diverse PI3K-regulated responses, including cell growth, cytoskeleton reorganization, RNA processing, protein synthesis, autophagy, and apoptosis (20, 21). By regulating cytoskeleton reorganization, PI3K signaling regulated by p85α facilitates various intracellular plasma membrane rearrangement events, including membrane fusion, vesicle formation, and vesicle movement. Cofilin, along with a Rho family GTPase CDC42, is a key cellular factor in this process (22, 23). The EGFR/PI3K signaling pathway has been shown to promote the entry of herpes simplex virus-1 (HSV-1) into host cells by regulating actin cytoskeleton reorganization (24), and recently, the PI3K/Akt signaling pathway has been involved in dengue virus (DENV) infection in a Rho GTPase- and actin-dependent manner (25). However, the role of actin cytoskeleton reorganization regulated by p85α or PI3K signaling in membrane fusion and syncytium formation during RSV infection and its molecular mechanisms remain unclear.

Our previous study found that a novel PI3K inhibitor PIK-24, designed and synthesized with PI3K protein as the target, significantly inhibited the activities of different subtypes of PI3K (PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ), with 50% inhibitory concentration (IC₅₀) values of 2,518.96, 4,563.06, 35.68, and 5.52 nM, respectively. The results of the antiviral activity assay showed that PIK-24 significantly inhibited RSV infection. The anti-RSV activity of PIK-24 was stronger than the positive drug ribavirin in vivo and in vitro. The antiviral mechanism of PIK-24 was to block PI3K signaling activation and actin cytoskeleton reorganization, thereby inhibiting the fusion process of RSV entry (26). However, it is unclear whether PIK-24 inhibits membrane fusion at the stage of syncytium formation and whether it targets the p85α subunit of PI3K to exert an anti-RSV effect. In this study, PIK-24 was used as a molecular probe to further investigate the association among RSV-induced syncytium formation, PI3K signaling, and actin cytoskeleton reorganization. The findings indicated that p85α was the potential target regulating RSV infection in the PI3K protein, and PIK-24 blocked the PI3K signaling activation and actin cytoskeleton reorganization by targeting PI3K p85α protein, thereby inhibiting membrane fusion during syncytium formation.

RESULTS

PIK-24 inhibits RSV infection in vitro.

The chemical structures of PIK-24, LY294002, PI-103, and ribavirin are shown in Fig. S1 in the supplemental material. The anti-RSV activity of PIK-24 was investigated by generating dose-response curves of three cell lines commonly used in RSV-related studies: HEp-2, HeLa, and A549. The results showed that PIK-24 inhibited RSV infection in a concentration-dependent manner in the three cell lines (Fig. 1A). The IC₅₀ values of PIK-24 were 1.40 ± 0.13 μM, 1.71 ± 0.14 μM, and 2.36 ± 0.48 μM, respectively (Table 1). Two additional PI3K inhibitors, LY294002 and PI-103, were also active in infectious cells (Fig. 1B and C). However, the antiviral activity of the two PI3K inhibitors was weaker than that of PIK-24 in the concentration range of 40 to 1.25 μM. Ribavirin, as a broad-spectrum antiviral inhibitor, was also less effective than PIK-24, with IC₅₀ values of 5.08 ± 0.13 μM, 4.13 ± 0.48 μM, and 4.50 ± 0.48 μM in the three cell lines, respectively (Fig. 1D). The cytotoxicity assay showed that PIK-24 and ribavirin had no obvious cytotoxicity below 40 μM. PIK-24 was significantly less toxic to various cell lines than LY294002 and PI-103 (Fig. 1E–H). The selectivity index (SI) values of PIK-24 to inhibit RSV infection (SI_HEp-2, >71.43; SI_Hela, >58.48; and SI_A549, >42.37) were also greater than those of LY294002, PI-103, and ribavirin (Table 1).

FIG 1.

PIK-24 inhibits RSV infection in vitro. (A–D) RSV-infected (MOI = 0.1) HEp-2, HeLa, or A549 cells were treated with PIK-24, LY294002, PI-103, or ribavirin (40, 20, 10, 5, 2.5, or 1.25 μM). After 48 h of incubation, the supernatant of each group was collected, and the virus titer was tested by immunofluorescence assay. (E–H) HEp-2, HeLa, or A549 cells with a cell density of 50% were exposed to different concentrations of PIK-24 (40, 20, 10, 5, 2.5, or 1.25 μM) and then incubated for 48 h. LY294002, PI-103, and ribavirin were treated similarly. The cytotoxicity of the compounds was measured by a CCK-8 assay. Data are means ± SD from three independent experiments. (*, P < 0.05; **, P < 0.01; ***, P < 0.001 compared to the viral control group).

TABLE 1.

Anti-RSV activities of PIK-24 in HEp-2, HeLa, and A549 cells^a

Compounds	HEp-2			Hela			A549
	CC50 (μM)	IC50 (μM)	SI	CC50 (μM)	IC50 (μM)	SI	CC50 (μM)	IC50 (μM)	SI
PIK-24	>100	1.40 ± 0.13	>71.43	>100	1.71 ± 0.14	>58.48	>100	2.36 ± 0.48	>42.37
Ribavirin	>100	5.08 ± 0.13	>19.69	>100	4.13 ± 0.48	>24.21	>100	4.50 ± 0.48	>22.22
LY294002	53.36 ± 5.77	7.65 ± 0.79	6.98	>100	7.62 ± 1.04	>13.12	>100	8.32 ± 0.60	>12.02
PI-103	41.56 ± 1.02	2.60 ± 0.66	15.98	82.02 ± 6.69	3.55 ± 0.71	23.10	>100	4.26 ± 0.27	>23.47

^aCC₅₀ was estimated by CCK-8 assay; IC₅₀was assessed by immunofluorescence assay; SI value was calculated by CC₅₀/IC₅₀.

PIK-24 acts in the late stage of RSV infection.

To investigate whether PIK-24 plays a role in the late stage of RSV infection, HEp-2 cells were treated with PIK-24 at 24 h after RSV infection and the amount of progeny virus was detected at 48 h postinfection using plaque assay. As shown in Fig. 2A, the number of progeny viruses was significantly reduced in infectious cells treated with PIK-24 or LY294002 under this administration mode. However, the delay of ribavirin treatment greatly impaired its inhibitory effect on RSV infection. The reason is that the antiviral mechanism of ribavirin is to inhibit the activity of IMP dehydrogenase (IMPDH) and prevent the synthesis of viral RNA or DNA. Therefore, it can be concluded that PIK-24 blocks the late stage of RSV infection.

FIG 2.

PIK-24 acts in the late stage of RSV infection by inhibiting syncytium formation. Confluent HEp-2 cell cultures were inoculated with RSV (MOI = 0.1) for 24 h. The monolayer was then treated with PIK-24 (20 μM) and cultured for an additional 24 h. LY294002 (20 μM) and ribavirin (20 μM) were treated similarly. (A) The supernatant of each group was subjected to plaque assay to evaluate progeny virus production. (B–D) The monolayer was then fixed, permeabilized, and stained with anti-respiratory syncytial virus fusion (F) glycoprotein antibody and Alexa Fluor 488 donkey antimouse IgG (H+L) to detect RSV-F protein. The number of syncytia and fluorescence intensity of RSV-F protein were determined using a fluorescence microscope. Data are means ± SD from three independent experiments. (***, P < 0.001 compared to the viral control group).

PIK-24 inhibits RSV-induced syncytium formation by blocking intercellular membrane fusion.

At the late stage of RSV infection, infected cells express the F protein on their surface, and the latter induces membrane fusion between adjacent cells to promote syncytium formation and viral spread (10). Here, the viral protein was detected, and the number of syncytia was counted microscopically in cells treated with PIK-24 at 24 h after RSV infection to see if PIK-24 inhibits syncytium formation. As shown in Fig. 2B–D, large syncytia were observed in the virus group. Correspondingly, a large amount of green fluorescence (RSV-F protein) was observed in the infected cells, most notably near syncytium formation. The number of syncytia sharply decreased after treatment with PIK-24 or LY294002 at 20 μM. However, no significant decrease in RSV-F protein was observed, indicating that the delay of PIK-24 or LY294002 treatment resulted in the loss of the inhibitory effect on RSV replication. Unlike PIK-24, ribavirin could still inhibit RSV replication to some extent. Hence, the number of syncytia and RSV-F protein observed in the ribavirin group was also reduced compared to the virus control group. According to the above results, it can be inferred that PIK-24 specifically blocks syncytium formation.

An optimized dual-luciferase reporter assay (27) confirmed whether PIK-24 specifically suppresses F protein-induced intercellular membrane fusion (Fig. 3A). In this assay, the target cells were cotransfected with pcDNA3.1(+) harboring human codon-optimized full length of RSV F gene and a pT7-Luc plasmid including the firefly luciferase gene under the control of T7 promoter. The effector cells were cotransfected with a pCAG-T7 Pol plasmid including the T7 RNA polymerase gene and a pRL-TK plasmid expressing renilla luciferase. During the membrane fusion between the target cells and the effector cells, T7 RNA polymerase in the effector cells diffused into the target cells, bound to the T7 promoter of pT7-Luc plasmid, and triggered the expression of firefly luciferase. The activity of firefly luciferase directly reflected the extent of cell-to-cell fusion. As shown in Fig. 3B, obvious signs of multinucleate syncytia could be detected in the nontreated pcDNA3.1(+)-F plasmid-transfected control group. Treatment with PIK-24 or LY294002 at 20 μM led to a marked reduction in the total syncytia. However, neither PIK-24 nor LY294002 had significant effects on the expression of F protein (Fig. 3C). The activity of firefly luciferase could be tested as early as 48 h in HEK293T cells after transfection with the above four plasmids, whereas signs of firefly luciferase activity in HEK293T cells treated with PIK-24 or LY294002 disappeared along with inhibition of syncytium formation (Fig. 3D). In contrast, ribavirin had no obvious effect on these phenomena. These results suggest that PIK-24 inhibits the cell-to-cell fusion induced by RSV during syncytium formation.

FIG 3.

PIK-24 inhibits RSV-induced cell-to-cell fusion. (A) Schematic diagram of a dual-luciferase reporter model used to quantify cell-to-cell fusion induced by plasmid encoding RSV-F protein. The transfection of HEK293T cells with the pcDNA3.1(+)-F, pT7-Luc, pRL-TK, or pCAG-T7 Pol plasmid was conducted according to the dual-luciferase reporter model. HEK293T cells were treated with PIK-24 (20 μM) at 6 h posttransfection. LY294002 (20 μM) and ribavirin (20 μM) were treated similarly. At 12 h posttransfection, the target cells were mixed with the effector cells in equal amounts and incubated continuously until 48 h. (B and C) The degree of lesion and fluorescence intensity of RSV-F protein were then measured as described above, and (D) the activities of firefly luciferase and renilla luciferase were detected using the dual-luciferase reporter assay system. Data are means ± SD from three independent experiments. (^###, P < 0.001 compared to the blank control group; **, P < 0.01 and *** P < 0.001 compared to the nontreated pcDNA3.1(+)-F plasmid-transfected control group).

PIK-24 blocks RSV-induced actin cytoskeleton reorganization.

During syncytium formation, the membrane fusion between adjacent cells is a cell membrane rearrangement process regulated by RSV-F protein and actin cytoskeleton (10–12, 28). To further elucidate the mechanism of the inhibitory effect of PIK-24 on RSV-induced intercellular membrane fusion, PIK-24 was initially used to interact with the purified F protein to determine whether PIK-24 inhibits the binding of F protein to the cell surface. In this assay, the fluorescence intensity (purified F protein) could reflect the binding activity of F protein to cells. As shown in Fig. 4A, 3,4-DCQAME (29), the established RSV fusion inhibitor, significantly reduced the production of green fluorescence. On the contrary, there was no significant difference in green fluorescence intensity among the PIK-24 group, LY294002 group, or virus group, indicating that PIK-24 did not affect the binding of F protein to the cell surface. Next, we further detected the effect of PIK-24 on the morphological changes of the actin cytoskeleton. The results showed that RSV-infected HEp-2 cells formed large numbers of filopodia on the cell surface. The cytoskeletal inhibitor cytochalasin D can bind to filamentous (F) actin and disrupts polymerization. In this assay, filopodia were impaired by the disruption of actin dynamics in the presence of cytochalasin D. As expected, both PIK-24 and LY294002 obtained similar results to cytochalasin D in inhibiting filopodia formation (Fig. 4B). The inhibitory effect of PIK-24 on filopodia formation was also confirmed in HEK293T cells transfected with pcDNA3.1(+)-F plasmid, ruling out cell-specific effects (Fig. 4C). Since filopodia are large assemblies of actin filaments (F-actin) formed by two parallel chains of head-tail polymers of globular actin (G-actin), the amount of F-actin in each group was further detected to analyze the filopodia quantitatively. Compared with the control group, an obvious upregulation of F-actin was observed in RSV-infected HEp-2 cells during syncytium formation. The total amount of F-actin was significantly decreased after PIK-24 or LY294002 treatment (Fig. 4D and F). Therefore, we infer that PIK-24 inhibits RSV-induced membrane fusion between adjacent cells via preventing actin cytoskeleton reorganization.

FIG 4.

PIK-24 blocks actin cytoskeleton reorganization and PI3K signaling activation during RSV-induced syncytium formation. (A) HEp-2 cells were treated with RSV-F protein (1 μg/mL) and PIK-24 (20 μM) for 2 h. LY294002 (20 μM) and 3,4-DCQAME (20 μM) were treated similarly. The fluorescence intensity of RSV-F protein was measured using a confocal microscope. (B) HEp-2 cells were inoculated with RSV (MOI = 1) for 24 h. The monolayer was then treated with PIK-24 (20 μM) and cultured for an additional 24 h. LY294002 (20 μM) and cytochalasin D (2 μM) were treated similarly. HEp-2 cells were then fixed, permeabilized, and stained with Alexa Fluor 488 phalloidin. The actin cytoskeleton morphology of each group was determined using a confocal microscope. (C) HEK293T cells were transfected with pcDNA3.1 (+)-F for 6 h and then treated with PIK-24 (20 μM). LY294002 (20 μM) and cytochalasin D (2 μM) were treated similarly. After 48 h of incubation, the actin cytoskeleton morphology of each group was determined using a confocal microscope, as described above. (D–I) HEp-2 cells were inoculated with RSV (MOI = 1) for 24 h. The monolayer was then treated with PIK-24 (20 μM) and cultured for an additional 24 h. LY294002 (20 μM) was treated similarly. Western blot analysis of the phosphorylation of cofilin and Akt or total amounts of Akt, Cdc42, cofilin, and F-actin. The activity of Cdc42 was measured by a pulldown assay. The interaction between Cdc42 and p85α was determined by a coimmunoprecipitation assay. Data are means ± SD from three independent experiments. Red arrows indicate RSV-F protein. Yellow arrows indicate filopodia. (^#, P < 0.05; ^##, P < 0.01; and ^###, P < 0.001 compared to the blank control group. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 compared to the RSV-F or viral control group).

PIK-24 blocks RSV-induced PI3K signaling activation.

To further elucidate the series of events in which PIK-24 blocks RSV-induced actin cytoskeleton reorganization through PI3K signaling, the effect of PIK-24 on the changes of PI3K signaling or actin cytoskeleton-related proteins during syncytium formation was tested. Since Akt has long been recognized as a reliable indicator of PI3K signaling activation, we initially determined whether PIK-24 blocked the phosphorylation of Akt. As determined by Western blot assay, the amount of phosphorylated Akt was evident at 48 h postinfection and returned to the background after PIK-24 or LY294002 treatment. The total Akt protein level did not display obvious alterations in all groups (Fig. 4D and G). The Rho family GTPases play a key role in actin cytoskeleton dynamics. Cdc42, as one of the most important members of the Rho family GTPases, controls the formation of filopodia (30, 31). Moreover, it has been shown that Cdc42 interacts with PI3K regulatory subunit p85α and acts as its downstream regulator to participate in actin cytoskeleton dynamics (32). Indeed, we utilized the anti-p85α antibody to immunoprecipitate endogenous p85α and assessed the number of p85α interacting with Cdc42 during syncytium formation. It was found that Cdc42 was readily detected from the precipitate with an anti-Cdc42 antibody. At 20 μM, PIK-24 decreased the coprecipitated Cdc42 by 82.57%, whereas LY294002 reduced the coprecipitated Cdc42 by only 45.39%. On the other hand, Cdc42 was immunoprecipitated from cell lysates, and the change in the amount of p85α in the immunoprecipitation complex detected with the anti-PI3K p85α antibody was consistent with previous results (Fig. 4E). As expected, Cdc42 was activated upon interaction with p85α and exercised the function of PI3K signaling in regulating actin cytoskeleton dynamics. The level of GTP-Cdc42 was reduced after PIK-24 or LY294002 treatment (Fig. 4D and H). The endogenous p85α and Cdc42 in the total cell lysate in each group remained unchanged. In addition, cofilin is a well-known regulator responsible for actin cytoskeleton remodeling. The function of cofilin is to sever F-actin to induce depolymerization of the actin filament (33). Cofilin has also been regulated by PI3K signaling or Rho family GTPases (24, 34, 35). In this study, cofilin was phosphorylated at 48 h after RSV infection, and its total protein expression level decreased slightly. PIK-24 or LY294002 resulted in the downregulation of cofilin phosphorylation, indicating that PIK-24 could inhibit upregulation of the inactive state of cofilin during syncytium formation (Fig. 4D and I). The dynamic changes of p85α, Akt, Cdc42, and cofilin closely corresponded to those of F-actin and filopodia formation. Collectively, these results indicate that the regulation of PI3K signaling and Cdc42 and cofilin activities by PIK-24 block the dynamic changes of the actin cytoskeleton during syncytium formation.

p85α regulates RSV infection in vitro and in vivo.

To confirm the PI3K subunit plays a key role in RSV infection, the small interfering RNA (siRNA) was used to knock down the regulatory and catalytic subunit genes of PI3K protein, including PIK3R1, PIK3R2, si-PIK3R3, PIK3CA, and PIK3CB. As shown in Fig. 5A and Fig. S2, si-PIK3R1, si-PIK3R2, si-PIK3R3, si-PIK3CA, and si-PIK3CB could effectively inhibit the expression of p85α, p85β, p55γ, p110α, and p110β at the concentration of 20, 20, 20, 50, and 50 nM, respectively. The inhibition rate of these siRNAs on the corresponding protein expression was more than 70%. The result of cytotoxicity assay showed that these siRNAs had no obvious toxicity to the cells at the corresponding concentrations (Fig. 5B). At a nontoxic dose, si-PIK3R1 had significant anti-RSV activity, with an inhibition rate of 76.39% against RSV infection. Conversely, si-PIK3R2, si-PIK3R3, si-PIK3CA, and si-PIK3CB did not affect RSV infection significantly (Fig. 5C). To confirm the observation, we further investigated the effects of si-PIK3R1, si-PIK3R2, si-PIK3R3, si-PIK3CA, and si-PIK3CB on the expression of three important RSV genes, NS1, NS2, and F. The expression levels of NS1, NS2, and F genes in si-PIK3R1-transfected infectious cells were decreased by 71.67%, 64.48%, and 59.09%, respectively. si-PIK3R2, si-PIK3R3, si-PIK3CA, and si-PIK3CB had no significant effect on the expression levels of viral genes (Fig. 5D–F). The reduction of RSV gene expression was further confirmed by immunofluorescence analysis of RSV-F protein (Fig. 5G and H). Moreover, we established a p85α^−/−C57BL/6 mouse model to investigate whether p85α regulates RSV infection in vivo. Knockout of p85α effectively promoted the recovery of body weight in RSV-infected mice (Fig. 6A). Importantly, the immunofluorescence staining, lung indices, and histopathological examination revealed that knockout of p85α significantly reduced the number of virus particles and relieved RSV-induced inflammation in the lung tissue (Fig. 6B–E). The above results show that the p85α regulatory subunit plays an important role in RSV infection.

FIG 5.

Knockdown of p85α inhibits RSV infection in vitro. HEp-2 cells were transfected with si-PIK3R1, si-PIK3R2, si-PIK3R3, si-PIK3CA, or si-PIK3CB for 48 h. (A) The total amounts of p85α, p85β, p55γ, p110α, and p110β were measured by Western blot analysis. (B) The cytotoxicity of si-PIK3R1, si-PIK3R2, si-PIK3R3, si-PIK3CA, or si-PIK3CB against HEp-2 cells was measured by CCK-8 assay. Knockdown HEp-2 cells were then infected with RSV (MOI = 0.1) and incubated for 24 or 48 h. Samples of each group were divided into three parts to determine (C) viral titers, (D–F) the mRNA expression levels of F, NS1, and NS2, and (G and H) the expression levels of RSV-F protein. Data are means ± SD from three independent experiments. (***, P < 0.001 compared to the viral control group).

FIG 6.

Knockout of p85α inhibits RSV infection in vivo (n = 6). WT or p85α^−/− C57BL/6 mice were intranasally infected with RSV. (A) Body weight in each group was measured daily for 6 d. On day 4 after RSV infection, the mice were sacrificed, and their lungs were excised, weighed, and divided into several parts to determine (B) lung indices, (C and D) immunofluorescence staining, and (E) histopathological examination. Yellow arrows indicate 4',6-diamidino-2-phenylindole (DAPI). Red arrows indicate RSV virions. (^###, P < 0.001 compared to the control group; **, P < 0.01 and ***, P < 0.001 compared to the viral control group).

To investigate the relationship between syncytium formation and p85α, we tested the effects of silencing the PIK3R1 gene on syncytium formation, PI3K signaling-related protein activity, and the morphology of the actin cytoskeleton. As shown in Fig. 7A, obvious green fluorescence could be detected in the area covered by syncytia, and the fluorescence was distributed in a diffused state. Compared with the Fwt group, the intensity and quantity of green fluorescence in si-PIK3R1-transfected HEK293T cells did not decrease significantly. However, the coverage of fluorescence was significantly reduced, which was caused by the suppression of syncytia. Treatment of HEK293T cells with si-PIK3R1 also significantly reduced luciferase activity (about 64.07%; Fig. 7B). Conversely, si-PIK3R2, si-PIK3R3, si-PIK3CA, and si-PIK3CB failed to exert significant inhibitory effects on syncytium formation, the coverage of fluorescence, and luciferase activity. Furthermore, the addition of si-PIK3R1 to HEp-2 cells significantly downregulated the activities of Cdc42 and Akt, reduced the phosphorylation of cofilin and the content of F-actin, and degraded intracellular actin fibers, thereby changing the morphology of the actin cytoskeleton (Fig. 7C–H). Since Akt, Cdc42, and cofilin are important factors that regulate PI3K signaling and actin cytoskeleton, silencing of the Cdc42, Akt, and cofilin genes in HEp-2 cells was performed, and the consistency of anti-RSV activity between these three and PIK-24 (or si-PIK3R1) was confirmed. As shown in Fig. S3, si-AKT1, si-CDC42, and si-CLF1 successfully inhibited the expression of the corresponding protein. The inhibitory effects of si-AKT1, si-CDC42, and si-CLF1 against RSV infection at corresponding nontoxic concentrations are similar to PIK-24 or si-PIK3R1. According to the above results, it is reasonable to infer that the anti-RSV activity and mechanism of si-PIK3R1 are similar to PIK-24.

FIG 7.

p85α regulates cell-to-cell fusion, PI3K signaling, and actin cytoskeleton morphology. HEK293T or HEp-2 cells were transfected with si-PIK3R1, si-PIK3R2, si-PIK3R3, si-PIK3CA, or si-PIK3CB for 48 h. (A and B) The transfection of HEK293T cells with the pcDNA3.1(+)-F, pT7-Luc, pRL-TK, or pCAG-T7 Pol plasmid was conducted according to the dual-luciferase reporter model as described above. At 48 h posttransfection, the fluorescence intensity of F protein was determined using a fluorescence microscope, and the activities of firefly luciferase and renilla luciferase were detected using the dual-luciferase reporter assay system. (C) Immunofluorescent analysis of actin cytoskeleton morphology. (D–H) Western blot analysis of the phosphorylation of cofilin and Akt or total amounts of Akt, Cdc42, cofilin, and F-actin. The activity of Cdc42 was measured by a pulldown assay. Data are means ± SD from three independent experiments. Yellow arrows indicate actin fibers. (^###, P < 0.001 and ***, P < 0.001 compared to the blank control group or nontreated pcDNA3.1(+)-F plasmid-transfected control group).

PIK-24 has an obvious binding activity with p85α.

Since p85α is the potential target of PI3K protein in regulating RSV infection, we further explored whether PIK-24 exerts antiviral effects by targeting p85α. As shown in Fig. 8A and B, the inhibition rate of PIK-24 alone or in combination with si-PIK3R1 against RSV infection in cells was 87.70% and 88.45%, respectively, and there was no significant difference between the two. On the contrary, overexpression of p85α in cells could effectively abolish the inhibitory effect of PIK-24 on RSV infection. Therefore, we speculate that p85α is the target protein of PIK-24 to inhibit RSV infection.

FIG 8.

PIK-24 has an obvious binding activity with p85α. (A) HEp-2 cells transfected with si-PIK3R1 or si-NC were infected with RSV (MOI = 0.1) and then treated with PIK-24 (20 μM). After 48 h of incubation, the supernatant of each group was collected, and the virus titer was tested by immunofluorescence assay. (B) HEK293T cells transfected with p85α or vector plasmid were infected with RSV (MOI = 0.1) and then treated with PIK-24 (20 μM). After 48 h of incubation, the supernatant of each group was collected, and the virus titer was tested by immunofluorescence assay. (C) The binding activity of PIK-24 and PI3K p85α protein was detected by MST assay. Data are means ± SD from three independent experiments. (***, P < 0.001 compared to the viral control group).

The microscale thermophoresis assay is a technique that can detect the interaction between small molecule compounds and proteins. Therefore, the microscale thermophoresis (MST) technology further investigated whether PIK-24 binds to p85α. In this assay, the fluorescence intensity of p85α protein labeled with molecular fluorescent probes was greater than 300, which met the range of fluorescence values required by the MST technology (200 to 2,000 counts). Moreover, the two are considered to bind each other only if both the response amplitude and the signal-to-noise ratio are greater than 5. Therefore, we could conclude from the results of the binding check assay that PIK-24 could specifically bind to p85α. Based on the binding check assay, the binding activity of PIK-24 and p85α protein was further quantitatively detected by the binding affinity assay. The binding activity curve is shown in Fig. 8C. The binding activity of PIK-24 and PI3K p85α protein was dose-dependent, and its Kd value was 0.50 ± 0.04 μM. Meanwhile, the response amplitude and the signal-to-noise ratio were 39.04 ± 2.89 and 11.16 ± 0.87, respectively. These results show that PIK-24 has obvious binding activity with p85α.

DISCUSSION

During infection of host cells, many viruses exploit a wide range of intracellular signaling pathways to support their replication. PI3K signaling is one of the most outstanding pathways and plays a critical role in regulating viral entry, pre-mRNA splicing, translation, or replication (36, 37). Like other viruses, RSV also employs different strategies to activate the PI3K signaling pathway in the early/late stage of infection. It has been found that the PI3K inhibitor LY294002 can inhibit RSV entry (38). Besides, the antiapoptotic signaling of host cells induced by RSV is mediated via activation of PI3K-dependent pathways, which promotes cellular survival and RSV replication (39, 40). Our previous study found that a novel PI3K inhibitor, PIK-24, inhibited RSV replication in cells with a low IC₅₀ value. Intragastric administration of RSV-infected mice with PIK-24 markedly reduced virus titer in the lung tissue. PIK-24 could inhibit membrane fusion between virus and cell membranes during RSV entry, mainly achieved by blocking PI3K signaling activation and actin cytoskeleton reorganization (26). The results indicated that PI3K signaling was involved in RSV-mediated membrane fusion. To our knowledge, membrane fusion also occurs during syncytium formation because RSV needs to induce membrane fusion between infected and uninfected cells to facilitate the spread of viral particles (10). As expected, PIK-24 could also specifically inhibit RSV-induced membrane fusion between adjacent cells and subsequent syncytium formation, indicating that PIK-24 exerted an antiviral effect mainly by blocking membrane fusion at different periods in the process of RSV infection. Moreover, the anti-RSV activity of PIK-24 was compared with two other classical PI3K inhibitors, LY294002 and PI-103. It was found that PIK-24 had a stronger anti-RSV activity than LY294002 and PI-103, with lower toxicity in different cell models. Unfortunately, PIK-24 was less active than the available fusion inhibitors (Presatovir and Sisunatovir) or nucleoprotein binder (EDP-938), which targets the RSV F or N protein and possesses antiviral properties in the nanomolar range (41–43). Nonetheless, it is undeniable that the RSV inhibitors targeting the host proteins have the advantage of overcoming the generation of drug-resistant mutations compared to RSV inhibitors targeting viral proteins. Given the potent anti-RSV activity of PIK-24 both in vitro and in vivo, PIK-24 has a promising development prospect as an RSV fusion inhibitor.

RSV-F protein and actin cytoskeleton are essential factors regulating syncytium formation during RSV infection (10–12, 28). In this study, PIK-24 did not affect the expression of RSV-F protein and the binding of RSV-F protein to the cell surface. Meanwhile, it had been previously confirmed that PIK-24 did not cause resistance mutations in the RSV-F protein (26). Therefore, it was reasonable to believe that the inhibitory effect of PIK-24 on syncytium formation was independent of the RSV-F protein. On the other hand, large amounts of filopodia were formed on the infected-cell surface at the stage of syncytium formation, indicating that RSV could trigger actin cytoskeleton reorganization to promote the rearrangement of the plasma membrane. Interestingly, the actin cytoskeleton reorganization induced by RSV entry mainly promoted the formation of stress fibers rather than filopodia (26), which might be the difference between the two membrane fusions (virus-to-cell fusion, cell-to-cell fusion). Moreover, the modulation of cytoskeleton reorganization is one prominent role of PI3K signaling (32, 44). The research was further expanded to analyze the role of PI3K signaling in RSV-induced actin cytoskeleton reorganization. Treatment of PIK-24 and LY294002 could effectively inhibit the formation of filopodia induced by RSV, as expected. Since the actin cytoskeleton also plays a role in phagocytosis, T cell immune regulation, cell movement, and immune escape, blocking the actin cytoskeleton reorganization may affect the phagocytosis of macrophage, immune function, and tumor generation and metastasis in vivo (45–51). However, these remain to be further researched. To conclude, it can be inferred that PIK-24 can block actin cytoskeleton reorganization by regulating PI3K signaling, thereby inhibiting syncytium formation.

As one of the most common RHO family GTPases that can regulate cytoskeletal dynamics, Cdc42 is mainly responsible for regulating filopodia formation (30, 31). With the deepening of research, it was found that Cdc42 binds to the PI3K regulatory subunit p85α to activate and exert its function (32). In this study, filopodia formation induced by RSV was accompanied by the activation of Cdc42 and interaction between Cdc42 and p85α. PIK-24 treatment could effectively abolish the generation of these events. The above results indicate that the activation of Cdc42 controlled by PI3K p85α is essential for membrane fusion during syncytium formation. In addition, cofilin is also a key factor that regulates the dynamics of the actin cytoskeleton in many cellular physiological processes. The most important physiological function of cofilin is to dissolve and depolymerize actin fibers (33). Cofilin works primarily in response to upstream regulators PI3K or RhoGTPases that serve to phosphorylate cofilin (24, 34, 35). Whether the serine residue position 3 is phosphorylated, cofilin can be divided into inactive form (phosphorylation) and active form (dephosphorylation). When cofilin is in a phosphorylated state, it will not be able to bind to actin, causing the aggregation of actin fibers. In contrast, the dephosphorylation of cofilin will be reactivated to dissolve actin fibers (52, 53). The experimental results showed that the upregulation of F-actin expression and filopodia formation induced by syncytium formation coincided with the phosphorylation of cofilin. Meanwhile, total-cofilin expression was slightly downregulated, indicating that the inactive form of cofilin was upregulated, in contrast to the upregulation of total-cofilin expression caused by RSV entry (26). The possible reason is that the biphasic change of cofilin corresponds to the remodeling pattern of actin cytoskeleton caused by different stages of RSV infection, both benefit viral infection and intracellular replication, which is consistent with studies that cofilin-mediated actin cytoskeleton dynamics regulate other viral infections, such as porcine hemagglutinating encephalomyelitis virus (PHEV) and HSV-1 (54, 55). PIK-24 treatment could prevent the phenomenon by inhibiting the activity of PI3K signaling. Therefore, it can be seen that PIK-24 blocks actin cytoskeleton reorganization by inhibiting PI3K signaling-mediated Cdc42 activation and cofilin function change, thereby preventing syncytium formation and RSV proliferation.

Among the subunits of PI3K protein, p85 regulates the activity of PI3K and is also a key target for viral infection of host cells. Several viruses, such as semliki forest virus (SFV) or ross river virus (RRV), have been proven to directly interact with p85 regulatory subunits through specific motifs (YXXM) in their viral proteins to stimulate the PI3K signaling pathway and increase glucose metabolism toward the synthesis of fatty acids, thereby promoting the replication of virions (56). There are also related viruses that activate the PI3K signaling pathway through interaction of their viral proteins with the p85 regulatory subunit to delay cell apoptosis and promote viral replication, such as Marek's disease virus (MDV) (57). In this study, given that PI3Kα and PI3Kβ are commonly expressed in host cells, the genes of PI3Kα and PI3Kβ protein-related subunits were silenced, and their effects on RSV infection were examined. The experimental results showed that p85α could control RSV infection in vitro, equivalent to PIK-24. Importantly, knockout of p85α also exhibited more potent anti-RSV activity in vivo, probably due to the effects of PI3K protein on the physiological process and the immune system in the organism. Furthermore, si-PIK3R1 effectively reduced the activities of Akt and Cdc42, the phosphorylation of cofilin, and the content of F-actin (dissolving actin fibers) in host cells, which may be the reason for p85α-regulated filopodia formation during RSV-induced syncytium formation. However, silencing PIK3R2, PIK3R3, PIK3CA, and PIK3CB genes had no significant effect on RSV infection. Further studies found that PIK-24 had bind activity with the p85α protein. Knockdown of p85α had no synergistic action on the inhibition of RSV infection by PIK-24, while overexpression of p85α protein in cells abolished the anti-RSV effect of PIK-24. The above results suggested that PIK-24 exerted an anti-RSV effect by targeting PI3K p85α protein instead of p85β, p55γ, p110α, and p110β. This phenomenon occurs because PI3Ks have multiple subtypes in host cells, and different viruses have been able to target different subunits of the PI3K protein through their long evolution, thereby maximizing the replication efficiency of virus particles (58, 59). Coincidentally, this study confirms the importance of p85α in RSV infection.

In conclusion, PIK-24, as a novel PI3K inhibitor, has potent anti-RSV activity, and its antiviral effect is stronger than that of the classic PI3K inhibitor LY294002, PI-103, and broad-spectrum antiviral drug ribavirin. The molecular mechanism of PIK-24 against RSV infection involves the inhibition of PI3K signaling activation, actin cytoskeleton reorganization, and cell-to-cell fusion (Fig. 9). This study finds for the first time that RSV utilizes the p85α regulatory subunit of PI3K protein to facilitate its proliferation. The anti-RSV events of PIK-24 confirm the feasibility of this idea. This study fully illustrates the molecular mechanism of PI3K signaling intervention in RSV infection and provides a promising leading compound for novel anti-RSV infection drug development.

FIG 9.

Schematic diagram of the regulatory effect of PIK-24 on PI3K signaling activation and actin cytoskeleton reorganization during RSV-induced syncytium formation. During RSV-induced syncytium formation, the virus induces Cdc42 activation, cofilin inactivation, and F-actin polymerization via the PI3K signaling pathway, which may promote cell-to-cell fusion and initiate syncytium formation. PIK-24 treatment significantly reverses all these effects by targeting the regulatory subunit p85α of PI3K, thereby exerting an antiviral effect.

MATERIALS AND METHODS

Compounds, reagents, and antibodies.

PIK-24 and 3,4-DCQAME were chemically synthesized in the laboratory. Ribavirin, LY294002, PI-103, and cytochalasin D were obtained from MedChemExpress (Monmouth Junction, NJ, USA). Anti-PI3 kinase p85 alpha, anti-PI3 kinase p85 beta, anti-PI3 kinase p55 gamma, anti-PI3 kinase p110 alpha, anti-PI3 kinase p110 beta, anti-p-Akt (Ser473), anti-Akt, anti-cofilin (phospho S3), anti-cofilin, anti-F-actin, anti-GAPDH, recombinant human PI3 kinase p85 alpha protein, and anti-respiratory syncytial virus fusion (F) glycoprotein antibody were purchased from Abcam (Cambridge, UK). Antirabbit IgG, HRP-linked antibody, antimouse IgG, HRP-linked antibody, Alexa Fluor 488 donkey antimouse IgG (H+L), and Alexa Fluor 488 phalloidin were acquired from Cell Signaling Technology (Danvers, MA, USA). Human respiratory syncytial virus (RSV) (A2) fusion glycoprotein (ECD, His tag) was purchased from Sino Biological, Inc. (Beijing, China). A RhoA/Rac1/Cdc42 activation assay combo biochem kit was obtained from Cytoskeleton, Inc. (Denver, CO, USA). The Dual-Lumi luciferase reporter gene assay kit was acquired from Beyotime Biotechnology (Shanghai, China). The Monolith protein labeling kit RED-NHS (2nd generation) was purchased from NanoTemper (Munich, Germany).

Cell culture, viral proliferation, and treatments.

HEp-2, HeLa, A549, and HEK293T cells were purchased from the American Type Culture Collection (ATCC). The four cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS).

Human respiratory syncytial virus (RSV A2 strain) was provided by the Medicinal Virology Institute of Wuhan University, China. RSV A2 was proliferated via HEp-2 cell infection. Briefly, HEp-2 cells were inoculated with RSV at 37°C and shaken gently every 15 min. After 2 h of incubation, the viral supernatant was removed, and the monolayer was washed 3 times in PBS and supplemented with a fresh maintenance medium (DMEM, 2% FBS, and 1% PS). HEp-2 cells were then incubated continuously until 48 h. After freeze-thaw cycle treatment, the suspension was collected and centrifuged at 3,000 × g for 15 min. Viral stocks were aliquoted and stored at −80°C. Viral titers were measured by plaque assay and expressed as PFU mL⁻¹.

Cells, plasmid-transfected cells, or knockdown cells were treated with different concentrations of PIK-24, LY294002, PI-103, cytochalasin D, or ribavirin in the absence or presence of RSV for 24 or 48 h.

Plasmids, siRNA, and transfection.

Human codon-optimized full-length RSV-F was cloned into the pcDNA3.1(+) vector. pT7-Luc plasmid, pCAG-T7 plasmid, and pRL-TK plasmid were purchased from MiaoLingBio (Hubei, China). p85α plasmid was constructed by MiaoLingBio.

siRNAs (si-PIK3R1, si-PIK3R2, si-PIK3R3, si-PIK3CA, and si-PIK3CB) were purchased from RiboBio Co. Ltd. (Guangzhou, China). The primer pairs are listed in Table S1.

According to the manufacturer's instructions, plasmids or siRNAs were transfected into the HEp-2 or HEK293T cells via lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA). Plasmids or siRNAs were diluted in Opti-MEM and then mixed with another Opti-MEM containing lipofectamine 2000. After 30 min of incubation, the transfection mixture was added to the monolayer. At 4 to 6 h posttransfection, the mixture was replaced with a maintenance medium. At 24 or 48 h posttransfection, the related experiment was carried out.

Cytotoxicity assay.

The cytotoxicity of compounds (PIK-24, LY294002, PI-103, and ribavirin) was measured by CCK-8 (Yeasen Biotechnology, Shanghai, China) assay. Briefly, HEp-2, HeLa, or A549 cells with a cell density of 50% in 96-well plates were treated with different concentrations (40, 20, 10, 5, 2.5, or 1.25 μM) of compounds and then incubated at 37°C for 48 h. Next, 10 μL of CCK-8 was added to each well. After 30 min of incubation at 37°C, absorbance values were detected in a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at 450 nm. Cell viability was expressed as a percentage of blank control, and the 50% cytotoxic concentration (CC₅₀) was acquired by regression analysis.

Anti-RSV activity assay.

The anti-RSV activity of compounds (PIK-24, LY294002, PI-103, and ribavirin) was evaluated by immunofluorescence assay. Briefly, HEp-2, HeLa, or A549 cell monolayers in 96-well plates were inoculated with RSV (multiplicity of infection [MOI] = 0.1) and simultaneously exposed to compounds at different concentrations (40, 20, 10, 5, 2.5, or 1.25 μM). After 48 h of incubation, the supernatant of each group was collected and added to new 96-well plates with cell monolayers. After 24 h of incubation, the cells were fixed in 4% paraformaldehyde for 15 min, permeabilized using 0.1% Triton X-100 for 10 min, and incubated in 5% BSA/PBS for 1 h at room temperature. The monolayers were stained with anti-RSV F antibodies overnight at 4°C and incubated in Alexa Fluor 488-conjugated secondary antibody at room temperature for 2 h. The cells were washed 3 times with PBS at the end of each process. The fluorescence intensity of each group was quantified with a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA), and the image was acquired using a fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany). The IC₅₀ was then obtained by regression analysis. The selective index (SI) was calculated by the ratio of CC₅₀ to IC₅₀ (CC₅₀/IC₅₀).

Plaque assay.

HEp-2 cells in 96-well plates were inoculated with RSV (MOI = 0.1) and incubated for 24 h. PIK-24 (20 μM) was then added to HEp-2 cells and cultured for another 24 h. Ribavirin (20 μM) and LY294002 (20 μM) were treated similarly. The supernatant of each group was collected.

Confluent HEp-2 cell monolayers in 24-well plates were incubated with approximately 50 to 100 PFU of viral supernatant in each group at 37°C for 2 h. After removing the unentered virus, the monolayer was washed with PBS 3 times and overlaid with a fresh maintenance medium containing 1.2% agarose. After 96 h of incubation, the monolayer was fixed in 4% formalin overnight. The agarose was then removed, and the monolayer was stained with 1% crystal violet. After 30 min of incubation, the plaques were then visualized and recorded.

Syncytium formation inhibition assay.

The inhibitory effect of PIK-24 on syncytium formation was evaluated by immunofluorescence assay. The monolayer of HEp-2 cells was inoculated with RSV (MOI = 0.1) and incubated for 24 h. After supplementing PIK-24 (20 μM), HEp-2 cells were cultured for another 24 h. Ribavirin (20 μM) and LY294002 (20 μM) were treated similarly. The number of syncytia and fluorescence intensity of RSV-F protein in each group were then detected by immunofluorescence assay.

Cell-to-cell fusion assay.

The transfection of HEK293T cells with the pcDNA3.1(+)-F, pT7-Luc, pRL-TK, or pCAG-T7 Pol plasmids was performed with the lipofectamine 2000 reagent. For one 24-well transfection, 0.5 μg of the pcDNA3.1(+)-F and pT7-Luc plasmids were diluted in 250 μL Opti-MEM and mixed thoroughly, then 2 μL lipofectamine 2000 reagent was added. After 30 min of incubation, the transfection mixture was added to each well containing HEK293T cells. A transfection mixture without pcDNA3.1(+)-F was added as the mock-transfected control. For another 24-well transfection, HEK293T cells were transiently cotransfected with pCAG-T7 Pol (0.5 μg) and pRL-TK (0.25 μg) plasmids. At 4 to 6 h after transfection, the supernatant was replaced with the fresh maintenance medium containing PIK-24 (20 μM), ribavirin (20 μM), or LY294002 (20 μM). After incubation for 12 h, the HEK293T cells of two 24-well plates were mixed in equal proportion and seeded into a new 96-well plate in the presence of PIK-24, ribavirin, or LY294002. At 48 h posttransfection, the degree of lesion and fluorescence intensity of RSV-F protein were then measured as described above, and the activities of firefly luciferase and renilla luciferase were detected using the dual-luciferase reporter assay system (Promega, WI, USA).

Real-time PCR assay.

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), quantified, and reverse-transcribed into cDNA using a PrimeScript RT reagent kit (TaKaRa Bio Inc., Kusatsu, Japan). PCR amplification was performed using the synthesized cDNA template in a fluorescence-based quantitative PCR system (Roche, Pleasanton, CA, USA). The primer pairs are shown in Table S2.

Western blot assay.

The monolayer was collected, suspended, and incubated in a cell lysis mixture (100× phenylmethylsulfonyl fluoride [PMSF], 20× PI, 20× PPI, and cell lysis solution) for 30 min. The total proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes, and probed with the primary antibodies. Detection was performed after incubation with HRP-conjugated secondary antibodies. The protein bands were visualized by enhanced chemiluminescence using Amersham Imager 600 (General Electric Co., Boston, MA, USA). The band intensity was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Pulldown assay.

According to the manufacturer's instructions, the activity of Cdc42 was detected using the RhoA/Rac1/Cdc42 activation assay combo biochem kit. After RSV, compounds, or siRNA treatment, the monolayer was placed on ice and washed with chilled PBS. The cells were lysed with 2 mL of cell lysis buffer supplemented with a 1× protease inhibitor cocktail. The cell lysate was harvested with a cell scraper and immediately clarified by centrifugation at 10,000 × g at 4°C for 1 min. Then, 800 μg of total cell protein was added to 10 μg of PAK-PBD beads. The mixture was incubated at 4°C on a rocker for 1 h and centrifuged at 5,000 × g for 1 min. Next, 90% of the supernatant was removed carefully, and the beads were washed once with 500 μL of wash buffer. A total of 20 μL of 2× Laemmli sample buffer was added and thoroughly resuspended the beads. The bead samples were boiled, and the activity of Cdc42 was analyzed by Western blot assay.

Coimmunoprecipitation assay.

The total protein was extracted as described above, and 300 μg of total protein was exposed to 5 μL of anti-Cdc42 or anti-p85α antibody for 1 h at 4°C. The mixture was treated with 20 μL of the resuspended volume of protein A/G PLUS-Agarose (Santa Cruz Biotechnology, Dallas, TX, USA) and incubated at 4°C on a rocker platform overnight. The immunoprecipitates were collected by centrifugation at 3,000 × g for 5 min at 4°C. After washing the pellet 4 times with PBS, the supernatant was discarded, and the pellet was resuspended in 40 μL of 2× electrophoresis sample buffer. The samples were boiled for 5 min and then measured by Western blot assay.

RSV-F protein binding assay.

The monolayer was treated with a mixture containing PIK-24 (20 μM) and RSV-F protein (1 μg/mL) and then incubated for 2 h. 3,4-DCQAME (20 μM) and LY294002 (20 μM) were treated similarly. The binding of F protein to HEp-2 cells was determined by immunofluorescence assay, and the fluorescence intensity was measured through a confocal microscope (Carl Zeiss AG, Oberkochen, Germany).

Actin cytoskeleton morphology analysis.

The monolayer was fixed with 4% paraformaldehyde for 60 min, treated with 0.1% Triton X-100 for 15 min, and exposed to PBS containing 5% BSA for 60 min. The monolayer was then stained with Alexa Fluor 488 phalloidin for 2 h at room temperature. The cells were washed 3 times with PBS at the end of each process. The actin cytoskeleton morphology in each group was determined using a confocal microscope.

Microscale thermophoresis assay.

The binding activity of PIK-24 and p85α protein was detected using MST assay. Briefly, the p85α protein was labeled and purified using the Monolith protein labeling kit RED-NHS (2nd generation; NanoTemper, Munich, Germany) according to the manufacturer's instructions. Then, 10 μL of the 300 μM dye solution was added to 90 μL of the 10 μM p85α protein. The dye-protein solution was incubated for 30 min at room temperature in the dark. After the labeling reaction incubation, the dye-protein solution was transferred to the B-column to remove the free dye. Next, PIK-24 was subjected to 2-fold serial dilution with MST buffer to different concentrations. The initial concentration of PIK-24 is 10 μM. Different concentrations of PIK-24 were mixed with an equal volume of purified dye protein. The mixture was transferred to the capillary, and the binding activity of PIK-24 and p85α protein was measured using Monolith NT.115 (NanoTemper, Munich, Germany).

Animals and experimental protocol.

Six-week-old wild type (WT) or p85α^−/− C57BL/6 mice (CAVENS, Jiangsu, China) were randomized into three separate cages (WT, WT+RSV, and p85α^−/−+RSV; n = 6) and intranasally infected with RSV. Body weights were measured every 24 h from −1 d before to 4 d after RSV infection. On day 4 post-RSV infection, the mice were sacrificed. The lung tissues were excised and weighed. The lung index was obtained via the formula: lung index = lung weight/body weight. The lung tissues were then used to perform immunofluorescence staining and histopathological examination. All animal trials strictly adhered to the Guidelines for Laboratory Animal Use and Care of the Chinese Centers for Disease Control and Prevention (CDC) and the Rules for Medical Laboratory Animals of the Ministry of Health, China. The animal protocols were approved by the National Institute for Communicable Disease Control and Prevention and the Ethics Committee of Jinan University.

Statistical analysis.

Results are expressed as means ± SD. Statistical analyses were carried out in GraphPad Prism v.5.0 software (GraphPad Software, La Jolla, CA, USA.). Differences between the groups were compared using the one-way ANOVA with Bonferroni's corrections or Student's t tests. P < 0.05 was considered statistically significant.