Porcine reproductive and respiratory syndrome virus inhibits MARC-145 proliferation via inducing apoptosis and G2/M arrest by activation of Chk/Cdc25C and p53/p21 pathway

MARC-145 cells, a subclone of African green monkey kidney-derived MA-104 cells, were purchased from the China Center for Type Culture Collection(Wuhan, China). Cells were cultured either in 6-well plates or flasks, according to the standard culturing procedure with Dulbecco’s modified eagle medium(DMEM, ThermoFisher, #12800017) plus 10% fetal bovine serum(FBS), 100 μg/mL streptomycin, and 100 U/mL penicillin(Sigma-Aldrich, MO, USA) at 37 °C with 5% CO₂. PRRSV 2 strains, SD16 (GenBank ID:JX087437.1), VR2332(GenBank ID:EF536003.1), CH-1a(GenBank ID: AY032626) and PRRSV 1 strain, GZ11-G1(GenBank ID:KF001144.1), were propagated and titrated in MARC-145 cells.

p53 antibody(#9282), Phospho-p53(Ser15) antibody(#9284), p21^Walf1/Cip1(12D1) rabbit mAb(#2947), cyclinB1 antibody(#4138), Cdc2(POH1) mouse mAb(#9116), phospho-Cdc2(Tyr15) (10A11) rabbit mAb(#4539), Cdc25C(5H9) rabbit mAb(#4688), phospho-Cdc25C(Ser216) (63F9) rabbit mAb(#4901), Myt1 antibody(#4282), Weel antibody(#4963), GAPDH(14C10) Rabbit mAb(#2118), and Alexa Fluor® 488 Phalloidin (#8878) (Phalloidin belongs to a class of toxins called phallotoxins. It functions by binding and stabilizing filamentous actin (F-actin) and effectively prevents the depolymerization of actin fibers. The properties of phalloidin make it a useful tool for investigating the distribution of F-actin in cells by labeling phalloidin with fluorescent analogs and using them to stain F-actin for light microscopy.) were all purchased from Cell Signaling Technology, Inc.(Danvers, MA, USA). Nocodazole(Nocodazole is a commonly used mitotic inhibitor which interferes with microtubule assembly thus interfering with mitosis duo to formation of multipolar spindles and leading to cell cycle arrest in G2/M [11]), mouse monoclonal anti-α-tubulin antibody, propidium iodide (PI), and Ribonuclease A (RNase A) were obtained from Sigma-Aldrich(St Louis, MO, USA). Mouse monoclonal anti-nucleocapsid(N) antibody(6D10) was previously made in our laboratory. Peroxidase-conjugated affinipure goat anti-mouse IgG(H + L), peroxidase-conjugated affinipure goat anti-rabbit IgG(H + L), and Cy™3-conjugated affiniPure goat anti-rabbit IgG(H + L) were purchased from Jackson ImmunoResearch Laboratories, Inc.(West Grove, PA, USA). Cell counting kit-8(CCK-8) was purchased from Beyotime Institute of Biotechnology(Shanghai, China). Alexa Fluor® 488 annexin V/Dead cell apoptosis kit was purchased from Invitrogen™ Life Technology(Grand Island, USA). DAPI, Dynabeads Protein G, FBS, and DMEM were purchased from ThermoFisher(Waltham, MA, USA).

To determine the optimal inoculation, the standard curve of the absorbance of 450 nm (OD450nm) and cell number was obtained following the instructions of cell counting Kit-8 (CCK-8). MARC-145 cells were seeded in 96-well plates with 1 × 10⁴ cells/well and cultured to reach approximately 80% confluence at 37 °C, 5% CO₂. Then, the cells were either mock-infected or infected with PRRSV SD16 at 0.01, 0.1, 1, or 5 multiplicity of infection (MOI) and reduplicated in 6 wells for every infective dose. At 0, 6, 12, 24, 36, 48, and 72 h after PRRSV infection, 10 μL CCK-8 solution was added, incubated for another 2 h, then OD450nm was measured with a Micro-Volume Spectrophotometer System(Epoch, BioTek Vermont, USA).

MARC-145 cells were seeded in 6-well plates with 2 × 10⁵ cells/well and cultured to reach approximately 80% confluence. The cells were either mock-infected or infected with PRRSV SD16 at 1 MOI. At 0, 12, 24, 36, and 48 h after PRRSV infection, cells were collected and washed with cold phosphate-buffered saline(PBS). Cells were then resuspended in 1×annexin-binding buffer, followed by addition of Alexa Fluor® 488 annexin V and PI working solutions according to the manufacturer’s instructions. The apoptotic cells were analyzed by flow cytometry(Beckman Coulter Cytomics Altra, Brea, CA, USA).

The cell cycle and nuclear DNA content were determined using PI staining and flow cytometry. Mock-infected or PRRSV-infected MARC-145 cells were collected, washed with PBS, and fixed with 70% cold ethanol. The cell pellets were resuspended in 1 mL PI solution containing 100 μg/mL PI, 100 μg/mL RNase A, and 0.1% Triton X-100 and incubated for 30 min at 4 °C. The DNA content was analyzed by flow cytometry(Beckman Coulter Cytomics Altra, Brea, CA, USA).

Mock-infected or PRRSV-infected MARC-145 cells were fixed with 4% paraformaldehyde for 10 min at RT, washed with PBS, permeabilized with 0.3% triton X-100/PBS for 3 min, then washed and blocked with 5% BSA/PBS. After washing, the cells were incubated with primary antibodies for 1 h at 37 °C, washed with PBS, and incubated with the corresponding secondary antibody. Finally, cells were stained with DAPI and visualized using Leica microsystems(Leica AF6000, Germany).

Cells mock-infected and PRRSV-infected were harvested using Trypsin-EDTA(0.25%) (ThermoFisher, USA) digestion. After washing with PBS, cell samples were treated with NP40 lysis buffer (Beyotime, China), and then protein concentrations were determined using the Pierce BCA protein assay kit(ThermoFisher, USA). Equal amounts of protein were loaded and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and then transferred to PVDF membranes(Millipore, USA) using BIO-RAD Mini Trans-blot. The membranes were blocked with 5% non-fat dry milk and then incubated with indicated primary antibodies overnight at 4 °C, followed by HRP-conjugated secondary antibodies. α-tubulin or GAPDH were used as loading control, and nocodazole-treated cells were used as positive control. The protein bands were visualized using ChemiDoc™ MP Imaging System(Bio-Rad,USA).

p21 and Cdc2-cyclinB1 interactions were analyzed using immunoprecipitation according to introduction of the Dynabeads Protein G. Cells of mock-infected, PRRSV-infected, or nocodazole-treated were lysed using ice-cold NP40 cell lysis buffer, and the supernatants were obtained by centrifugation. Dynabeads-Ab complex was prepared by incubating Cdc2 mouse mAb with Dynabeads Protein G using a Catch and Release(version 2.0) reversible immunoprecipitation system(ThermoFisher, USA). Then, the supernatants were added to the tubes containing Dynabeads-Ab complex and incubated overnight at 4 °C. After washing with PBS, p21^Walf1/Cip1 rabbit mAb and cyclinB1 antibody were used to detect the Dynabeads-Ab-Ag complex by western blot.

MARC-145 cells were seeded in 6-well plates at a density of 2 × 10⁵ cells/well, and cultured to reach approximately 80% confluence. PRRSV strains SD16, GZ11-G1, VR-2332, or CH-1a were used to infect the cells at 1 MOI. At 48 h after viral infection, cells were collected and cyclinB1 and p-Cdc2 (Tyr15) expression were analyzed by western blot.

Although PAM is the primary target cell of PRRSV, it is a terminally differentiated cell and can not divide and proliferate. So MARC-145 cell line was used in the presented study. To determine the optimal infective dose, a standard curve of OD450nm and cell number was produced according to CCK-8 instructions. The OD450nm was detected every half hour for 4 h after CCK-8 solution was added. The results indicate that the optimal time for detection is 2 h after adding CCK-8 solution. The slope equation “y = 0.3098 ln(x)-2.4347” was generated with R² = 0.998 (Fig. 1a). The equation was used to determine the numbers of normal MARC-145 cells and PRRSV-infected MARC-145 cells with 0.01, 0.1, 1, and 5 MOI at 6, 12, 24, 36, 48, and 72 h after infection where x and y are cell number and OD450nm, respectively. As shown in Fig. 1b, from 6 h to 24 h after seeding, cells were in logarithmic growth phase with or without PRRSV inoculation, and PRRSV infection showed little effect on cell proliferation, which was sustainable about 24 h or longer. However, the total cell number reduced greatly at 36, 48, and 72 h with an infective dose of 1 MOI. Because the total cell number decreased quickly between 24 and 36 h after infection with 5 MOI, 1 MOI was used in the following experiments. After infection with 1 MOI, MARC-145 cells showed typical cytopathic effects(CPE) and the CPE became stronger and stronger from 24 h to 48 h post-infection (Fig. 1c).

PRRSV infection can induce cell apoptosis both in vivo and in vitro. Cell apoptosis has been reported in alveolar macrophages, porcine intravascular monocytes, lymphocytes, and testicular germ cells of infected pigs which corresponds to a sharp reduction in these cell numbers in PRRSV positive swine [5, 12, 13]. We infected MARC-145 cells with PRRSV SD16 at 1 MOI and then examined cell apoptosis using an Alexa Fluor® 488 annexin V/Dead cell apoptosis kit. Our results show that the numbers of early and total apoptotic cells increased significantly after PRRSV infection. With the development of infection, more and more apoptotic cells were observed in MARC-145 cells infected with PRRSV when compared with those of mock-infected cells (Fig. 2a). At 48 h after PRRSV infection, the percentages of early and late apoptotic cells in PRRSV-infected MARC-145 cells were remarkably higher than those in mock-infected cells (24.1% ± 0.6% and 7.4% ± 0.6% versus 7.9% ± 0.5% and 1.8% ± 0.2%, respectively) (Fig. 2b).

It is well known that many viruses can induce cell cycle arrest in various kinds of cells [10, 14]. The decrease in cell numbers in PRRSV-infected cells promptes us to determine whether PRRSV infection is associated with an arrest of cell division during a specific phase in the cell cycle in addition to inducing cell apoptosis. To address this question, cell cycle analysis of mock-infected and 1 MOI PRRSV-infected MARC-145 cells was performed at 0, 12, 24, 36, and 48 h post-infection by PI staining and flow cytometry. Representative cell cycle profiles and histograms of mock- and PRRSV-infected cells are presented in Fig. 3a and b, respectively. As shown in Fig. 3a, mock-infected MARC-145 cells maintained a normal cell cycle profile, and more than 75% cells were in G0/G1 phase when cells were in the state of contact inhibition after culture 48 h. However, PRRSV infection disturbed the normal cell cycle, some cells were arrested at G2/M phase and can not enter the next cell cycle, which resulted in the accumulation of cells in the G2/M phase. This phenomenon became more and more obvious with the development of virus infection. In addition, the percentages of PRRSV-infected cells in S phase also increased from 24 h to 48 h post infection and had a significant difference at 36 h and 48 h post-infection. At 48 h, the cells in G0/G1 phase decreased greatly, while cells in the G2/M phase increased significantly (Fig. 3b). These results demonstrate that PRRSV infection promoted the cycle progression of MARC-145 cells from G0/G1 phase to G2/M phase and then arrest in G2/M phase.

The life cycle of a dividing cell can be split into four stages: G1, S, G2 and mitosis(M), with cells that are no longer cycling being said to be quiescent or in G0 phase. Progression from one stage to the next is controlled by the activities of kinase complexes made up of cyclins bound to cyclin-dependent kinases (Cdk). Mitosis is thought to be triggered by Cdk1 (also known as Cdc2 or p34^cdc2 kinase) whose activation begins when it binds to its regulatory subunit cyclinB1. It accumulates in S and G2 phases to form a mitosis-promoting factor (MPF) with Cdc2 and is then ubiquitinated and degraded by the anaphase-promoting complex (APC) after the cells pass through mitosis [15]. To determine whether PRRSV infection affects cyclinB1 expression, the expression of cyclinB1 protein was detected by western blot at different times post-infection. Results showed that the expression of cyclinB1 in PRRSV infected MARC-145 cells increased significantly at 24 (p < 0.01) and 48 h (p < 0.001) post-infection compared with those of mock-infected cells (Fig. 4a and b). CyclinB1 shuttles between the nucleus and cytoplasm during interphase, and is known to be localized in the cytoplasm at the G2 phase and to be transported into the nucleus during the M phase [16]. Several reports have shown that virus infection-induced cell cycle arrest in G2 is due to the prevention of nuclear localization of cyclinB1 [17, 18]. In this study, although the expression of cyclinB1 was found in the cytoplasm and the nucleus in PRRSV-infected cells at 48 h, its expression was obviously higher than that in mock-infected cells. What’s more, in white light, the infected cells showed typtical CPE (Fig. 4b). These results clearly indicate that G2/M phase arrest induced by PRRSV infection does not result from loss of cyclinB1 or from interference with its nuclear translocation.

The Cdc2 kinase is the key regulator of the G2/M phase. Before mitosis, cyclinB-Cdc2 complexes are held in an inactive state by phosphorylation of Cdc2 at Thr14 and Tyr15, which is catalyzed by the protein kinases Wee1 (which phosphorylates Tyr15 only) and Myt1 (which phosphorylates both Thr14 and Tyr15). Cdc2 activation at the onset of mitosis results from the concurrent inhibition of Wee1 and Myt1 and stimulation of the phosphatase Cdc25C [15]. The increase in Tyr15 phosphorylation on Cdc2 is associated with multiple cell cycle arrests, especially G2/M phase arrest [19, 20]. To determine whether PRRSV infection also affects Cdc2 activity, the expressions of Cdc2 and p-Cdc2(Tyr15) were detected with western blot. Results revealed that the expression of Cdc2 in PRRSV-infected cells was higher than that in mock-infected cells at 24 h and the expression of p-Cdc2(Tyr15) in PRRSV-infected cells was significantly higher than that in mock-infected cells at 48 h post-infection (Fig. 5a), which indicated the G2/M phase arrest caused by PRRSV infection is related with expression increase of p-Cdc2(Tyr15). The increase of p-Cdc2(Tyr15) and its distribution in PRRSV-infected MARC-145 cells at 48 h post-infection was further confirmed by immunofluorescence analysis (Fig. 5b).

Considering that Cdc2 kinase activity is negatively regulated by kinases Wee1 and Myt1 and positively regulated by phosphatase Cdc25C, we further investigated the expression of these G2/M cell cycle-regulatory proteins during PRRSV infection. As shown in Fig. 6a, Wee1 expression levels were higher in PRRSV-infected cells than those in mock-infected cells at 24 h(1.728-fold) and 48 h(1.885-fold) post-infection, and Myt1 expression levels were also higher in PRRSV-infected cells than those in mock-infected cells at 24 h(1.249-fold) and 48 h(1.6635-fold) post-infection. These data suggest that Wee1 and Myt1 are involved in the regulation of G2/M arrest induced by PRRSV infection. In addition, we found that Wee1 expression in nocodazole-treated cells also increased greatly, but Myt1 expression was hardly detected, which may be a result of protein degradation during M phase.

Cdc25C is a Cdc2-specific phosphatase. Studies have suggested that phosphorylation of Cdc25C on Ser216 by Chk1 or Chk2 leads to 14–3-3 protein binding, resulting in the sequestration of Cdc25C in the cytoplasm and cytoplasmic accumulation of phospho-Cdc25C (Ser216) denies access to its substrate Cdc2 subunit and prevents cells from going into mitosis by keeping the MPF inactive, resulting in the arrest of cells at G2/M [21]. We analyzed the expression of Cdc25C and phosphorylated Cdc25C(p-Cdc25C). As displayed in Fig. 6b, PRRSV-infected cells showed increased levels of the Ser216-phosphorylated form of Cdc25C compared with those in mock-infected controls at 24 and 48 h post-infection. The expression and distribution of p-Cdc25C(Ser216) was also analyzed using IFA (Fig. 6c), p-Cdc25C(Ser216) expression was obviously detectable and mainly located in the cytoplasm of PRRSV-infected cells, in contrast to its expression in mock-infected cells. These results collectively suggest that PRRSV infection gives rise to the level of Cdc2 phosphorylation increase in PRRSV-infected cells by enhancing Wee1 and Myt1 expression and prevents Cdc2 dephosphorylation by inhibiting Cdc25C activity, which inhibits of the activity of Cdc2 in infected cells and results in the arrest of cells at G2/M phase.

The DNA damage checkpoint kinases, Chk1 and Chk2, play important roles in regulating the G2/M checkpoint via the phosphorylation of Cdc25C at Ser216 through an ATM/ATR-dependent pathway [22, 23] . Given that we have found increased p-Cdc25C(Ser216) levels in PRRSV-infected cells, we further analyzed Chk1 activation with western blot. As expected, PRRSV infection significantly enhanced Chk1 activation by increasing phosphorylation of Chk1 at Ser345 in PRRSV infected cells (Fig. 7).

p53 is a transcription factor that is induced in response to DNA damage and/or cellular stress, which controls the G2/M checkpoint by allowing sufficient repairs to occur before the cell enters mitosis [24]. Ser15 phosphorylation of p53(Ser18 phosphorylation in mice) can lead to stability increase of p53, a common event in DNA damage and other stress responses [25, 26]. Phosphorylation of p53 usually correlates with the ability of p53 to transactivate a number of downstream genes to mediate either cell cycle arrest or apoptosis. p21 is a cyclin-dependent kinase inhibitor located in the downstream of the p53 gene that can inhibit the activity of the Cdc2-cyclinB1 complex. p53 also regulates the G2/M checkpoint through induction of 14–3-3 sigma(σ), a protein that protects damaged cells from entry into mitosis by binding and sequestering Cdc2-cyclinB1 complexes in the cytoplasm [27]. To investigate the relationship between G2/M arrest induced by PRRSV infection and the p53 signaling pathway, we examined the expressions of p53, p-p53(Ser15), 14–3-3σ, and p21 using western blot and p-p53(Ser15) with IFA. The results show that the expression of 14–3-3σ and p21 increased significantly at 24 and 48 h after PRRSV infection, while p-p53(Ser15) and p53 expression was only upregulated at 48 h after PRRSV infection (Fig. 8a and b). This indicates that the cell cycle G2/M arrest caused by PRRSV infection is also associated with p53 signal pathway.

We further conducted immunoprecipitation assay using Cdc2 antibody to precipitate p21. The result confirms the interaction between p21 and Cdc2-cyclinB1 in MARC-145 cells infected by PRRSV (Fig. 8c). These results reveal that activation of the p53/p21 signaling pathway may also be one reason for G2/M arrest of PRRSV-infected cells.

To determine whether different PRRSV strains can induce MARC-145 cell cycle arrest, we used PRRSV 2 strains SD16, VR2332, CH-1a and PRRSV 1 strain GZ11-G1 infected MARC-145 cells. At 48 h post-infection, cells were collected and cyclinB1 and p-Cdc2 (Tyr15) expression were detected with western blot. As expected, PRRSV 1 and PRRSV 2 strains infection all induces cyclinB1 and p-Cdc2(Tyr15) expression increase, which indicates that PRRSV induces MARC-145 cell cycle arrest is common (Fig. 9).