JNK and PI3k/Akt signaling pathways are required for establishing persistent SARS-CoV infection in Vero E6 cells

Abstract

Persistence was established after most of the SARS-CoV-infected Vero E6 cells died. RNA of the defective interfering virus was not observed in the persistently infected cells by Northern blot analysis. SARS-CoV diluted to 2 PFU failed to establish persistence, suggesting that some particular viruses in the seed virus did not induce persistent infection. Interestingly, a viral receptor, angiotensin converting enzyme (ACE)-2, was down-regulated in persistently infected cells. G418-selected clones established from parent Vero E6 cells, which were transfected with a plasmid containing the neomycin resistance gene, were infected with SARS-CoV, resulting in a potential cell population capable of persistence in Vero E6 cells. Our previous studies demonstrated that signaling pathways of extracellular signal-related kinase (ERK1/2), c-Jun N-terminal protein kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3'-kinase (PI3K)/Akt were activated in SARS-CoV-infected Vero E6 cells. Previous studies also showed that the activation of p38 MAPK by viral infection-induced apoptosis, and a weak activation of Akt was not sufficient to protect from apoptosis. In the present study, we showed that the inhibitors of JNK and PI3K/Akt inhibited the establishment of persistence, but those of MAPK/ERK kinase (MEK; as an inhibitor for ERK1/2) and p38 MAPK did not. These results indicated that two signaling pathways of JNK and PI3K/Akt were important for the establishment of persistence in Vero E6 cells.

Fig. 1

Indirect immunofluorescence staining of SARS-CoV proteins. For the establishment of persistent infection, the virus was infected to Vero E6 cells at 10 m.o.i. At 10 days p.i., intracellular viral antigens were detected by indirect immunofluorescence staining using rabbit anti-SARS-CoV antibody, which recognizes SARS-CoV proteins (unpublished data). Briefly, cells were spotted onto a 12-well Teflon-coated slideglass, air dried in a biosafety cabinet under UV irradiation, and fixed with 100% pre-chilled acetone. Aliquots of 25 μl of 1:160 diluted anti-SARS-CoV antibody were placed on the coated wells and incubated at 37 °C for 30 min in a moist chamber. After washing with PBS, a fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG antibody was added at a dilution of 1:40, and incubated for 30 min at 37 °C.

Fig. 2

Northern blot analysis of viral mRNAs. Vero E6 cells were inoculated with SARS-CoV at 10 m.o.i. Total RNA was extracted with Isogen (Nippon Gene, Tokyo, Japan) from SARS-CoV-infected cells at 24 h and 11 days p.i. and from mock-infected cells. RNAs were electrophoresed on 1% agarose gels in the presence of formaldehyde, blotted onto the nitrocellulose membrane. The probe was constructed based on the PCR product of mRNA9. Briefly, viral RNA was reverse-transcribed using SuperScriptIII (Invitrogen, Carlsbad, CA, USA) and the reverse primer, 5′-AGTCAGTCTAATACGACTCACTATAGGGTCCTAAGAAGCTATTAAAATTAGC-3′(29,687 to 29,721 nt of GenBank accession numberNC_004718). The underline indicates the promoter sequence of T7 RNA polymerase. PCR amplification was performed using High Fidelity Platinum Taq DNA polymerase (Invitrogen) using the reverse primer and forward primer, 5′-TGGCTAGCGGAGGTGGTGAAACTGCCC-3′ (28,751 to 28,777 nt). The RNA probe was transcribed from the PCR product using T7 RNA polymerase (Ambion, Austin, TX, USA) under incorporating digoxigenin (DIG RNA labeling kit, Boehringer Mannheim GmbH, Mannheim, Germany). Hybridization and wash were performed as described previously . Left and right panels indicated short and long detection times using the LAS-3000 mini system (Fuji Photo Film Co. Ltd, Tokyo, Japan), respectively.

Fig. 3

Down-regulation of ACE-2 by SARS-CoV infection. Western blot of acute infection (8, 18, and 24 h.p.i.) and persistent infection (passage 7) were performed using anti-ACE-2 antibody. Whole-cell extracts were electrophoresed in 10–20% gradient polyacrylamide gel and transferred onto PVDF membranes (Immobilon-P, Millipore, Bedford, MA, USA). We applied two sets of samples to the polyacrylamide gels, and the blots were divided into two sheets for detection by a ProtoBlot II AP system (Promega Co., Madison, WI, USA), as described previously , , . Mouse anti-human ACE-2 ectodomain monoclonal antibody was purchased from R and D systems Inc (Minneapolis, MN, USA) and used at a dilution of 1:1000. Mouse anti-beta actin antibody was purchased from Sigma (St. Louis, MO, USA) and used at a dilution of 1:5000.

Fig. 4

Establishment of persistent SARS-CoV-infected cells by diluted viruses. Aliquots of 1.5 × 10⁵ Vero E6 cells were inoculated with 2 × 10⁻¹, 2 × 10⁰, 2 × 10¹, and 2 × 10² PFU of SARS-CoV. The cells were stained with 0.05% crystal violet after fixing with 20% formaldehyde.

Fig. 5

Persistent SARS-CoV infection of cloned Vero E6 cells. A plasmid, pcDNA3, was transfected into Vero E6 cells using FuGene6 (Roche Diagnostics, Penzberg, Germany), and cloned cells were picked after neomycin selection (500 μg/ml). The 91 cloned cells were inoculated with SARS-CoV at 10 m.o.i. At 20 days p.i., the 24-well plates were fixed and stained.

Fig. 6

Effects of signaling pathways on establishing the persistence of Vero E6 cells. SB203580 as a p38 MAPK inhibitor, PD098059 as a MEK inhibitor, and SP600125 as a JNK inhibitor were purchased from Calbiochem (San Diego, CA, USA). LY294002 as a PI3K inhibitor was purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). All reagents were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 or 20 mM. All test wells, including mock-treated controls, were treated with DMSO. Vero E6 cells were pre-treated with SB203580, PD98059, SP600125, and LY294002 at concentrations from 10 to 50 μM for 1 h, and then inoculated with SARS-CoV at 2 m.o.i. The plates were fixed and stained at 20 days p.i.

Fig. 7

Phosphorylation status of Akt in viral infected cells. Western blot of acute infection (8, 18, and 24 h.p.i.) and persistent infection (passage 7) were performed using anti-phospho Akt antibody. The rabbit anti-phospho Akt (Ser473) antibody was purchased from Cell Signaling Technology Inc. and used at a dilution of 1:1000. SARS-CoV was infected to cloned Vero E6 cells at 10 m.o.i. The Western blot of the protein samples at 20 h.p.i. was performed using anti-phospho Akt antibody.