Cyclosporine inhibits mouse cytomegalovirus infection via a cyclophilin-dependent pathway specifically in neural stem/progenitor cells - PubMed (original) (raw)
Cyclosporine inhibits mouse cytomegalovirus infection via a cyclophilin-dependent pathway specifically in neural stem/progenitor cells
Hideya Kawasaki et al. J Virol. 2007 Sep.
Abstract
The potential of neural stem and progenitor cell (NSPC) transplantation in neurodegenerative disease raises a concern about immunosuppressive agents and opportunistic neurotropic pathogens that may interfere with engraftment. Cytomegalovirus (CMV) is an important opportunistic pathogen infecting the central nervous system, where it may remain latent for life, following transplacental transmission. Cyclosporine (Cs), an immunosuppressive drug used in organ transplantation, where its use is associated with CMV reactivation, suppressed murine CMV (MCMV) infection in cultured NSPCs but not in fibroblasts. This activity of Cs appears to be mediated via cyclophilin (CyP) rather than via calcineurin. First, the calcineurin-specific inhibitor FK506 failed to suppress replication. Second, the CyP-specific inhibitor NIM811 strongly suppressed replication in NSPC. NSPCs maintained in the presence of NIM811 retained viral genomes for several weeks without detectable viral gene expression or obvious deleterious effects. The withdrawal of NIM811 reactivated viral replication, suggesting that the inhibitory mechanism was reversible. Finally, inhibition of endogenous CyP A (CyPA) by small interfering RNA also inhibited replication in NSPCs. These results show that MCMV replication depends upon cellular CyPA pathways in NSPCs (in a specific cell type-dependent fashion), that CyPA plays an important role in viral infection in this cell type, and that inhibition of viral replication via CyP leads to persistence of the viral genome without cell damage. Further, the calcineurin-signaling pathway conferring immunosuppression in T cells does not influence viral replication in a detectable fashion.
Figures
FIG. 1.
Isolation and assay of NSPC. (A) Phase-contrast micrograph of a single neural stem cell. (B) Phase-contrast micrograph of a neurosphere after 7 days of proliferation in response to EGF and bFGF. (C to H) Secondary neurospheres were immunostained with antibodies specific to the neural stem cell markers such as nestin (C), Musashi-1 (D), and CD133 (E). Phase-contrast micrograph of each marker's neurosphere (nestin [F], Musashi-1 [G], and CD133 [H]). (I to K) NSPC from neurospheres were dissociated and plated onto glass coverslips that had been coated with poly-
d
-lysine and were differentiated into neurons (β-III tubulin) (I), astrocytes (glial fibrillary acidic protein [GFAP]) (J), and oligodendrocytes (Olig-2) (K). (L to N) NSPC were dissociated and plated onto glass coverslips that had been coated with poly-
d
-lysine and treated with Cs (0.5 μM) and were differentiated into neurons (L), astrocytes (M), and oligodendrocytes (N). Bar, 20 μm.
FIG. 2.
Suppression of MCMV replication by Cs in NSPC cultures. (A and B) Phase-contrast micrograph (top) and immunofluorescence image for GFP (bottom) of NSPC cultures at 7 dpi with MCMV (RM4503) at an MOI of 1 and left untreated (A) or treated with 0.5 μM Cs (B). (C) Flow cytometric analysis of the percentage of MCMV IE1 antigen-positive cells in NSPC cultures infected at an MOI of 1 and left untreated (control) or treated with 0.5 μM Cs (**, P < 0.01). (D) Virus titers determined by plaque assay in NSPC cultures infected at an MOI of 1 and left untreated (control) or treated with 0.5 μM Cs (**, P < 0.01). (E) Percentage of IE1-positive cells (7 dpi) in infected NSPC cultures after different input doses of virus (MOIs of 0.1, 1, or 10) and left untreated (control) or treated with 0.5 μM Cs and assayed (**, P < 0.01; *, P < 0.05). (F) Percentage of IE1-positive cells (7 dpi) in NSPC cultures infected at an MOI of 1 and left untreated (control) or treated with 0.5 μM Cs from mice at embryonic day 15 (E15), postnatal day 7 (P7), and postnatal day 21 (P21) (**, P < 0.01).
FIG. 3.
(A) MEF were infected with MCMV (RM4503) at an MOI of 1 and left untreated (control) or treated with Cs at 0.5 or 5 μM. Photomicrographs showed fluorescent image (green, GFP) and phase-contrast images. (B) Comparison of time course of IE1 antigen-positive cells in MEF left untreated (control) or treated with Cs at 0.5 or 5 μM. (C) Virus titers in infected MEF cultures left untreated (control) or treated with Cs at 0.5 μM, analyzed by plaque assay.
FIG. 4.
The inhibitory effect of Cs on MCMV replication is independent of calcineurin function and dependent on CyP. (A) Phase-contrast micrographs (top) and GFP fluorescence (bottom) analyses of MCMV infection at 7 dpi in control cells and in the presence of 0.5 μM FK506, NIM811, and PSC833, as indicated. (B) Comparison of the percentage of MCMV IE1-antigen positive cells (7 dpi) in untreated NSPC cultures and in cultures maintained in Cs or FK506, with inhibitors at 0.1, 0.5, or 1.0 μM as determined by flow cytometry. (C) Comparison of the percentage of MCMV IE1-antigen positive cells at 7 dpi in untreated NSPC cultures and in cultures maintained in NIM811 or PSC833, with inhibitors at 0.1, 0.5, or 1.0 μM as determined by flow cytometry. (D) Comparison of virus titers in control and infected NSPC cultures treated with FK506, PSC833, or NIM811 (0.5 μM) until 7 dpi (**, P < 0.01). (E to H) Viability of NSPC treated with increasing doses of Cs (E), FK506 (F), NIM811 (G), or PSC833 (H) for 7 days, as determined by cell count and PI uptake (104 cells/ml).
FIG. 5.
Reporter cell assay analysis. (A to E) NSPC and EL-4 cells were transfected with pNF-AT-luc (A, B, and C) or pAP-1-luc (D), NF-κB-luc (E), or pRL-TK reporter plasmid as a control, followed by treatment with different doses of Cs (A, C, D, and E) and FK506 (B and C). At 24 h after transfection, the luciferase activities of whole lysates were measured. (F and G) NSPC were transfected with MCMV IE-promoter-β-Gal, followed by treatment with different doses of Cs (F) and NIM811 (G). At 24 h after transfection, the β-Gal activities of whole lysates were measured. All of the data represent the means of the relative luciferase and β-galactosidase activities in three independent experiments.
FIG. 6.
Detection of viral transcripts after infection with NIM811. NSPC were infected at an MOI of 1 PFU per cell with MCMV in the presence or absence of NIM811 (1 μM). Whole-cell RNA was harvested at 3 hpi, treated with DNase at 6 hpi, and reverse transcribed with oligo(dT). PCRs were performed with primer sets specific for ie1, ie3, and HPRT as described in Materials and Methods. Amplified products were separated on 2% agarose gels and visualized by ethidium bromide staining. NIM811 (1 μM) inhibited ie1/ie3 mRNA expression at 6 hpi in NSPC.
FIG. 7.
MCMV persistent infection in the presence of Cs or NIM811 and reactivation after the removal. (A) NSPC were cultured in the presence of Cs (0.5 μM) for 7 days, when cultures were washed, split, and cultured in the presence or absence of Cs for analysis of MCMV IE1 antigen-positive cells. The arrow indicates the day when Cs was removed from cultures. (B and C) GFP fluorescence micrographs and phase-contrast micrographs of NSPC. NSPC were infected with MCMV (RM4503) at an MOI of 0.1 and cultured for 2, 4, or 6 weeks, when the cells were divided into cultures without or with NIM811 (1.0 μM) and cultured for an additional 2 weeks as indicated below the panels. (B and C) Continual NIM811-treated NSPC (B) and NIM811-released NSPC (C). Phase-contrast micrographs (top) and GFP fluorescence micrographs (bottom) are shown. (D to G) Comparison of the percentage of IE1-positive cells in cultures infected with MCMV (RM4503) at an MOI of 0.1 and treated with NIM811 (1 μM). Cultures were released from suppression by NIM811 every 2 weeks up to 8 weeks postinfection (2 weeks of culture [D], 4 weeks of culture [E], 6 weeks of culture [F], and 8 weeks of culture [G]). The data are expressed as ± the standard deviation of at least three independent experiments (**, P < 0.01).
FIG. 8.
Function of CyPA in MCMV infection in NSPC. (A) The expression and knockdown of CyPA and CyPB by each specific siRNA and si-control were confirmed by immunoblot analysis in total cell lysate of NSPC. β-Actin was detected as an internal control by immunoblot analysis. (B) Inhibition of endogenous CyPA inhibits MCMV infection in NSPC significantly at 7 dpi (MOI = 1) (n = 9; **; P < 0.001). (C) Expression and knockdown of CyPA by each specific siRNA and si-control were confirmed by immunoblot analysis in total cell lysates of NIH 3T3 cells. β-Actin was detected as an internal control by immunoblot analysis. (D) Inhibition of endogenous CyPA did not inhibit MCMV IE1 expression in NIH 3T3 cells at 3 dpi (MOI = 1) (n = 3).
FIG. 9.
(A) Two-color flow cytometry of MCMV IE1 antigen and CyPA (5 dpi). (B) Upregulation of CyPA by CMV infection in NSPC (3 dpi). The expression of CyPA and IE1 of MCMV was confirmed by immunoblot analysis. β-Actin was detected as an internal control. (C to F) Reversal of MCMV inhibition with Cs and NIM811 by adding recombinant CyPA in medium. (C) NSPC infected with MCMV (RM4503) at an MOI of 1 were cultured in the presence of Cs alone or together with human recombinant CyPA. Comparison of the percentage of IE1-positive cells in control untreated (left) and Cs-treated (0.5 μM) cultures in the absence (middle) or presence of CyPA (100 ng/ml) (right), as assessed by flow cytometry. (E) Comparison of the percentage of IE1-positive cells in control, untreated (left) and NIM811-treated (0.5 μM) cultures in the absence (middle) or presence of CyPA (100 ng/ml) (right), as assessed by flow cytometry. (D) CyPA (1, 10, or 100 ng/ml) reversed the suppression of MCMV infection by Cs (0.5 μM) examined by flow cytometry. (F) CyPA (1, 10, or 100 ng/ml) reversed the suppression of MCMV infection by NIM811 (0.5 μM) examined by flow cytometry. (G) Comparison of CyPA and YY1 expression between MEF and NSPC. Each total cell lysate of a similar number of cells (104) from MEF and NSPC was compared and detected by blotting with antibody to CyPA and YY1.
FIG. 10.
Model for inhibition mechanism of Cs and NIM811 to MCMV infection. Forming of complex of Cs and CyP or complex of NIM811 and CyP inhibits CyP functions to lead the suppression of MCMV ie1/ie3 transcripts and MCMV proliferation upstream of the MCMV IE-promoter (pretranscription level) (red). This complex does not inhibit AP-1, NF-κB, or MCMV IE promoter activity in NSPC (black line). In T cells, the Cs/CyP or FK506/FKBP complex directly binds to calcineurin and inhibits the phosphatase activity of calcineurin, resulting in the silencing of transcription factor NF-AT (blue line). The inhibition of MCMV infection by Cs and NIM811 uses CyP-mediated pathway independent of calcineurin/NF-AT pathway.
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