Simian virus 40 infection triggers a balanced network that includes apoptotic, survival, and stress pathways

Abstract

The infection process by simian virus 40 (SV40) and entry of its genome into nondividing cells are only partly understood. Infection begins by binding to GM1 receptors at the cell surface, cellular entry via caveolar invaginations, and trafficking to the endoplasmic reticulum, where the virus disassembles. To gain a deeper insight into the contribution of host functions to this process, we studied cellular signaling elicited by the infecting virus. Signaling proteins were detected by Western blotting and immunofluorescence staining. The study was assisted by a preliminary proteomic screen. The contribution of signaling proteins to the infection process was evaluated using specific inhibitors. We found that CV-1 cells respond to SV40 infection by activating poly(ADP-ribose) polymerase 1 (PARP-1)-mediated apoptotic signaling, which is arrested by the Akt-1 survival pathway and stress response. A single key regulator orchestrating the three pathways is phospholipase C-gamma (PLCgamma). The counteracting apoptotic and survival pathways are robustly balanced as the infected cells neither undergo apoptosis nor proliferate. Surprisingly, we have found that the apoptotic pathway, including activation of PARP-1 and caspases, is absolutely required for the infection to proceed. Thus, SV40 hijacks the host defense to promote its infection. Activities of PLCgamma and Akt-1 are also required, and their inhibition abrogates the infection. Notably, this signaling network is activated hours before T antigen is expressed. Experiments with recombinant empty capsids, devoid of DNA, indicated that the major capsid protein VP1 alone triggers this early signaling network. The emerging robust signaling network reflects a delicate evolutionary balance between attack and defense in the host-virus relationship.

FIG. 1.

Activation of PARP-1 and Caspases. (A) PARP-1 was immunoprecipitated from total cell extracts at the designated time points with anti-PARP-1 antibodies. Western detection was performed with anti-PAR antibody. PARP-1 is 116 kDa. The arrow points to active, poly(ADP-ribosylated) PARP-1. IgG served as a loading control. (B) PARP-1 is required for the infection to proceed. PARP^+/+ and PARP^−/− cell lines were assayed for T-antigen expression 48 h after infection. Nuclear extracts were prepared, and T antigen was analyzed by Western blotting. Emerin detection served as a loading control. In this experiment we used an exceptionally high MOI of 500 because mouse cells are nonpermissive for productive SV40 infection as they do not support T-antigen-dependent viral DNA replication (9). (C) Cleavage by caspases. The Western blot shows the uncleaved 116-kDa protein and the 89-kDa caspase cleavage product. Etoposide and cisplatin were added at 15 and 30 μM. Western blotting was performed 48 h after the addition of etoposide and cisplatin and 6 h after infection by SV40. The arrow indicates active, poly(ADP-ribosylated) PARP-1. (D) Cellular distribution of PARP-1. Cells were fixed with 4% formaldehyde at 6 h postinfection and stained with polyclonal anti-PARP-1 and Cy3. Images in the top panels were taken at magnification of ×60 with zoom 3; lower images are at a magnification of ×40. Control and infected cells were at the same confluence. (E) Detection of active PARP-1 in total cell lysates. Western blotting was performed following loading of 20 μg of total protein per lane. The arrow points to higher-molecular-weight species of PARP-1 with extensive poly(ADP-ribosylation). The 89-kDa caspase cleavage product is significantly reduced in the presence of a 70 μM concentration of the pan-caspase inhibitor Z-VAD-FMK (Z-V-F). This experiment was reproduced five times. (F) Quantification of PARP-1 and active poly(ADP-ribosylated) PARP-1 following SV40 infection.

FIG. 2.

Caspase activation. (A) The left panels show caspases that are activated following SV40 infection, as indicated by the cleavage products: 20-kDa fragment of caspase-10, 14.5-kDa fragment of caspase-6, and 17-kDa fragment of caspase-3. Arrows on the right panel point to the expected locations of cleavage products of caspase-9 (37 kDa), caspase-7 (20 kDa), and caspase-8 (18 kDa). (B) Activation of caspase-6 by caspase-10. Caspase-6 cleavage is inhibited following the addition of caspase-10 inhibitor (left). Positive control for caspase-6 activation by etoposide treatment (30 μM) is seen on the right. The cells were harvested 48 h after etoposide addition when ∼50% were dead. (C) Expression of T antigen in infected CV-1 cells in the presence of the following inhibitors: all caspases, 70 μM Z-VAD-FMK; caspase-3, 20 μM Ac-DMQD-CHO; caspase-6, 20 μM aldehyde; caspase-10, 5 μM Z-AEVD-FMK. Images were obtained by fluorescence microscopy at a magnification of ×40.

FIG. 3.

SV40-infected cells do not undergo apoptosis or DNA damage during the first 24 h following infection. SV40-infected cells and controls were assayed by TUNEL staining. SV40-infected cells were assayed at the indicated time points. Etoposide-treated cells are shown at 48 h after addition. At that time ∼75% of the cells were TUNEL positive, indicating DNA damage, and ∼50% were dead, measured by trypan blue staining. DNase I-treated cells were photographed 1 h after Triton X-100 permeabilization.

FIG. 4.

Activation of survival pathway and stress response. (A) Western blotting showing activation of Akt-1 by phosphorylation (top), upregulation of Akt-1 protein (middle), and inhibition of BAD and caspase-9 by phosphorylation. Lamin B served as a loading control. (B) Detection of phospho-Akt-1 by immunostaining. Cells were fixed with 4% formaldehyde at 6 h postinfection and stained with polyclonal anti-P-Ser473-Akt-1 and Cy3. Images were taken at a magnification of ×40. (C) Modulations of Hsp/c70 protein level. HS indicates a positive control, showing the heat shock response of CV-1 cells that were placed at 55° for 1 h. Lamin B served as a loading control. (D) Confocal microscopy of Hsp/c70. Cells were fixed at the designated time points and stained with monoclonal anti-Hsp/c70 and Cy5. Photographs were taken at a magnification of ×60 with zoom 2.

FIG. 5.

Infected cells do not proliferate in the absence of T antigen. (A) Flow cytometry of cells infected with the constructs designated on the right. The cells were fixed in 100% ethanol, treated with RNase for 1 h, and analyzed by fluorescence-activated cell sorting (FACS) following staining with propidium iodide. (B) Graphical representation of S-phase cells; data are the average of three independent infection experiments.

FIG. 6.

PLCγ is a key element in SV40-induced signaling. (A) Activation of PLCγ, seen by appearance of the phosphorylated species. To clarify the bands, this image was auto level adjusted using Adobe Photoshop CS2. Quantification of PLCγ and its phosphorylated form is shown below. (B) Quantification of the levels of active PARP-1, P-Akt-1, and Hsp/c70 in the presence and absence of the PLCγ inhibitor U73122 (10 μM). The level of P-Akt-1 was also measured in the presence of PI3K inhibitor LY294002 (50 μM). The data points were normalized relative to lamin B, which served as a loading control. The average of three experiments with standard deviations are shown for each data point. (C) Expression of T antigen in infected cells in the presence of the following inhibitors: for PLCγ, 10 μM U73122; PI3K, 50 μM LY294002; Akt-1, 20 μM Tricibine V. All the inhibitors were added 1 h before adsorption. At these concentrations no cytotoxicity was observed during 3 days. Images were taken after 2 days.

FIG. 7.

SV40-triggered signaling network. Thick arrows designate pathways essential for productive SV40 infection, as confirmed by specific inhibitors. Thin arrows indicate known pathways, not confirmed in the present study. The role of GM1 in the activation of PLCγ is hypothetical.