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Pseudorabies virus induces apoptosis in tissue culture cells

Summary.  The process of cell death as a result of exposure to pseudorabies virus (PRV) in cultured cells was examined and specific features characteristic of apoptosis were observed. At early times of infection, externalization of membrane phospholipid phophatidylserine was detected by flow cytometry analysis. During the infection process, caspase 3-like protease activity was induced and the activity increased in a time dependent manner. Cellular DNA degradation was demonstrated by agarose gel electrophoresis. Morphologic changes of the nucleus that included chromatin condensation and margination to the periphery of the nucleus were evident in electron microscopy analysis. These biochemical and morphologic changes demonstrated that, during PRV replication, the host cell was induced to undergo apoptosis.

A wide variety of viruses have been reported to induce apoptosis [23,27] and an increasing number of viral components have been shown to induce or inhibit apoptosis [13,19]. The anti-apoptotic activities enable the virus to block premature death of the host cell for the propagation of progeny viruses or establishment of persistence, and the pro-apoptotic activities ensure efficient dissemination of progeny viruses to neighboring cells. Pseudorabies virus (PRV) belongs to the alphaherpes virus subfamily and the association of PRV infection with apoptosis has not been established. In this work, we showed that PRV induces programmed cell death in Madin-Darby bovine kidney (MDBK) cells.
The Indiana-Funkhauser strain of PRV was used to infect MDBK cells cultivated in Eagle's minimum essential medium supplemented with 10% fetal bovine serum. The experiments were carried out at a multiplicity of infection (m.o.i.) of 10 and the virus titer was quantitated by a plaque assay previously described [5]. One of the earliest morphologic changes during apoptosis is the translocation of membrane phospholipid phophatidylserine (PS) from the inner to the outer leaflet of the plasma membrane [17]. Simultaneous staining of cells with annexin V, which binds PS, and propidium iodide (PI), which is excluded by viable cells, allows differentiation of cells undergoing apoptosis (annexin V positive, PI negative) or necrosis (annexin V positive, PI positive) [28]. Previously, we have demonstrated that progeny PRV start to appear 8 h after infection of MDBK cells [4]. Therefore, we analysed the externalization of PS of MDBK cells during the early stages of PRV infection. Infected cultures at different times postinfection (p.i.) were analysed by flow cytometry after staining with FITC-annexin V and/or PI. Dexamethasone, which has been widely used to induce apoptosis in many cell types, was included as a positive control [26]. In comparison to untreated cells, the dexamethasone-treated and PRV-infected cultures showed an increased number of annexin V positive and PI negative cells soon after exposure to dexamethasone or PRV (Fig. 1), indicating an increased externalization of PS in viable cells.
During apoptosis, one or more cascades of proteolytic enzymes are activated. Caspase 3, which is a key component of the apoptotic pathway, is responsible for proteolytic cleavage of a large number of substrates [7,22]. A commercially available kit was used to monitor caspase 3-like activity in mock-infected and PRV-infected MDBK cells. Two independent experiments were conducted and similar results were obtained. One of the experiments is presented in Fig. 2 and the results demonstrated that as PRV infection progressed, caspase 3-like activity increased, as shown by cleavage of the substrate, DEVD-pNA. At all time points tested, caspase 3-like activity was higher in PRV-infected cell lysates than in mock-infected cell lysates. The caspase 3-like activity was significantly reduced in the presence of the caspase 3 inhibitor, DEVD-fmk.
Degradation of cellular DNA inside a cell is a hallmark of apoptosis. Experiments were carried out to visualize cellular DNA fragmentation triggered by PRV. Total cell DNA was isolated from PRV-infected MDBK cells at different times p.i. and subjected to agarose-gel electrophoresis. The uninfected cell DNA appeared as high molecular weight form and the PRV-infected cell contained a significant proportion of lower molecular weight DNA, which appeared as a  In this assay, a colorimetric tetra-peptide labeled with p-nitroanilide (DEAD-pNA) is cleaved by proteases with caspase-3-like activity and samples are read in a spectrophotometer at 405 nm [11]. The experiment was carried out in duplicate and DEVD-fmk was included in one of the assays  infection with transmissible gastroenteritis virus (TGEV) [8]. The results showed that both TGEV and PRV induced DNA degradation in ST cells. However, internucleosomal DNA laddering that was prominent with the TGEV-infected ST cells was not readily detected with the PRV-infected cells (Fig. 3b).
Morphologic changes associated with apoptosis can also be visualized by transmission electron microscopy (EM). At different times p.i., MDBK cells exposed to PRV were prepared for EM examination. Nuclear chromatin condensation and margination, commonly associated with apoptosis, were observed in the infected cells. The EM images of uninfected and PRV-infected MDBK cells were presented for comparison (Fig. 4). The condensed chromatin appeared as fragmented electron dense masses that were marginalized to the periphery of the nucleus. Intra-cellular and extra-cellular viruses associated with PRV-infected cells were readily observed at 12 h p.i. At 48 h p.i., this chromatin condensation pattern was evident in all cells where intracellular viral particles were observed.
In this report, we examined whether PRV can induce apoptosis in MDBK cells during virus replication. Several features characteristic of apoptosis were detected. The morphologic changes detected by flow cytometry and electron microscopy showed externalization of PS and chromatin condensation and margination, respectively. Cellular DNA fragmentation, considered a hallmark of programmed cell death as the result of endonucleolytic activity, can exist in several forms, including large fragments of 30-500 kilo-bp, 180-200 bp oligomeric internucleosomal fragments and single-stranded DNA breaks [3,21,29,30]. The relationships between these types of DNA degradation are not clear. Agarose gel electrophoresis analysis demonstrated that PRV could induce DNA fragmentation in MDBK as well as in ST cells. However, PRV did not induce a distinct oligonucleosomal DNA cleavage pattern as exhibited by other alphaherpes viruses, e.g. herpes simplex virus type 1 [15,16], varicella-zoster virus [24] and bovine herpes virus type 1 [12]. Activation of caspase 3-like activity, commonly associated with programmed cell death, was detected. Our experiments showed that caspase 3-like activity increased in a time dependent manner after PRV infection of MDBK cells. Thus, the morphologic and biochemical results from the above experiments demonstrated that PRV can induce apoptosis in cultured cells.
PRV can undergo a lytic or a latent infection when exposed to a susceptible host. As shown in this study, progeny viruses were produced during a lytic infection which resulted in death of the host cell. During a latent infection, the host cell survives and harbors the PRV genome. As such, the apoptotic process inducible by PRV must be inhibited during the establishment of a latent infection.
Therefore, understanding how PRV induces apoptosis will provide insights into the events involved in PRV latency.