(A) The collected cytosolic proteins were probed with antibodies specific to mitochondrial marker protein COX IV to verify the absence of contamination with mitochondrial proteins. intestine tissues of pigs inoculated with human NV, and progressive histopathological lesions were detected in immunodeficient mice naturally infected with MNV (13, 49). In addition, infected cells dying by apoptosis were detected in multiple organs of the RHDV-infected rabbits. KRT17 The finding of apoptotic cells at sites with pronounced tissue damage suggested involvement of apoptosis in pathogenesis of the RHDV-induced disease (3, 28). Although the functional importance of apoptosis in calicivirus replication is PHA690509 not understood, it has been suggested that caliciviruses use this self-destructive cellular process to facilitate the dissemination of viral progeny in the host (3, 47). The molecular mechanisms employed by caliciviruses to trigger the apoptotic cascade still remain unknown; however, it has been shown that induction of apoptosis in FCV-infected cells involves the mitochondrial pathway (35). The intrinsic or mitochondrial apoptotic pathway is dependent on the release into the cytosol of apoptotic factors sequestered by the mitochondria, such as cytochrome and Smac/DIABLO, and is directly PHA690509 linked to permeability of the organelle outer membrane. Loss of the mitochondrial membrane integrity and cytochrome release result in apoptosome formation PHA690509 and processing of procaspase-9 followed by downstream activation of executioner caspase-3. The catalytic activity of caspase-9 can be inhibited by a group of proteins known as inhibitors of apoptosis (IAPs). The 16.5-kDa mouse survivin protein encoded by the BIRC5 gene is the smallest member of the IAP family. Survivin interacts with cofactor molecules, such as the X-linked mammalian IAP protein (XIAP) and the hepatitis B virus X-interacting protein (HBXIP), to specifically inhibit caspase-9 activation (4, 6). It has been shown that a number of viruses are capable of upregulating survivin in order to delay apoptosis in infected cells (9, 11, 39, 51, 53, 54). In this study, we first established that replication-dependent apoptosis occurred in MNV-1-infected RAW264.7 cells. Then, in order to study the apoptotic cascade in detail, we investigated the expression profiles of MNV-1-infected versus mock-infected RAW264.7 cells. Here, we report that the replication of a virus (MNV-1) in cultured cells is associated with downregulation of survivin expression and results in induction of apoptosis through the mitochondrial pathway. MATERIALS AND METHODS Cells and virus. The murine macrophage-like cell line RAW264.7 was obtained from ATCC (Manassas, VA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing penicillin (250 systems/ml) and streptomycin (250 g/ml) and supplemented with 5% heat-inactivated fetal bovine serum. A characterized plaque-purified isolate of MNV-1 previously, specified MNV-1.CW1P3, was used seeing that the foundation of trojan PHA690509 in this research (46). Plaque and Propagation titration assays of MNV-1 in Organic264.7 cells were completed as defined previously (46). The MNV-1 virions had been purified using ultracentrifugation within a CsCl gradient regarding to protocols released somewhere else (50). The purified trojan was dialyzed against 1,000 amounts of phosphate-buffered saline (PBS) right away. For time training course tests, the viral share was serially diluted in DMEM to get the desired inocula for the multiplicity of an infection (MOI) of 4. Organic264.7 cells (5 107) were inoculated with either diluted MNV-1 or DMEM (mock an infection control), incubated at 37C, and collected at each best period stage for even more American blot or RNA appearance analyses. DAPI staining. To imagine nuclear morphology, mock- and MNV-1-contaminated Organic264.7 cells were fixed with methanol at 28 h postinfection (h p.we.) and incubated with 4,6-diamidino-2-phenylindole (DAPI; Roche Molecular Biochemicals, Indianapolis, IN) staining alternative (1 g/ml in PBS buffer) for 20 min at area temperature. After many washes with PBS buffer, stained cells had been visualized using.