scholarly journals Simian Varicella Virus Induces Apoptosis in Monkey Kidney Cells by the Intrinsic Pathway and Involves Downregulation of Bcl-2 Expression

2009 ◽  
Vol 83 (18) ◽  
pp. 9273-9282 ◽  
Author(s):  
Subbiah Pugazhenthi ◽  
Donald H. Gilden ◽  
Sreekala Nair ◽  
Anne McAdoo ◽  
Mary Wellish ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Active Form ◽  
Vero Cells ◽  
Monkey Kidney ◽  
Kidney Cells ◽  
Intrinsic Pathway ◽  
Caspase 9 ◽  
Simian Varicella Virus ◽  
Varicella Virus ◽  
Infected Cells

ABSTRACT Simian varicella virus (SVV) causes varicella in primates, becomes latent in ganglionic neurons, and reactivates to produce zoster. SVV produces a cytopathic effect in monkey kidney cells in tissue culture. To study the mechanism by which SVV-infected cells die, we examined markers of apoptosis 24 to 64 h postinfection (hpi). Western blot analysis of virus-infected cell lysates revealed a significant increase in the levels of the cleaved active form of caspase-3, accompanied by a parallel increase in caspase-3 activity at 40 to 64 hpi. Caspase-9, a marker for the intrinsic pathway, was activated significantly in SVV-infected cells at all time points, whereas trace levels of the active form of caspase-8, an extrinsic pathway marker, was detected only at 64 hpi. Bcl-2 expression at the mRNA and protein levels was decreased by 50 to 70% throughout the course of virus infection. Release of cytochrome c, an activator of caspase-9, from mitochondria into the cytoplasm was increased by 200% at 64 hpi. Analysis of Vero cells infected with SVV expressing green fluorescent protein (SVV-GFP) at 64 hpi revealed colocalization of the active forms of caspase-3 and caspase-9 and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining with GFP. A significant decrease in the bcl-2 mRNA levels along with an abundance of mRNA specific for SVV genes 63, 40, and 21 was seen in the fraction of Vero cells that were infected with SVV-GFP. Together, these findings indicate that SVV induces apoptosis in cultured Vero cells through the intrinsic pathway in which Bcl-2 is downregulated.

scholarly journals Array Analysis of Simian Varicella Virus Gene Transcription in Productively Infected Cells in Tissue Culture

2005 ◽  
Vol 79 (9) ◽  
pp. 5315-5325 ◽  
Author(s):  
Steven B. Deitch ◽  
Donald H. Gilden ◽  
Mary Wellish ◽  
John Smith ◽  
Randall J. Cohrs ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Cdna Probe ◽  
Vero Cells ◽  
Open Reading Frames ◽  
Simian Varicella Virus ◽  
Varicella Zoster ◽  
Varicella Virus ◽  
Culture Cells ◽  
Infected Cells ◽  
Reading Frames

ABSTRACT Simian varicella virus (SVV) is a neurotropic alphaherpesvirus of monkeys that is a model for varicella pathogenesis and latency. Like human varicella-zoster virus (VZV), SVV causes chicken pox (varicella), becomes latent in ganglia along the entire neuraxis, and reactivates to produce shingles (zoster). We developed macroarrays to determine the extent of viral transcription from all 70 predicted SVV open reading frames (ORFs) in infected cells in tissue culture. Cloned fragments (200 to 400 bp) from the 5′ and 3′ ends of each ORF were PCR amplified, quantitated, spotted onto nylon membranes, and fixed by UV cross-linking. Using a cDNA probe prepared from poly(A)+ RNA extracted from SVV-infected Vero cells at the height of the cytopathic effect (3 days after infection) and chemiluminescence for detection, transcripts corresponding to all SVV ORFs were identified. The abundance of each SVV transcript was compared with that previously demonstrated for VZV in infected tissue culture cells.

scholarly journals Cloning and Characterization of Rat Caspase-9: Implications for a Role in Mediating Caspase-3 Activation and Hippocampal Cell Death after Transient Cerebral Ischemia

2002 ◽  
Vol 22 (5) ◽  
pp. 534-546 ◽  
Author(s):  
Guodong Cao ◽  
Yumin Luo ◽  
Tetsuya Nagayama ◽  
Wei Pei ◽  
R. Anne Stetler ◽  
...  
Keyword(s):  
Neuronal Death ◽  
Hippocampal Neurons ◽  
Caspase 3 ◽  
Neuronal Survival ◽  
Global Ischemia ◽  
Adult Brain ◽  
Caspase 8 ◽  
Intrinsic Pathway ◽  
Caspase 9 ◽  
Transient Global Ischemia

Delayed hippocampal neurodegeneration after transient global ischemia is mediated, at least in part, through the activation of terminal caspases, particularly caspase-3, and the subsequent proteolytic degradation of critical cellular proteins. Caspase-3 may be activated by the membrane receptor-initiated caspase-8–dependent extrinsic pathway and the mitochondria-initiated caspase-9–dependent intrinsic pathway; however, the precise role of these deduced apoptosis-signaling pathways in activating caspase-3 in ischemic neurons remains elusive. The authors cloned the caspase-9 gene from the rat brain and investigated its potential role in mediating ischemic neuronal death in a rat model of transient global ischemia. Caspase-9 gene expression and protease activity were extremely low in the adult brain, whereas they were developmentally upregulated in newborn rats, especially at postnatal 12 weeks, a finding consistent with the theory of an essential role for caspase-9 in neuronal apoptosis during brain development. After 15-minute transient global ischemia, caspase-9 was overexpressed and proteolytically activated in the hippocampal CA1 neurons at 8 to 72 hours of reperfusion. The temporal profile of caspase-9 activation coincided with that of cytochrome c release and caspase-3 activation, but preceded CA1 neuronal death. Immunoprecipitation experiments revealed that there was enhanced formation of Apaf-1/caspase-9 complex in the hippocampus 8 and 24 hours after ischemia. Furthermore, intracerebral ventricular infusion of the relatively specific caspase-9 inhibitor N-benzyloxycarbonyl-Leu-Glu-His-Asp-fluoro-methylketone before ischemia attenuated caspase-3–like activity and significantly enhanced neuronal survival in the CA1 sector. In contrast, inhibition of caspase-8 activity had no significant effect on caspase-3 activation or neuronal survival. These results suggest that the caspase-9–dependent intrinsic pathway may be the primary mechanism responsible for the activation of caspase-3 in ischemic hippocampal neurons.

scholarly journals The mitochondrial pathway of apoptosis is triggered during feline calicivirus infection

2006 ◽  
Vol 87 (2) ◽  
pp. 357-361 ◽  
Author(s):  
Alessandro Natoni ◽  
George E. N. Kass ◽  
Michael J. Carter ◽  
Lisa O. Roberts
Keyword(s):  
Caspase 3 ◽  
Chromatin Condensation ◽  
Mitochondrial Pathway ◽  
Feline Calicivirus ◽  
Upper Respiratory Tract ◽  
Caspase 9 ◽  
The Family ◽  
Infected Cells ◽  
Upper Respiratory ◽  
Subsequent Activation

Feline calicivirus (FCV) belongs to the family Caliciviridae and is an important pathogen of the upper respiratory tract of cats. Recent studies have shown that cells infected with FCV undergo apoptosis, as evidenced by caspase activation, chromatin condensation and cleavage of poly(ADP-ribose) polymerase. Here, the upstream events were investigated in order to define the molecular mechanism of apoptosis in FCV-infected cells. It was shown that FCV induced translocation of phosphatidylserine to the cell outer membrane and release of cytochrome c from mitochondria at about 6–8 h post-infection. These events were preceded by the loss of mitochondrial membrane potential and Bax translocation from the cytosol to mitochondria between 4 and 6 h after infection. Release of cytochrome c from mitochondria triggered the activation of caspase-9 and the subsequent activation of the executioner caspase, caspase-3. These results suggest that the mitochondrial pathway of apoptosis is triggered during FCV infection.

Inhibition of vimentin synthesis and disruption of intermediate filaments in simian virus 40-infected monkey kidney cells

1984 ◽  
Vol 4 (9) ◽  
pp. 1880-1889
Author(s):  
A Ben-Ze'ev
Keyword(s):  
Intermediate Filament ◽  
Blot Hybridization ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Monkey Kidney ◽  
In Vitro Translation ◽  
Translation System ◽  
Kidney Cells ◽  
Infected Cells ◽  
Filament Network

The organization, synthesis, and phosphorylation of vimentin were studied at various times after infection of monkey kidney cells with simian virus 40. Late after infection (between 36 and 48 h postinfection) there is a dramatic reduction in vimentin synthesis that is paralleled by a specific disruption of the intermediate filament network. At the same time there is no apparent alteration of the organization or the synthesis of the actin-containing filaments and of the microtubules. The inhibition of vimentin synthesis is also reflected by the level of vimentin mRNA activity in the infected cells, as assayed in a cell-free in vitro translation system, and vimentin mRNA concentration as revealed by RNA blot hybridization to cloned vimentin cDNA. The level of vimentin phosphorylation also decreases dramatically but at a much earlier time after infection (between 14 and 24 h postinfection), when mitosis in the infected cells is blocked. Although the decrease in vimentin synthesis in simian virus 40-infected cells is paralleled by the alterations in the organization of the intermediate filament network, the phosphorylation of vimentin correlates with the cell cycle, as it does in other systems. A possible feedback control mechanism of vimentin synthesis by alterations in the organization of the intermediate filament network is discussed.

scholarly journals Inhibition of vimentin synthesis and disruption of intermediate filaments in simian virus 40-infected monkey kidney cells.

1984 ◽  
Vol 4 (9) ◽  
pp. 1880-1889 ◽  
Author(s):  
A Ben-Ze'ev
Keyword(s):  
Intermediate Filament ◽  
Blot Hybridization ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Monkey Kidney ◽  
In Vitro Translation ◽  
Translation System ◽  
Kidney Cells ◽  
Infected Cells ◽  
Filament Network

The organization, synthesis, and phosphorylation of vimentin were studied at various times after infection of monkey kidney cells with simian virus 40. Late after infection (between 36 and 48 h postinfection) there is a dramatic reduction in vimentin synthesis that is paralleled by a specific disruption of the intermediate filament network. At the same time there is no apparent alteration of the organization or the synthesis of the actin-containing filaments and of the microtubules. The inhibition of vimentin synthesis is also reflected by the level of vimentin mRNA activity in the infected cells, as assayed in a cell-free in vitro translation system, and vimentin mRNA concentration as revealed by RNA blot hybridization to cloned vimentin cDNA. The level of vimentin phosphorylation also decreases dramatically but at a much earlier time after infection (between 14 and 24 h postinfection), when mitosis in the infected cells is blocked. Although the decrease in vimentin synthesis in simian virus 40-infected cells is paralleled by the alterations in the organization of the intermediate filament network, the phosphorylation of vimentin correlates with the cell cycle, as it does in other systems. A possible feedback control mechanism of vimentin synthesis by alterations in the organization of the intermediate filament network is discussed.

scholarly journals Cryptosporidium parvum at Different Developmental Stages Modulates Host Cell Apoptosis In Vitro

2004 ◽  
Vol 72 (10) ◽  
pp. 6061-6067 ◽  
Author(s):  
Raffaella Mele ◽  
Maria Angeles Gomez Morales ◽  
Fabio Tosini ◽  
Edoardo Pozio
Keyword(s):  
Cell Culture ◽  
Host Cell ◽  
Cryptosporidium Parvum ◽  
Cell Apoptosis ◽  
Caspase 3 ◽  
Active Form ◽  
Annexin V ◽  
Apoptotic Cells ◽  
Host Cell Apoptosis ◽  
Infected Cells

ABSTRACT We studied apoptosis in a human ileocecal adenocarcinoma tumor cell line (HCT-8) infected with Cryptosporidium parvum, from 2 to 72 h postinfection (h.p.i.). At 2 h.p.i., the percentage of annexin V-positive cells in the cell culture had increased to 10% compared to 2.5% in noninfected control culture; sorted infected cells expressed mRNA of FasL, the active form of caspase 3, and high caspase 3 activity, whereas the noninfected neighboring cells sorted from the same culture showed no signs of apoptosis. At 24 h.p.i., the percentages of early (annexin V positive) and late (DNA fragment) apoptotic cells were 13 and 2%, respectively, in the entire cell culture, and these percentages were not statistically significant in comparison with those from noninfected control cultures. At this time, sorted infected cells expressed the inactive form of caspase 3, a low caspase 3 activity, and the antiapoptotic protein Bcl-2. Noninfected cells sorted from the same culture showed expression of the active form of caspase 3, a moderate caspase 3 activity, and no Bcl-2 expression. At 48 h.p.i., the percentages of early and late apoptotic cells and caspase 3 activity had increased in the total cell culture, and both sorted infected and noninfected cells showed the active form of caspase 3. These results show that C. parvum, depending on its developmental stage, can inhibit (at the trophozoite stage) or promote (at the sporozoite and merozoite stages) host cell apoptosis, suggesting that it is able to interact with and regulate the host-cell gene expression.

scholarly journals Simian varicella virus gene 28 and 29 promoters share a common upstream stimulatory factor-binding site and are induced by IE62 transactivation

2006 ◽  
Vol 87 (6) ◽  
pp. 1501-1508 ◽  
Author(s):  
Yang Ou ◽  
Wayne L. Gray
Keyword(s):  
Binding Site ◽  
Intergenic Region ◽  
Latent Infection ◽  
Vero Cells ◽  
Upstream Stimulatory Factor ◽  
Simian Varicella Virus ◽  
Factor Binding Site ◽  
Factor Binding ◽  
Varicella Virus ◽  
Stimulatory Factor

Simian varicella virus (SVV) is a neurotropic alphaherpesvirus that causes a natural, varicella-like disease in non-human primates. After resolution of the primary disease, SVV, like its human counterpart, varicella-zoster virus (VZV), establishes latent infection in the neural ganglia of the host. In this study, gene expression of SVV open reading frames (ORFs) 28 and 29, which encode the viral DNA polymerase and DNA-binding protein, respectively, was characterized during lytic infection of Vero cells. The results indicate that the intergenic region controlling gene 28 and 29 expression includes overlapping, divergent promoters. The ORF 28 and 29 promoters are active in SVV-infected Vero cells, but not in uninfected cells. The SVV immediate-early gene 62 (IE62) product transactivates ORF 28 and 29 expression, and a cellular upstream stimulatory factor-binding site is important for efficient IE62 induction of genes 28 and 29. DNA sequence analysis of the 185 bp intergenic region identified putative cellular transcription factor-binding sites. Transcriptional analysis mapped ORF 28 and 29 RNA start sites. A recombinant SVV was employed to demonstrate that the ORF 29 promoter can express a heterologous gene (green fluorescent protein) when inserted into a novel site (the ORF 12/13 intergenic region) within the SVV genome. The findings demonstrate similarities between SVV and VZV ORF 28/29 expression and indicate that the simian varicella model may be useful to investigate the differential regulation of viral genes during lytic and latent infection.

scholarly journals Functional Analysis of the Inhibitor of Apoptosis (iap) Gene Carried by the Entomopoxvirus of Amsacta moorei

2005 ◽  
Vol 79 (4) ◽  
pp. 2335-2345 ◽  
Author(s):  
Qianjun Li ◽  
Peter Liston ◽  
Richard W. Moyer
Keyword(s):  
Caspase 3 ◽  
Autographa Californica ◽  
Inhibitor Of Apoptosis ◽  
Intrinsic Pathway ◽  
Abortive Infection ◽  
Caspase 9 ◽  
Rapid Induction ◽  
Human Caspase ◽  
Proapoptotic Gene

ABSTRACT The entomopoxvirus from Amsacta moorei (AmEPV) contains none of the commonly recognized vertebrate poxvirus apoptotic suppressor genes. However, AmEPV carries a single inhibitor of apoptosis (iap) gene (AMViap) not present in vertebrate poxviruses. The AMViap gene was active when coexpressed with the Drosophila proapoptotic gene hid in Ld652 cells and can rescue cells from apoptosis as shown by increased number of surviving cells and reduced levels of caspase-3-like activity. We also showed that expression of the AMViap gene rescued polyhedron production in Autographa californica M nucleopolyhedrovirus (AcMNPV)Δp35-infected Sf9 cells during an otherwise abortive infection induced by apoptosis. Surprisingly, deletion of the AMViap gene from the AmEPV genome led to only a modest (10-fold) loss of virion production in infected Ld652 cells, indicating that the AMViap gene is nonessential for virus replication under these conditions. However, infection of Ld652 cells by AmEPV lacking a functional iap gene led to a more rapid induction of cytotoxicity and increased levels of caspase-3-like activity. Similar results were observed and were more pronounced in infected Sf9 and S2 cells. The purified AMVIAP protein also inhibits the enzymatic activities of human caspase-9 and caspase-3 in vitro. Our results indicate that while the AMViap gene was active in controlling apoptosis through the intrinsic pathway, the virus likely encodes additional proteins that also regulate apoptosis.

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