SARS–associated Coronavirus Replication in Cell Lines

Kaye, Matthew; Druce, Julian; Tran, Thomas; Kostecki, Renata; Chibo, Doris; Morris, Jessica; Catton, Mike; Birch, Chris

doi:10.3201/eid1201.050496

Cited by 127 publications

(136 citation statements)

References 12 publications

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“…In order to confirm that the interaction between nsp8 and ORF6 occurs during SARS-CoV infection, co-immunoprecipitation experiments were performed with lysates obtained from SARS-CoV infected cells. Infection of Vero E6 cells with an isolate of SARS-CoV (strain HKU 39849) was carried out in a physical containment level 4 (PC4) laboratory as previously described (Kaye et al, 2006). The cells were harvested at 0 or 24 h post-infection (h.p.i.)…”

Section: Resultsmentioning

confidence: 99%

“…For co-immunoprecipitation experiments performed with SARS-CoV infected Vero E6 cells, Vero E6 cells were plated in 25 cm 2 flasks and infected with an isolate of SARS-CoV (strain HKU39849) as previously described (Kaye et al, 2006). The cells were harvest at 0 or 24 h.p.i.…”

Section: Expression Of Viral Proteins In Mammalian Cells and Co-immuomentioning

confidence: 99%

“…Indirect immunofluorescence was performed using SARS-CoV infected Vero E6 cells as previously described (Tan et al, 2004c). Briefly, Vero E6 cells were plated onto 4-well chamber slides (Lab-Tek) and infected with an isolate of SARS-CoV (strain HKU39849) as previously described (Kaye et al, 2006). 42 h post-infection, chamber slides were washed twice in phosphate-buffered saline (PBS); cells were then fixed and permeabilized with methanol for 5 min and subsequently subjected to gamma-irradiation to neutralize infectivity of the virus.…”

Section: Immunofluorescence Analysismentioning

confidence: 99%

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The nonstructural protein 8 (nsp8) of the SARS coronavirus interacts with its ORF6 accessory protein

et al. 2007

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Kumar, P., et al. (2007). The nonstructural protein 8 (nsp8) of the SARS coronavirus interacts with its ORF6 accessory protein. Virology, 366(2): 293-303 http://dx. Abstract In order to investigate the genomic organization of the Trichoplusia ni Single Capsid Nucleopolyhedrovirus (TnSNPV), a 2,966 basepairs (bp) genomic fragment was sequenced. The fragment was found to contain five open reading frames (ORFs) homologous to baculovirus genes, including p26, fibrillin (p10), AcMNPV ORF-29, late expression factor 6 (lef-6) and the C-terminal portion of p74, on either strand of DNA. Predicted amino acid sequences for the ORFs were compared and identity values of between 12% and 54% were observed. TnSNPV has previously been tentatively identified as a member of the Group II NPVs. Clustering and arrangement of the TnSNPV genes were similar to the clustering reported for SeMNPV, confirming TnSNPV as a Group II NPV.

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Section: Resultsmentioning

confidence: 99%

Section: Expression Of Viral Proteins In Mammalian Cells and Co-immuomentioning

confidence: 99%

Section: Immunofluorescence Analysismentioning

confidence: 99%

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The nonstructural protein 8 (nsp8) of the SARS coronavirus interacts with its ORF6 accessory protein

et al. 2007

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“…In this system, there was increased caspase-8 activation, but no change in Bad or Bcl-2 levels, suggesting that the death-receptor (extrinsic) apoptosis pathway was activated (Law et al, 2005). We chose to study the apoptotic activity of the 3a protein in the Huh7 human hepatocellular carcinoma cell line, as liver cells have been shown to be permissive for SARS-CoV infection and replication (Kaye, 2006). Furthermore, these cells are negative for caveolin, unlike Vero or A549 cells (Damm et al, 2005).…”

Section: Sars-cov 3a Protein Activates the Intrinsic Pathway Of Apoptmentioning

confidence: 99%

Severe acute respiratory syndrome coronavirus 3a protein activates the mitochondrial death pathway through p38 MAP kinase activation

Padhan

Minakshi

Towheed

et al. 2008

Journal of General Virology

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The molecular mechanisms governing severe acute respiratory syndrome coronavirus-induced pathology are not fully understood. Virus infection and some individual viral proteins, including the 3a protein, induce apoptosis. However, the cellular targets leading to 3a protein-mediated apoptosis have not been fully characterized. This study showed that the 3a protein modulates the mitochondrial death pathway in two possible ways. Activation of caspase-8 through extrinsic signal(s) caused Bid activation. In the intrinsic pathway, there was activation of caspase-9 and cytochrome c release from the mitochondria. This was the result of increased Bax oligomerization and higher levels of p53 in 3a protein-expressing cells, which depended on the activation of p38 MAP kinase (MAPK) in these cells. For p38 activation and apoptosis induction, the 3a cytoplasmic domain was sufficient. In direct Annexin V staining assays, the 3a protein-expressing cells showed increased apoptosis that was attenuated with the p38 MAPK inhibitor SB203580. A block in nuclear translocation of the STAT3 transcription factor in cells expressing the 3a protein was also observed. These results have been used to present a model of 3a-mediated apoptosis. INTRODUCTIONThe aetiological agent for severe acute respiratory syndrome (SARS) was identified as a novel coronavirus (SARS-CoV) (Peiris et al., 2003). SARS-CoV has a polyadenylated, positive-sense RNA genome of approximately 30 kb (Marra et al., 2003). In addition to the prototypic coronavirus genes, the SARS-CoV genome also contains nine unique open reading frames (ORFs) (Marra et al., 2003). Of these, orf3a is the largest and encodes a protein of 274 aa, variously called ORF3A (Ito et al., 2005), X1 (Rota et al., 2003) or U274 (Tan et al., 2004b). The 3a protein has been predicted to contain an N-terminal signal peptide followed by three transmembrane domains and a C-terminal cytoplasmic domain of approximately 150 aa (Zeng et al., 2004).The 3a protein is associated with virus particles produced following infection of Vero E6 or Caco-2 cells (Ito et al., 2005;Shen et al., 2005) and can assemble into virus-like particles when co-expressed with the membrane and envelope proteins in insect cells (Shen et al., 2005). In vitro studies have also shown the 3a protein to interact with the viral envelope, membrane and spike proteins (Tan et al., 2004b;Tan, 2005) and the cellular protein caveolin-1 (Padhan et al., 2007). A deletion of orf3a was shown to reduce virus titres, but not to eliminate virus replication (Yount et al., 2005), and convalescent sera from SARS patients have antibodies to the 3a protein (Tan et al., 2004a), suggesting that it is expressed during virus infection of the host. The 3a protein was shown to upregulate the expression of fibrinogen in A549 lung epithelial cells and to possess an ionchannel activity selective for monovalent cations (Lu et al., 2006). Ectopic expression of the 3a protein has been shown to induce apoptosis in Vero E6 cells through activation of caspase-8, chromatin c...

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“…The disease was first documented during late 2002 and the outbreak was effectively controlled by mid-2003 through rigorous quarantine measures (Zhong et al, 2003). However, the ability of the virus in infecting multiple cell types (Hattermann et al, 2005;Kaye, 2006) and animals (Martina et al, 2003) implied the risk of viral circulation in animal reservoirs and hence the re-emergence of the virus in human population. Understanding the molecular mechanisms of viral pathogenesis may provide important information for rational design of antiviral drugs, which may be important to combat against another possible SARS epidemic.…”

Section: Introductionmentioning

confidence: 99%

Transcriptional profiling of Vero E6 cells over-expressing SARS-CoV S2 subunit: Insights on viral regulation of apoptosis and proliferation

et al. 2008

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We have previously demonstrated that over-expression of spike protein (S) of severe acute respiratory syndrome coronavirus (SARS-CoV) or its C-terminal subunit (S2) is sufficient to induce apoptosis in vitro. To further investigate the possible roles of S2 in SARS-CoV-induced apoptosis and pathogenesis of SARS, we characterized the host expression profiles induced upon S2 over-expression in Vero E6 cells by oligonucleotide microarray analysis. Possible activation of mitochondrial apoptotic pathway in S2 expressing cells was suggested, as evidenced by the up-regulation of cytochrome c and down-regulation of the Bcl-2 family anti-apoptotic members. Inhibition of Bcl-2-related anti-apoptotic pathway was further supported by the diminution of S2-induced apoptosis in Vero E6 cells over-expressing Bcl-xL. In addition, modulation of CCN E2 and CDKN 1A implied the possible control of cell cycle arrest at G1/S phase. This study is expected to extend our understanding on the pathogenesis of SARS at a molecular level.

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SARS–associated Coronavirus Replication in Cell Lines

Abstract: Virus can replicate in several common cell lines, sometimes without cytopathic effect.

Cited by 127 publications

References 12 publications

The nonstructural protein 8 (nsp8) of the SARS coronavirus interacts with its ORF6 accessory protein

The nonstructural protein 8 (nsp8) of the SARS coronavirus interacts with its ORF6 accessory protein

Severe acute respiratory syndrome coronavirus 3a protein activates the mitochondrial death pathway through p38 MAP kinase activation

Transcriptional profiling of Vero E6 cells over-expressing SARS-CoV S2 subunit: Insights on viral regulation of apoptosis and proliferation

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