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Classic swine fever virus NS2 protein leads to the induction of cell cycle arrest at S-phase and endoplasmic reticulum stress.

Tang QH, Zhang YM, Fan L, Tong G, He L, Dai C - Virol. J. (2010)

Bottom Line: A significant increase in cyclin A transcriptional levels was observed in NS2-expressing cells but was accompanied by a concomitant increase in the proteasomal degradation of cyclin A protein.Therefore, the induction of cell cycle arrest at S-phase by CSFV NS2 protein is associated with increased turnover of cyclin A protein rather than the down-regulation of cyclin A transcription.These findings provide novel information on the function of the poorly characterized CSFV NS2 protein.

View Article: PubMed Central - HTML - PubMed

Affiliation: College of Veterinary Medicine, Northwest A & F University, Yangling, Shaanxi, China.

ABSTRACT

Background: Classical swine fever (CSF) caused by virulent strains of Classical swine fever virus (CSFV) is a haemorrhagic disease of pigs, characterized by disseminated intravascular coagulation, thrombocytopoenia and immunosuppression, and the swine endothelial vascular cell is one of the CSFV target cells. In this report, we investigated the previously unknown subcellular localization and function of CSFV NS2 protein by examining its effects on cell growth and cell cycle progression.

Results: Stable swine umbilical vein endothelial cell line (SUVEC) expressing CSFV NS2 were established and showed that the protein localized to the endoplasmic reticulum (ER). Cellular analysis revealed that replication of NS2-expressing cell lines was inhibited by 20-30% due to cell cycle arrest at S-phase. The NS2 protein also induced ER stress and activated the nuclear transcription factor kappa B (NF-kappaB). A significant increase in cyclin A transcriptional levels was observed in NS2-expressing cells but was accompanied by a concomitant increase in the proteasomal degradation of cyclin A protein. Therefore, the induction of cell cycle arrest at S-phase by CSFV NS2 protein is associated with increased turnover of cyclin A protein rather than the down-regulation of cyclin A transcription.

Conclusions: All the data suggest that CSFV NS2 protein modulate the cellular growth and cell cycle progression through inducing the S-phase arrest and provide a cellular environment that is advantageous for viral replication. These findings provide novel information on the function of the poorly characterized CSFV NS2 protein.

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The analysis of the stages of cell division of expressing CSFV NS2 protein by flow cytometry. (A) Histograms from flow cytometry data for propidium iodide staining. (B) Analysis of the percentage of cells in each phase of the cell cycle from flow cytometry data.
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Figure 3: The analysis of the stages of cell division of expressing CSFV NS2 protein by flow cytometry. (A) Histograms from flow cytometry data for propidium iodide staining. (B) Analysis of the percentage of cells in each phase of the cell cycle from flow cytometry data.

Mentions: To determine whether the growth inhibition of CSFV NS2-expressing cells was due to the arrest of the cell cycle at a certain phase(s) of cell division, flow cytometric analysis was performed based on DNA content in nuclei stained with PI. The proportions of G0/G1 phase, S-phase and G2/M phases for the control cells were 57.96%, 35.98% and 6.06%, respectively. For SUVEC expressing GFP, the proportions of the phases were G0/G1: 60.84%, S-phase: 34.19%, and G2/M: 4.96%, whereas for GFP-NS2-expressing SUVEC stable cells, the proportions were G0/G1: 52.96%, S-phase: 42.29% and G2/M: 4.76%, consistently, for the NS2-GFP-expressing SUVEC stable cells, the proportions were G0/G1: 54.40%, S-phase: 41.40% and G2/M: 4.19% (Fig. 3A). Apoptosis was also analyzed by flow cytometry but no differences were observed between untransfected cells or pEGFP-C1 transfected cells and cells expressing GFP-NS2 or NS2-GFP fusion protein. A sub-G0/G1 peak was not detected by flow cytometry for both the GFP-NS2-expressing, NS2-GFP-expressing and control cells and suggests that the CSFV NS2 protein induced cell cycle arrest in the S-phase, rather than inducing apoptosis. The results showed that relative to control cells, NS2 expression causes a significant increase in the proportion of cells in the S-phase accompanied by a decrease in the cell proportion in the G0/G1 phase (Fig. 3B). Taken together, these results strongly suggest that NS2 protein causes the inhibition of cell growth by the induction of cell cycle arrest in the S-phase.


Classic swine fever virus NS2 protein leads to the induction of cell cycle arrest at S-phase and endoplasmic reticulum stress.

Tang QH, Zhang YM, Fan L, Tong G, He L, Dai C - Virol. J. (2010)

The analysis of the stages of cell division of expressing CSFV NS2 protein by flow cytometry. (A) Histograms from flow cytometry data for propidium iodide staining. (B) Analysis of the percentage of cells in each phase of the cell cycle from flow cytometry data.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2819037&req=5

Figure 3: The analysis of the stages of cell division of expressing CSFV NS2 protein by flow cytometry. (A) Histograms from flow cytometry data for propidium iodide staining. (B) Analysis of the percentage of cells in each phase of the cell cycle from flow cytometry data.
Mentions: To determine whether the growth inhibition of CSFV NS2-expressing cells was due to the arrest of the cell cycle at a certain phase(s) of cell division, flow cytometric analysis was performed based on DNA content in nuclei stained with PI. The proportions of G0/G1 phase, S-phase and G2/M phases for the control cells were 57.96%, 35.98% and 6.06%, respectively. For SUVEC expressing GFP, the proportions of the phases were G0/G1: 60.84%, S-phase: 34.19%, and G2/M: 4.96%, whereas for GFP-NS2-expressing SUVEC stable cells, the proportions were G0/G1: 52.96%, S-phase: 42.29% and G2/M: 4.76%, consistently, for the NS2-GFP-expressing SUVEC stable cells, the proportions were G0/G1: 54.40%, S-phase: 41.40% and G2/M: 4.19% (Fig. 3A). Apoptosis was also analyzed by flow cytometry but no differences were observed between untransfected cells or pEGFP-C1 transfected cells and cells expressing GFP-NS2 or NS2-GFP fusion protein. A sub-G0/G1 peak was not detected by flow cytometry for both the GFP-NS2-expressing, NS2-GFP-expressing and control cells and suggests that the CSFV NS2 protein induced cell cycle arrest in the S-phase, rather than inducing apoptosis. The results showed that relative to control cells, NS2 expression causes a significant increase in the proportion of cells in the S-phase accompanied by a decrease in the cell proportion in the G0/G1 phase (Fig. 3B). Taken together, these results strongly suggest that NS2 protein causes the inhibition of cell growth by the induction of cell cycle arrest in the S-phase.

Bottom Line: A significant increase in cyclin A transcriptional levels was observed in NS2-expressing cells but was accompanied by a concomitant increase in the proteasomal degradation of cyclin A protein.Therefore, the induction of cell cycle arrest at S-phase by CSFV NS2 protein is associated with increased turnover of cyclin A protein rather than the down-regulation of cyclin A transcription.These findings provide novel information on the function of the poorly characterized CSFV NS2 protein.

View Article: PubMed Central - HTML - PubMed

Affiliation: College of Veterinary Medicine, Northwest A & F University, Yangling, Shaanxi, China.

ABSTRACT

Background: Classical swine fever (CSF) caused by virulent strains of Classical swine fever virus (CSFV) is a haemorrhagic disease of pigs, characterized by disseminated intravascular coagulation, thrombocytopoenia and immunosuppression, and the swine endothelial vascular cell is one of the CSFV target cells. In this report, we investigated the previously unknown subcellular localization and function of CSFV NS2 protein by examining its effects on cell growth and cell cycle progression.

Results: Stable swine umbilical vein endothelial cell line (SUVEC) expressing CSFV NS2 were established and showed that the protein localized to the endoplasmic reticulum (ER). Cellular analysis revealed that replication of NS2-expressing cell lines was inhibited by 20-30% due to cell cycle arrest at S-phase. The NS2 protein also induced ER stress and activated the nuclear transcription factor kappa B (NF-kappaB). A significant increase in cyclin A transcriptional levels was observed in NS2-expressing cells but was accompanied by a concomitant increase in the proteasomal degradation of cyclin A protein. Therefore, the induction of cell cycle arrest at S-phase by CSFV NS2 protein is associated with increased turnover of cyclin A protein rather than the down-regulation of cyclin A transcription.

Conclusions: All the data suggest that CSFV NS2 protein modulate the cellular growth and cell cycle progression through inducing the S-phase arrest and provide a cellular environment that is advantageous for viral replication. These findings provide novel information on the function of the poorly characterized CSFV NS2 protein.

Show MeSH
Related in: MedlinePlus