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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 effect of CSFV NS2 expression on porcine cyclin A and GRP78 transcription in cultured SUVEC cells. Total RNA was extracted from cells expressing either GFP alone, GFP-NS2 fusion, NS2-GFP fusion or untransfected cells. Real-time RT-PCR analysis of (A) cyclin A and (B) GRP78 mRNA levels were normalized to the corresponding CT value for porcine β-actin mRNA. The basal expression level in untransfected controls was assigned a value of 1 for each experiment.
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Figure 5: The effect of CSFV NS2 expression on porcine cyclin A and GRP78 transcription in cultured SUVEC cells. Total RNA was extracted from cells expressing either GFP alone, GFP-NS2 fusion, NS2-GFP fusion or untransfected cells. Real-time RT-PCR analysis of (A) cyclin A and (B) GRP78 mRNA levels were normalized to the corresponding CT value for porcine β-actin mRNA. The basal expression level in untransfected controls was assigned a value of 1 for each experiment.

Mentions: Since cyclin A is required for the entry into G2/M phase, the cyclin A protein levels in NS2 expressing cell lines and control cells were analyzed by western blot assay. Cyclin A expression level was significantly decreased in both cell lines that expressed NS2 protein compared with control cells. Furthermore, when the GFP-NS2-expressing and NS2-GFP-expressing cell lines were pre-treated with MG132, blocking proteasomal degradation of proteins, the relative intensity of cyclin A western blot signal was significantly increased (Fig. 4), suggesting that cyclin A is rapidly degraded in the presence of NS2. To further support these findings, quantitative real-time RT-PCR was employed and showed that cyclin A mRNA levels in the GFP-NS2-expressing and NS2-GFP-expressing cell lines were significant higher than in control cells (Fig. 5A). This suggests that the induction of cell cycle arrest in the S-phase by CSFV NS2 protein is associated with the increase of cyclin A proteasomal degradation rather than a decrease of cyclin A transcription. Moreover, transcription of GRP78, the ER molecular chaperone was also increased in GFP-NS2-expressing cell lines compared with controls (Fig. 5B). Analysis of the activity of NF-κB in GFP-NS2-expressing and NS2-GFP-expressing cell lines using a TransAMTM NF-κB p50 Transcription Factor Assay Kit demonstrated that the NF-κB was significantly activated compared with control cells (Fig. 6). Together, these analyses have shown that expression of CSFV NS2 results in the up-regulation of GRP78, cyclin A and NF-κB suggesting a role of NS2 in ER stress activation. Additionally, NS2 induces cell cycle arrest at the S-phase and is associated with the increased proteasomal degradation of cyclin A.


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 effect of CSFV NS2 expression on porcine cyclin A and GRP78 transcription in cultured SUVEC cells. Total RNA was extracted from cells expressing either GFP alone, GFP-NS2 fusion, NS2-GFP fusion or untransfected cells. Real-time RT-PCR analysis of (A) cyclin A and (B) GRP78 mRNA levels were normalized to the corresponding CT value for porcine β-actin mRNA. The basal expression level in untransfected controls was assigned a value of 1 for each experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: The effect of CSFV NS2 expression on porcine cyclin A and GRP78 transcription in cultured SUVEC cells. Total RNA was extracted from cells expressing either GFP alone, GFP-NS2 fusion, NS2-GFP fusion or untransfected cells. Real-time RT-PCR analysis of (A) cyclin A and (B) GRP78 mRNA levels were normalized to the corresponding CT value for porcine β-actin mRNA. The basal expression level in untransfected controls was assigned a value of 1 for each experiment.
Mentions: Since cyclin A is required for the entry into G2/M phase, the cyclin A protein levels in NS2 expressing cell lines and control cells were analyzed by western blot assay. Cyclin A expression level was significantly decreased in both cell lines that expressed NS2 protein compared with control cells. Furthermore, when the GFP-NS2-expressing and NS2-GFP-expressing cell lines were pre-treated with MG132, blocking proteasomal degradation of proteins, the relative intensity of cyclin A western blot signal was significantly increased (Fig. 4), suggesting that cyclin A is rapidly degraded in the presence of NS2. To further support these findings, quantitative real-time RT-PCR was employed and showed that cyclin A mRNA levels in the GFP-NS2-expressing and NS2-GFP-expressing cell lines were significant higher than in control cells (Fig. 5A). This suggests that the induction of cell cycle arrest in the S-phase by CSFV NS2 protein is associated with the increase of cyclin A proteasomal degradation rather than a decrease of cyclin A transcription. Moreover, transcription of GRP78, the ER molecular chaperone was also increased in GFP-NS2-expressing cell lines compared with controls (Fig. 5B). Analysis of the activity of NF-κB in GFP-NS2-expressing and NS2-GFP-expressing cell lines using a TransAMTM NF-κB p50 Transcription Factor Assay Kit demonstrated that the NF-κB was significantly activated compared with control cells (Fig. 6). Together, these analyses have shown that expression of CSFV NS2 results in the up-regulation of GRP78, cyclin A and NF-κB suggesting a role of NS2 in ER stress activation. Additionally, NS2 induces cell cycle arrest at the S-phase and is associated with the increased proteasomal degradation of cyclin A.

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