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Constant up-regulation of BiP/GRP78 expression prevents virus-induced apoptosis in BHK-21 cells with Japanese encephalitis virus persistent infection.

Lyoo HR, Park SY, Kim JY, Jeong YS - Virol. J. (2015)

Bottom Line: In JEV acute infection on normal BHK-21 cells, silencing CHOP expression through specific siRNA blocked cell death almost completely.Meanwhile, depletion of BiP by specific siRNA unlocked CHOP expression in cBS6-2 and cBS6-3 cells, resulting in massive cell death.Fulminant apoptotic cell death for both cell clones on tunicamycin treatment revealed that the JEV persistently infected cells still contained functional arms for cell fate decisions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, College of Sciences, Kyung Hee University, Seoul, 130-701, Republic of Korea. miracleiris@khu.ac.kr.

ABSTRACT

Background: Persistent infection of the Japanese Encephalitis Virus (JEV) has been reported in clinical cases, experimental animals, and various cell culture systems. We previously reported the establishment of spontaneous JEV persistent infection, assisted by defective interfering particle accumulation and/or attenuated helper viruses, in BHK-21 cells devoid of virus-induced apoptosis, cBS6-2 and cBS6-3. However, cell-specific factors may play important roles in controlling JEV replication and have never been assessed for this specific phenomenon. Recent evidence suggests that viruses have evolved various mechanisms to cope with endoplasmic reticulum stress signaling pathways for their efficient amplification and transmission, including the unfolded protein response (UPR).

Results: To identify the host cell factors that affect JEV persistence, we investigated the expression of essential UPR factors in cBS6-2 and cBS6-3 cells. Of the selected UPR factors tested, the most noticeable deviations from those of the normal BHK-21 cells with JEV acute infection were as follows: the suppression of C/EBP homologous binding protein (CHOP) and the constant up-regulation of immunoglobulin binding protein (BiP) expression in cBS6-2 and cBS6-3 cells. In JEV acute infection on normal BHK-21 cells, silencing CHOP expression through specific siRNA blocked cell death almost completely. Meanwhile, depletion of BiP by specific siRNA unlocked CHOP expression in cBS6-2 and cBS6-3 cells, resulting in massive cell death. Fulminant apoptotic cell death for both cell clones on tunicamycin treatment revealed that the JEV persistently infected cells still contained functional arms for cell fate decisions.

Conclusions: BHK-21 cells with JEV persistent infection strive against virus-induced apoptosis through constant up-regulation of BiP expression, resulting in the complete depletion of CHOP even with apparent virus amplification in the cells. Accordingly, the attenuation of virus replication as well as the modifications to cell metabolism could be additional factors contributing to the development of JEV persistent infection in mammalian cells.

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Effects of BiP silencing on persistently JEV-infected cell clones. (A) Persistently JEV-infected cell clones, cBS6-2 and cBS6-3, were transfected with specific siRNA for BiP or non-specific scramble siRNA at the indicated concentration for 40 hr. The cell lysates were collected to measure the expression levels of BiP, CHOP, caspase-3, JEV NS3, and β-actin as an internal control. (B) The number of viable cells from the sample of (A) was determined using trypan blue exclusion. *P < 0.05.
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Fig4: Effects of BiP silencing on persistently JEV-infected cell clones. (A) Persistently JEV-infected cell clones, cBS6-2 and cBS6-3, were transfected with specific siRNA for BiP or non-specific scramble siRNA at the indicated concentration for 40 hr. The cell lysates were collected to measure the expression levels of BiP, CHOP, caspase-3, JEV NS3, and β-actin as an internal control. (B) The number of viable cells from the sample of (A) was determined using trypan blue exclusion. *P < 0.05.

Mentions: The cBS6-2 and cBS6-3 cells were transfected with specific siRNA for BiP and harvested 40 hr later. The effect of BiP silencing was examined by Western blotting with antibodies against BiP, CHOP, caspase-3, and JEV NS3. The expression of BiP was suppressed almost completely by siBiP in both cBS6-2 and cBS6-3 cells, while the CHOP expression was clearly induced (Figure 4A). In addition, the numbers of viable cells decreased significantly in both cell lines according to cleavage of caspase-3 (Figure 4A and B). The results suggest that the constant overexpression of BiP in the JEV persistently infected cells somehow holds back CHOP expression, resulting in the prevention of virus-induced apoptosis. This observation is consistent with reports that the inhibition of CHOP guarantees a higher survival rate both in vivo and in vitro even though CHOP is not the sole factor promoting cell death undergoing ER stress [38,39]. Furthermore, these results revealed that resistance against virus-induced apoptosis of the cBS6-2 and cBS6-3 cells did not result from the lower level of virus replication efficiency; rather it was ascribable primarily to the active participation of cellular factors in the UPR.Figure 4


Constant up-regulation of BiP/GRP78 expression prevents virus-induced apoptosis in BHK-21 cells with Japanese encephalitis virus persistent infection.

Lyoo HR, Park SY, Kim JY, Jeong YS - Virol. J. (2015)

Effects of BiP silencing on persistently JEV-infected cell clones. (A) Persistently JEV-infected cell clones, cBS6-2 and cBS6-3, were transfected with specific siRNA for BiP or non-specific scramble siRNA at the indicated concentration for 40 hr. The cell lysates were collected to measure the expression levels of BiP, CHOP, caspase-3, JEV NS3, and β-actin as an internal control. (B) The number of viable cells from the sample of (A) was determined using trypan blue exclusion. *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4352245&req=5

Fig4: Effects of BiP silencing on persistently JEV-infected cell clones. (A) Persistently JEV-infected cell clones, cBS6-2 and cBS6-3, were transfected with specific siRNA for BiP or non-specific scramble siRNA at the indicated concentration for 40 hr. The cell lysates were collected to measure the expression levels of BiP, CHOP, caspase-3, JEV NS3, and β-actin as an internal control. (B) The number of viable cells from the sample of (A) was determined using trypan blue exclusion. *P < 0.05.
Mentions: The cBS6-2 and cBS6-3 cells were transfected with specific siRNA for BiP and harvested 40 hr later. The effect of BiP silencing was examined by Western blotting with antibodies against BiP, CHOP, caspase-3, and JEV NS3. The expression of BiP was suppressed almost completely by siBiP in both cBS6-2 and cBS6-3 cells, while the CHOP expression was clearly induced (Figure 4A). In addition, the numbers of viable cells decreased significantly in both cell lines according to cleavage of caspase-3 (Figure 4A and B). The results suggest that the constant overexpression of BiP in the JEV persistently infected cells somehow holds back CHOP expression, resulting in the prevention of virus-induced apoptosis. This observation is consistent with reports that the inhibition of CHOP guarantees a higher survival rate both in vivo and in vitro even though CHOP is not the sole factor promoting cell death undergoing ER stress [38,39]. Furthermore, these results revealed that resistance against virus-induced apoptosis of the cBS6-2 and cBS6-3 cells did not result from the lower level of virus replication efficiency; rather it was ascribable primarily to the active participation of cellular factors in the UPR.Figure 4

Bottom Line: In JEV acute infection on normal BHK-21 cells, silencing CHOP expression through specific siRNA blocked cell death almost completely.Meanwhile, depletion of BiP by specific siRNA unlocked CHOP expression in cBS6-2 and cBS6-3 cells, resulting in massive cell death.Fulminant apoptotic cell death for both cell clones on tunicamycin treatment revealed that the JEV persistently infected cells still contained functional arms for cell fate decisions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, College of Sciences, Kyung Hee University, Seoul, 130-701, Republic of Korea. miracleiris@khu.ac.kr.

ABSTRACT

Background: Persistent infection of the Japanese Encephalitis Virus (JEV) has been reported in clinical cases, experimental animals, and various cell culture systems. We previously reported the establishment of spontaneous JEV persistent infection, assisted by defective interfering particle accumulation and/or attenuated helper viruses, in BHK-21 cells devoid of virus-induced apoptosis, cBS6-2 and cBS6-3. However, cell-specific factors may play important roles in controlling JEV replication and have never been assessed for this specific phenomenon. Recent evidence suggests that viruses have evolved various mechanisms to cope with endoplasmic reticulum stress signaling pathways for their efficient amplification and transmission, including the unfolded protein response (UPR).

Results: To identify the host cell factors that affect JEV persistence, we investigated the expression of essential UPR factors in cBS6-2 and cBS6-3 cells. Of the selected UPR factors tested, the most noticeable deviations from those of the normal BHK-21 cells with JEV acute infection were as follows: the suppression of C/EBP homologous binding protein (CHOP) and the constant up-regulation of immunoglobulin binding protein (BiP) expression in cBS6-2 and cBS6-3 cells. In JEV acute infection on normal BHK-21 cells, silencing CHOP expression through specific siRNA blocked cell death almost completely. Meanwhile, depletion of BiP by specific siRNA unlocked CHOP expression in cBS6-2 and cBS6-3 cells, resulting in massive cell death. Fulminant apoptotic cell death for both cell clones on tunicamycin treatment revealed that the JEV persistently infected cells still contained functional arms for cell fate decisions.

Conclusions: BHK-21 cells with JEV persistent infection strive against virus-induced apoptosis through constant up-regulation of BiP expression, resulting in the complete depletion of CHOP even with apparent virus amplification in the cells. Accordingly, the attenuation of virus replication as well as the modifications to cell metabolism could be additional factors contributing to the development of JEV persistent infection in mammalian cells.

Show MeSH
Related in: MedlinePlus