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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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Persistently JEV-infected cell clones show apoptosis resistance even with active virus replication. (A) BHK-21 cells either mock-infected or infected with JEV at an MOI of 1 and harvested 72 hr later. PI cell clones were harvested at 72 hr after freshly seeding. Prepared cells were stained with annexin V/propidium iodide, and individual flow cytometric dot plots were displayed. (B) The extra- and intracellular virus samples were collected at the indicated p.i. time and counted by plaque-forming assay.
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Fig1: Persistently JEV-infected cell clones show apoptosis resistance even with active virus replication. (A) BHK-21 cells either mock-infected or infected with JEV at an MOI of 1 and harvested 72 hr later. PI cell clones were harvested at 72 hr after freshly seeding. Prepared cells were stained with annexin V/propidium iodide, and individual flow cytometric dot plots were displayed. (B) The extra- and intracellular virus samples were collected at the indicated p.i. time and counted by plaque-forming assay.

Mentions: Many viruses that are originally cytopathic have been found to lose their cytopathicity when the persistent infection is established [2]. JEV infection induces severe cytopathic effects in various cell culture systems, including BHK-21 cells, and researchers have documented the ER stress response and subsequent apoptosis in the JEV-infected cells [31,32]. In order to assess how much of the JEV persistently infected BHK-21 cell population is destined to apoptosis while continuously producing infectious virus particles, the two BHK-21 cell clones with JEV persistent infection—cBS6-2 and cBS6-3—were subjected to flow cytometry analysis after annexin V/propidium iodide staining. The number of apoptotic cells and the late apoptotic or necrotic cells increased significantly upon wild-type JEV infection in normal BHK-21 cells (Figure 1A). On the contrary, the number of cBS6-2 or cBS6-3 cells with JEV persistent infection undergoing apoptosis seemed to remain below the basal level shown in the naïve BHK-21 cells. The amount of intracellular infectious JEV particles produced in the persistently infected cells was 5- to 7-fold less than that of an acute infection (Figure 1B). All cells subjected to the flow cytometry were plated and analyzed at the same time points. The cells were grown under the same culture conditions, and no notable difference in the cell numbers of each population was observed.Figure 1


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)

Persistently JEV-infected cell clones show apoptosis resistance even with active virus replication. (A) BHK-21 cells either mock-infected or infected with JEV at an MOI of 1 and harvested 72 hr later. PI cell clones were harvested at 72 hr after freshly seeding. Prepared cells were stained with annexin V/propidium iodide, and individual flow cytometric dot plots were displayed. (B) The extra- and intracellular virus samples were collected at the indicated p.i. time and counted by plaque-forming assay.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Persistently JEV-infected cell clones show apoptosis resistance even with active virus replication. (A) BHK-21 cells either mock-infected or infected with JEV at an MOI of 1 and harvested 72 hr later. PI cell clones were harvested at 72 hr after freshly seeding. Prepared cells were stained with annexin V/propidium iodide, and individual flow cytometric dot plots were displayed. (B) The extra- and intracellular virus samples were collected at the indicated p.i. time and counted by plaque-forming assay.
Mentions: Many viruses that are originally cytopathic have been found to lose their cytopathicity when the persistent infection is established [2]. JEV infection induces severe cytopathic effects in various cell culture systems, including BHK-21 cells, and researchers have documented the ER stress response and subsequent apoptosis in the JEV-infected cells [31,32]. In order to assess how much of the JEV persistently infected BHK-21 cell population is destined to apoptosis while continuously producing infectious virus particles, the two BHK-21 cell clones with JEV persistent infection—cBS6-2 and cBS6-3—were subjected to flow cytometry analysis after annexin V/propidium iodide staining. The number of apoptotic cells and the late apoptotic or necrotic cells increased significantly upon wild-type JEV infection in normal BHK-21 cells (Figure 1A). On the contrary, the number of cBS6-2 or cBS6-3 cells with JEV persistent infection undergoing apoptosis seemed to remain below the basal level shown in the naïve BHK-21 cells. The amount of intracellular infectious JEV particles produced in the persistently infected cells was 5- to 7-fold less than that of an acute infection (Figure 1B). All cells subjected to the flow cytometry were plated and analyzed at the same time points. The cells were grown under the same culture conditions, and no notable difference in the cell numbers of each population was observed.Figure 1

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