Singapore grouper iridovirus (SGIV) induced parapotosis-like cell death in host cells via the

activation of MAPK signalings

Qiwei Qin

South China Sea Institute of OceanologyChinese Academy of Sciences

2013.7.27

programmed cell death ， PCD

PCD is natural and genetically regulated process of removing unneeded

or damaged cells, and it plays critical roles in the balance between host

defense and the invading pathogen during pathogen-host co-evolution.

Research background

(Dale E. Bredesen, 2007)

Morphological and biochemical characteristics of apoptosis and parapoptosis (Sperandio et al., 2000)

Virus infection induced PCD

Virus infection induced apoptosis. Virus infection induced autophagic PCD

So far, no report on virus infection induced parapoptosis !

MAPK signaling pathways and PCD

MAPKs have been identified: extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinases (JNK) and p38 MAPK （ Wada et al, 2004 ）。

Mitogen-activated protein kinase (MAPK) cascades are evolutionary conserved intracellular signaling pathways that regulate gene expression, mitosis, proliferation, metabolism and programmed cell death.

MAPKs and virus infection

MAPK

(Han, Nat Immunol, 2006)

TLR could recognize the PAMPs of pathogens (virus, bacterial) and trigger the host immune responses, including the activation of MAPK signaling pathways.

Virus infection could activate MAPK pathways in mammalian and humans.

Aquatic animal viruses activate MAPK signaling pathways ？？

Iridovirus is one of the most important infectious pathgens for maricultured grouper, Epinephelus spp ！

Singapore grouper iridovirus (SGIV) isolated from diseased grouper is a novel species of ranavirus, family iridovidae（ Qin et al., 2003).

1. How the SGIV interact with grouper host cells causing cell death ？

2 、 What kind of host cell signaling pathways involve in the virus infection ？

Huang et al, Aquaculture, 2009

SGIV infected grouper cells

Lai et al, J Fish Dis. 2008

GIV infected barramundi cells

Apoptosis Non-apoptosis

Whether SGIV infection induced PCD is cell type dependent ？

Research results

1.SGIV infection induced different type of cell death and nucleus morphology in host and non-host two fish cells

Cell types CharacteristicsSGIV-EAGS rounded, congregated, remain attach, condensed

nuclei, non-apoptotic bodies

SGIV-FHM rounded, detached, apoptotic bodies observed

Cells Characteristics

SGIV-FHM DNA fragementation, DNA ladder, TUNEL positive

SGIV-EAGS no fragementation,no DNA ladder, TUNEL negetive

DNA fragmentation and DAN content analysis of SGIV-infected two fish cells

SGIV-infectedEAGS: appearance of cytoplamic

vaculoes, distended endoplasmic reticulum (ER), large cluster of swollen mitochondria.

SGIV-FHM: ER swelling

Examination of PS externalization and caspae activities

The exposure of PS on cell surface is a general marker of apoptotic cells. The exposure of PS only occurred on the FHM cell surface during SGIV infection.

Caspase-3 and -9 were activated in SGIV-infected FHM cells, but not in infected EAGS cells.

UV-inactivated SGIV had not effect on EAGS cells, but still evoked obviously

apoptosis in FHM cells.

Virus replication was essential for SGIV induced nonapoptotic cell death, but not for apoptosis.

SGIV infection activated MAPK signaling molecules in grouper cells

ERK, p38 MAPK and JNK molecules were phosphorylated at the different stages during SGIV infection in host EAGS cells.

Inhibition of ERK and JNK activities could delay the CPE and reduce the virus production.

MAPK signaling pathways were involved in SGIV replication in grouper host cells

Inhibition of JNK pathway could inhibit SGIV gene transcription and protein synthesis. No obvious changes for inhibition of p38 MAPK.

JNK but not p38 MAPK is important for SGIV transcription and protein synthesis

SGIV infection evoked increased the expression of IL-8, IRF-1 and TNFa, inhibition of JNK resulted in the reduction of 3 immune genes, inhibition of p38 MAPK only reduced the expression of TNFa.

Fish immune genes were regulated by MAPKs

IL-8 IRF-1 TNF-a

Conclusions

SGIV infection induced nonapoptotic cell death-parapotosis in grouper host cells.

MAPK signaling pathways involved in the SGIV infection and replication in host cells.

ERK 、 JNK signals are essential for virus replication and virus induced parapotosis

SGIV-activated MAPK pathways could modulate the expression of immune genes and inflammatory cytokines

Related publications:

1. Huang X., Huang Y., Ouyang Z., Cai J., Qin Q. 2011. J Gen Virol., Jun; 92:1292-301.

2. Huang X., Huang Y., Ouyang Z., Xu L, Yan Y, Cui H, Han X, Qin Q. 2011. Apoptosis, Aug;16(8):831-45.

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Acknowledges: National Basical Research Program(973 program); NSFC; CAS

Thank you very much Thank you very much

for your attentionfor your attention ！！