Mohammad Khalid Parvez
King Saud University College of Pharmacy, Saudi Arabia
Mohammad Khalid Parvez is currently working in King Saud University College of Pharmacy which is located in Saudi Arabia
Background & aim:
Hepatitis E virus (HEV) has recently emerged to cause chronic infection, including neuropathy and arthralgia in some patients. Though in vitro modulation of cellular MAPK-ERK cascade by HEV-ORF3 protein is suggested to have a role in host pathobiology, activation of JNK1/2 has not been studied so far. To address this issue, we used the HEV genomic replicon system as well as expression vectors. Material & methods: In vitro transcribed pSKE2-WT (HEV-SAR55) replicon as well as expression vectors pTriEX-ORF2 and pTriEx-ORF3 were transfected into human hepatoma S10-3 cells(0.5×106 cells/well; 12-well plate; in triplicate). Following 24 hr incubation, RNA/DNA receiving cells were re-seeded in 8-chamber slides as well as in two sets(RNA-I & RNA-II and DNA-I & DNA-II) in 96-well culture plates. On day4 post-transfection, HEV replication was determined by double immune-staining for ORF2(chimp-sera/Alexa488-conjugated goat anti-human IgG) and ORF3(Rabbit-anti-ORF3/Alexa568-conjugated goat anti-rabbit) expressions in RNA-transfected cells or single staining for ORF2 and ORF3 in DNA-transfected cells. For a time-course (day2, 4, 6 and 8) JNK1/2 profiling, ELISA-based detection of JNK1/2 protein phosphorylation was performed, using BIOMOL Super Array Case-Kit. Results: While positive staining for ORF2/ORF3 in HEV RNA-transfected cells confirmed an active viral replication, the ELISA-based relative quantitation of JNK1/2 phosphoprotein in the same culture showed an optimal phosphorylation on day4. The JNK1/2 phosphorylation in RNA receiving cells was elevated by ~54% as compared to negative-control. Further, ~66% activation of phospho-JNK1/2 was observed in ORF3 over-expressing cells, compared to mock-control. Notably, these experiments when repeated in serum-free media under the same conditions did not show significant differences in the results. Conclusion(s): Our data therefore, shows regulation of MAPK-JNK1/2 by HEV-ORF3 in hepatocytes, mimicking HEV infection. This may have a possible pro-cell survival role in persistent infection in immunosuppressed individuals or extra hepatic manifestations of HEV. Molecular studies are underway to further validate these results.