Universitas Indonesia Conferences, Asian Federation for Pharmaceutical Sciences (AFPS) 2019

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Characterisation of the signalling properties of the HCMV-encoded receptor UL78
Irfan Muris Setiawan, Jelle van den Bor, Timo De Groof, Marco Siderius, Martine Smit

Last modified: 2019-06-16


Background: The genome of HCMV encodes four viral G Protein-Coupled Receptors (GPCRs) namely US28, US27, UL33, and UL78. US28 is activated by various chemokines and activates various inflammatory and proliferative signalling pathways in a constitutively active manner. UL78 is considered an orphan receptor since no ligands were identified to bind this receptor yet and little is known about the UL78 signalling properties. Objective: The aim of this study is to determine the signalling properties of UL78 Material and Method: DNA constructs of HA tagged UL78 and C-terminal mutant of UL78 receptor were amplified via PCR, the DNA was inserted to pcDEF3 vector plasmid and transformed into competent cells DH5 alpha. Purification of the DNA was done following Thermo Scientific GeneJET Plasmid miniprep kit protocol. HEK 293T cells were transfected with UL78 WT, HA-UL78, UL78-HA and HA-US28 as positive control, the expression level of the receptors were detected by Immunofluorescence and ELISA stained with anti HA (Roche) and anti UL78 antibody (Covance). Reporter gene assay were performed in transiently transfected HEK 293T cells with 100 ng/well of receptor and 1000 ng/well reporter plasmid. Control was transfected with 1000 ng/well pcDEF3 and reporter plasmid. An additional plate was prepared for ELISA to control receptor expression level. Results: Immunofluorescence assay and ELISA showed that All DNA construct of UL78 and C-terminal mutant of UL78 are expressed in transiently transfected HEK293T cells and the expression was in dose dependent manner. Several reporter gene assays were performed to determine signalling activity of UL78 and UL78 marginally activated NFAT and NF-kb reporter. The truncation of PDZ binding motif in C-terminal region of UL78 did not influence the activation of NFAT. Conclusion: UL78 is expressed in transiently transfected HEK 293T cells and  marginally activates NFAT reporter.


Keywords: Human Cytomegalovirus, UL78, G Protein-Coupled Receptors, Reporter gene, NFAT