Dengue virus (DENV) infection causes dengue, the most prevalent arboviral infection in the world and modelling suggests it infects 390 million people each year. Although, there is no specific treatment or safe vaccine. Dengue has hallmark signs of vascular leakage, fever, and thrombocytopenia, where vascular insufficiency can lead to hypovolemic shock and death.
Disease severity and patient outcome have been correlated to the changes in regulators and activators of the complement system through analysis of patient sera and GWAS. Specifically, reduced alternative pathway regulatory proteins, such as Factor H (FH), the most important complement alternative pathway regulator, are correlated with increased disease severity.
Previously our lab has demonstrated that DENV infection induces FH mRNA production in primary cells but no significant change in the concentration of secreted FH. Here we have demonstrated that HeLa can be used to reproduce this phenomenon.
Further, ELISA and Western blot of cell lysates demonstrates that FH protein is increased in DENV-infected cells but localised on or within the cell. To investigate the underlying mechanisms by which DENV facilitates FH retention by cells, the abundance of the two most prominent cell surface FH ligands, sialic acids (Sia) and heparan sulphate (HS) were quantitated using a novel fluorescence-based technique and UV spectroscopy respectively. No significant difference in the abundance of these FH ligands was found on the surface of DENV- compared to mock-infected cells. Membrane manipulation by enzymatic cleavage of HS and Sia from cell surfaces also yielded no difference in the amount of FH released from DENV infected cells compared to controls. Thus, DENV infection causes retention of FH by infected cells but not due to increased binding to Sia or HS. FH may be binding other known ligands such as oxidised fatty acids, C-reactive protein or C3 within the cell which is now being investigated.
Our results demonstrate dysregulation of FH ability to be secreted despite increased FH mRNA transcription due to cellular retention. Thus, restoring the regulatory capacity of FH by facilitating release and circulation from cells may be an effective therapeutic avenue to treat dengue.