Caratterizzazione della proteina US21 del Citomegalovirus umano: dalle funzioni al target antivirale

The Human Cytomegalovirus (HCMV) is an opportunistic beta-herpesvirus with a double-stranded DNA that has one of the largest viral genomes known (235 kb). The genome is composed by two regions: the UL domain, encoding for core genes, essential for viral replication in cultured fibroblasts, and the US domain, including multiple gene families non-essential for the viral replication in fibroblasts, but thought to be involved in the regulation of the viral cell tropism, spread and persistence in the natural host, as well as in counteracting host immune responses. Among the US gene families, the US12 gene family consists of 10 tandemly arranged genes (from US12 to US21) each encoding a predicted 7TMDs protein. However, in spite of the likely evolutionary importance of the US12 genes to HCMV biology, only few functions have been associated to the family members to date. Among the predicted US12 proteins, the US21 shows the highest level of identity with some cellular transmembrane BAX inhibitor motif-containing (TMBIM) proteins, which are involved in the regulation of cellular Ca2+ homeostasis and adaptive cell responses to stress conditions. In regard to, we reported previously that the US21 protein is an HCMV-encoded viroporin acting as a leaking calcium channel able to reduce the Ca2+ content of the endoplasmic reticulum (ER) store, and that its expression protects primary human fibroblasts against apoptosis provoked by different pro-apoptotic agents. In this thesis work, the functions of the pUS21 viroporin were investigated in other cell types. To this end, a cell system was set up in the human osteosarcoma U2OS cell line to achieve a tetracycline-inducible expression of pUS21. In functional calcium imaging experiments, the expression of pUS21 was observed to decrease in the amount of Ca2+ released from the ER intracellular store, thus confirming that pUS21 exert a Ca2+-channel activity even in U2OS cells. Then, we observed a decrease of U2OS cells susceptibility to intrinsic apoptosis, since cells expressing pUS21 and exposed to staurosporine showed lower levels of effector caspases in comparison to cells that do not express the protein, thus confirming that pUS21 protects cells against apoptosis even in cell types different from fibroblasts. Moreover, the expression of pUS21 increased the intracellular content of ATP, likely as a result of a stimulation of energy metabolism. Furthermore, using the predicted structural model of pUS21, a potential drug target in a virtual high-throughput screening (VHTS) of a library of approved calcium channel blockers (CCBs), four different molecules were selected for their potential binding within the pUS21 pore. Noteworthy, the four selected CCBs reduced the replication of HCMV in a dose-dependent manner with an EC50 range of 2-5 μM when examined in an antiviral assay in vitro. Taken together, the results of this thesis work confirm the viroporin and anti-apoptotic activity of pUS21 in different cell types, thus suggesting that it may dysregulate Ca2+ homeostasis in a cell-type independent manner. Moreover, pUS21 was validated as a potential antiviral drug target for repurposing approved CCBs against HCMV infection.