The US21 viroporin of human cytomegalovirus regulates cell adhesion and migration

Background. During coevolution with its host, the Human Cytomegalovirus (HCMV) has invested a large part of its protein coding potential to ensure the dysregulation of the majority of cellular homeostatic circuits. Among the viral genes involved, the US12 gene family includes a set of 10 contiguous tandemly arranged genes (US12 to US21) and constitutes about 5% of HCMV’s genetic content. The identification of putative seventransmembrane hydrophobic domains in each of the US12 ORFs predicts a common structural framework that associates these proteins with cellular membranes. However, despite the confirmed evolutionary importance of the US12 proteins to HCMV biology, only a few functions in the regulation of virus cell tropism, virion composition, and immunoevasion have been associated with the family to date. Previously, we characterized the function of the US21 protein as a HCMV homolog of cellular TMBIM proteins and an ER-resident virus-encoded calcium-permeable channel able to dysregulate intracellular Ca2+ homeostasis and that inhibit apoptosis (Luganini et al., PNAS, 115, E12370, 2018). Aim of the study. Given the role of Ca2+ in controlling cell adhesion and motility, in this study we investigated whether the pUS21-mediated Ca2+ release from intracellular stores might influence these important cytobiological responses. Resuts and conclusions. Using engineered human cell lines to express pUS21 in an inducible manner, that allows to investigate cell responses without non-specific toxic effects due to a prolonged pUS21 expression, we observed a significant increase in the migration rate of cells expressing pUS21wt, while its mutation in the critical D201 residue that define the pUS21’s Ca2+ channel leaking function, affected the ability to stimulate cell motility, thus suggesting an involvement of its ability to reduce the Ca2+ content in intracellular stores. To test this hypothesis, we performed migration assays in the presence of an inhibitor of calpain 2, a Ca2+ -activated cysteine protease, that regulates the disassembly of cellular focal adhesions and promotes cell motility. The addition of the calpain 2 inhibitor abrogated the pUS21-mediated increase of migration in cells expressing this HCMV protein. As a further confirmation of a calpain 2 involvement, its biochemical activity was measured in cell expressing either pUS21wt or its mutated forms in the two critical amino acid residues that define the TMBIM architecture of pUS21, D178 and D201. An increase of calpain 2 activity was observed in extracts from cells expressing pUS21wt or pUS21- D178N, while the expression of pUS21-D201N was unable to stimulate calpain 2 activity, thus confirming an involvement of pUS21 Ca2+ channel function in the control of calpain 2 activation. Moreover, the functional relationship between pUS21 and calpain 2 was further suggested by the observation that talin 1, a known calpain proteolytic substrate, interact with pUS21 as observed by mass spectrometry and co-immunoprecipitation. Together, these findings highlight a novel role of the pUS21 viroporin of HCMV as a regulator of focal adhesion dynamics, cell adhesion, and migration as a consequence of the dysregulation that pUS21 exerts on the localized Ca2+ dependent activation of calpain 2 and the subsequent talin activity at newly forming protrusions.