The US12 protein is the second viroporin encoded by the US12 gene family of Human Cytomegalovirus: when two is better than one

Background and aim: The US12 gene family of Human Cytomegalovirus (HCMV) includes a set of 10 contiguous tandemly arranged genes (US12 to US21) each predicted to encode a membrane-associated 7TMDs protein. Some US12 members show low level of homology to the cellular Transmembrane Bax-Inhibitor 1 Motif-containing (TMBIM) proteins that modulates cellular Ca2+ homeostasis and adaptive responses to stress conditions. However, despite the confirmed evolutionary importance of the US12 genes to HCMV biology, only a few functions have been associated with the gene family. Previously, we observed that inactivation of individual US12 genes in a clinical strain of HCMV does not affect viral replication in fibroblasts, while the disruption of US16, US18, US20, and US21 members prevented viral growth in endothelial and epithelial cells, thus suggesting a role of these US12 genes in regulating HCMV cell tropism (Bronzini et al., J. Virol. 2012; Cavaletto et al., J.Virol. 2015; Luganini et al., J. Virol. 2017). Moreover, we characterized the US21 protein as an ER-resident viroporin that dysregulates intracellular Ca2+ homeostasis, inhibits apoptosis (Luganini et al., PNAS 2018), and stimulates adhesion and motility of HCMV-infected cells (Luganini et al., submitted 2022). Here, we report on the functional characterization of another US12 family member, the US12 protein as the second viroporin of the gene family. Methodology: The BAC-Recombineering technology was employed to generate recombinant viruses on the HCMV TR background: TRUS12HA (HA tag at the C-terminus of the ORF), US12NV5-CHA (V5 tag at N-terminus and HA tag at C terminus), TRΔUS12 (deletion of the entire US12 ORF), and TRUS12stop (stop codon at the 5th codon of the ORF). The Trans-Membrane Domain (TMD) topology of pUS12 was predicted using five different algorithms, and a selective epitope accessibility immunofluorescence assay was then used to verify the in silico predictions. The Tetracycline-Regulated Expression (TREx) 293 cell system was used to achieve tetracycline-inducible expression of pUS12HA. Intracellular Ca2+ content was determined by ratiometric cytosolic Ca2+ ([Ca2+]i) measurement with the Fura-2 AM probe. Results and conclusions: Using recombinant TR-HCMV encoding different tagged versions of the US12 ORF, we observed that pUS12 is a 7TMD protein (with the N-terminal in the cytosolic side and the C-terminal in the luminal side of the membrane) and its expressed as early as 24h p.i. pUS12 co-localizes with Golgi-derived membranes and accumulates late in infection at the periphery of the cytoplasmic Virion Assembly Complex (cVAC). Recombinant TR-HCMV in which the US12 ORF was removed (TRΔUS12) or inactivated (TRUS12stop) exhibited major growth defects in endothelial and epithelial cells, whereas in fibroblasts their replication was not significantly different from that of parental TRwt. Calcium imaging experiments then showed that the tetracycline-induced expression of pUS12 in TREx-293 cells significantly decreased the amount of releasable Ca2+ from intracellular stores, thus indicating that pUS12 acts as a Ca2+-conducting viroporin. In addition to pUS21, pUS12 therefore represents the second protein of the US12 family able to hijack cell’s Ca2+ homeostasis. Studies are ongoing to determine the cytobiological consequences of the pUS12-mediated alteration of intracellular Ca2+ content.