Characterization of the Expression of US20 gene product of Human Cytomegalovirus

The Human Cytomegalovirus (HCMV) is an ubiquitous pathogen able to infect the overwhelming majority of population; it is responsible for generally asymptomatic and persistent infections in healthy people, but it can cause severe diseases in immunocompromised (such as in transplant recipients or AIDS patients) and immunoimmature (such as the fetus) individuals. Functional post-genomic profiling of the whole HCMV coding potential allowed to define which viral genes are essential or dispensable for its productive replication in the standard host cell model, represented by human fibroblasts. It resulted that in HCMV genome about 50 core genes, mainly located in the UL domain, encode the basic functions required at this regard; in contrast, the US region contains several gene families non essential for viral replication in primary fibroblasts, and thought to be important for regulating HCMV replication and persistence in other cell types and/or in the natural host. The US12 gene family, in particular, contains a set of 10 tandemly arranged non-essential genes (from US12 to US21) encoding predicted seven-transmembrane domain (7TMD) proteins whose specific functions remain to be largely elucidated. The aim of this thesis work was to investigate the expression pattern, cellular localization, topology and post-translational modification of the protein encoded by US20, a member of this gene family. We initially searched for pUS20 homologous and we found a limited level of amino acid sequence homology with members of the Transmembrane Bax Inhibitor containing Motif (TMBIM) family of anti-apoptotic proteins. To characterize the expression of pUS20, we generated a recombinant virus expressing the US20 ORF fused with a C terminal HA epitope tag, and we showed that US20 protein is expressed as a doublet, already detectable at 24 h.p.i. and insensitive to Foscarnet, thus indicating that US20 gene is expressed with an Early (E) gene kinetics. The generation of a further recombinant virus harbouring the US20 protein with two different tags at the N- and the C- termini allowed to determine that the doublet was not the result of a segmented expression of the US20 ORF. In contrast, the treatment with Endoglycosidase H converted the doublet into a single band, thus confirming the bioinformatics prediction that the occurrence of two bands was due to differential glycosylation. In different types of infected cells, pUS20HA showed a time-dependent localization, with a diffuse cytoplasmic distribution at early times and the subsequent accumulation, late in infection, in distinct peripheral cytoplasmic structures outside the cytoplasmic Virus Assembly Compartment (cVAC). To this regard, we observed a colocalization of pUS20 with the ER marker Calreticulin, thus indicating that pUS20 associates with ER-derived membranes over the entire HCMV replication cycle. Finally, an epitope accessibility assay allowed us to confirm that pUS20 is indeed a 7TMD protein, as we predicted by bioinformatics analysis. Hence, the results of this work led to the conclusion that the US20 gene of HCMV encodes a partially glycosylated 7TMD protein, with homology with TMBIM family members, that is expressed with an E kinetics and localizes within cytoplasmic structures derived from the ER compartment outside the cVAC. These results allowed us to gain initial insights on this protein features and pave the way for its functional characterization.