Expression and characterization of US12 protein of Human Cytomegalovirus

The Human cytomegalovirus (HCMV) is an opportunistic double-stranded DNA virus with one of the largest viral genomes known (235 kb). Its genome encodes about 170 canonical ORFs and is composed of two regions: the Unique Long (UL) and Unique Short (US) regions. The UL region contains about 50 herpesvirus-common genes, which encode those basic functions required for viral DNA replication and its assembly into viral particles. These core genes, as expected, are essential for the in vitro replication of HCMV in cultures of primary fibroblasts. The US region, on the contrary, includes multiple gene families that are CMV-specific, non-essential for HCMV replication in primary fibroblasts, and therefore thought to be involved in regulating the spread and persistence of HCMV in the natural host, in counteracting the intrinsic, innate and adaptive host immune responses, and in contributing to the viral infection in cell types other than fibroblasts. Among these US gene families, the US12 family includes a set of 10 tandemly arranged related genes (from US12 to US21) that encode putative multipass transmembrane proteins with high levels of conservation among different fresh clinical isolates of the virus and specific to CMVs of higher primates, thus allowing to hypothesize roles in regulating virus-host interactions during the infection of the natural hosts. However, the characterization of individual US12 members has yet to be completed. In this thesis work, by means of bioinformatics tools and the subsequent experimental validation was investigated the expression pattern, cellular localization and topology of the protein encoded by the US12 gene, the first member of this gene family. The importance of the US12 protein in the context of the HCMV replication in endothelial and epithelial cells was analyzed as well. In fibroblasts infected with a recombinant HCMV expressing a tagged version of the US12 ORF, pUS12 showed a time-dependent localization, with a diffuse cytoplasmic distribution already detectable at 24 h post-infection and the subsequent accumulation, later in infection, in Golgi-derived membranes with a peculiar ring-like pattern at the periphery of the cytoplasmic Virus Assembly Compartment (cVAC). An epitope accessibility assay upon selective cell permeabilization, then allowed to confirm that pUS12 is indeed a 7 Trans Membrane Domain (7TMD) protein, as predicted by the bioinformatics analysis, with a cytoplasmic N-terminus and a luminal C-terminus. Finally, a defective growth phenotype was observed in endothelial and epithelial cells infected with genetically US12-deficient viruses. Hence, the US12 gene of HCMV encodes a 7TMD protein expressed with an E kinetics that localizes within cytoplasmic structures derived from the Golgi compartment inside the cVAC, and that is essential for the viral replication in those cells that are main targets of HCMV replication in vivo.