The US20 protein of Human Cytomegalovirus is required for viral replication in endothelial cells

The Human Cytomegalovirus (HCMV) is a widespread pathogen able to infect the overwhelming majority of population; it is responsible for generally asymptomatic and persistent infections in healthy people, whilst in immunocompromised and immunoimmature individuals it can cause severe diseases. Functional post-genomic profiling of HCMV allowed to identify which viral genes are essential or dispensable for its productive in vitro replication in the standard cell culture system, represented by primary fibroblast. In HCMV genome, about 50 core genes, mainly located in UL domain, encode the essential functions required for HCMV replication. Conversely, in the US region of the genome, there are several gene families whose members are non essential for viral replication in fibroblasts, but thought to be involved in regulating different aspects of HCMV infection, such as the replication and persistence in other cell types. Among them, the US12 family contains a series of ten tandemly arranged, non-essential genes (from US12 to US21), predicted to encode putative seven trans-membrane domains proteins whose functions still remain to be defined. In this regard, work from our group demonstrated that the lack of US16 ORF impairs the replication of a clinical isolate of HCMV in both endothelial and epithelial cells, with an early block of the replicative cycle prior to immediate early (IE) gene expression (Bronzini et al., 2012). Given this, the aim of this thesis work was to investigate the impact of the lack of the US20 gene in the context of HCMV replication in relevant cell types, and to define in which phase of viral replicative cycle the US20 gene is involved. To this end, we first evaluated the growth kinetics in fibroblasts (HFF), epithelial (ARPE-19) and endothelial cells (HMVEC) of different US20-deficient viruses generated in the TR clinical strain of HCMV by the BAC recombineering technology. We observed that US20-null viruses exhibited a strong defective growth phenotype in HMVEC, but not in HFF and ARPE-19. Immunofluorescence analysis and virus growth kinetics assays allowed to determine a similar defective phenotype of genotipically negative US20 viruses in other types of endothelial cells, such as HUVECs and LECs, thus indicating that the defective phenotype does not depend on the origin of the endothelial cell. Evaluation of the kinetics of viral DNA synthesis by qPCR showed that the genome replication of a US20-deficient virus in HMVECs was substantially absent over the entire replication cycle. Moreover, immunoblot assays performed in infected HMVECs showed that the expression of representative immediate early (IE) proteins was strongly reduced in cells infected with a US20- null virus compared to wt, while that of representative early (E) and late (L) proteins was substantially suppressed. Real-time RT-PCR analysis allowed to demonstrate that the lack of E and L protein accumulation was due to an impairment of E and L gene transcription. Attachment and entry assays then allowed to determine that the lack of the US20 gene does not affect the ability of US20-negative HCMV virus to attach and enter into endothelial cells, thus further sustaining the view that US20 regulates a post-entry event in HMVECs. Finally, we concluded that pUS20 is required for efficient HCMV replication in endothelial cells and exerts a role at a stage of the virus replication cycle subsequent to entry, but prior to E gene expression and viral DNA synthesis.