Host Pathogen interaction during HCMV infection

Human Cytomegalovirus (HCMV) exerts complex effects on the host immune system through expression of several interfering functions. Although a remarkable number of the genes implicated in immune evasion have been identified, due to the huge coding potential of the HCMV genome and to the numerous ORFs with unassigned functions the list is predicted to grow. As the humoral response is known to play an important role in the control of HCMV infection and outcomes, we started a characterization of the antibody repertoire in patients infected by the virus. Through a combined approach of Reverse Vaccinology, recombinant mammalian protein expression, immunoblotting and protein microarrays, a total of 25 viral proteins were found to be recognized by Immunoglobulin (human plasma/Cytogam®) of HCMV infected patients. Among them, the majority were known to be involved in the control of host immune response, while others were only predicted ORFs. Based on bioinformatics and literature data we decided to focus our work on the characterization of UL10 and UL139 proteins. UL10 is a member of the HCMV RL11 family. The characteristics of the UL10 protein, a predicted heavily glycosylated Ig-like membrane protein, that is known to be dispensable for viral replication in cultured cells, suggested a possible role in host-cell interaction. Ex-vivo cell based assays (Rosetting assay) and flow cytometry experiments on both lymphoid cell lines and primary blood cells showed that the protein interacts with a ubiquitous receptor on the surface of human lymphocytes. Proliferation assays performed on PBMC purified from blood of healthy donors showed that the protein significantly reduced the proliferation of stimulated T-cells. Further ongoing experiments aim to identify the cellular receptor and characterize downstream signaling functions. Uncovering the role of the UL10 protein during 28 HCMV infection could allow new insights into the modulation of the immune response triggered by the virus and ultimately the development of new therapies. UL139 is a predicted ORF coding for a type I membrane glycoprotein. It is located in a polymorphic locus deleted in laboratory adapted strains of HCMV. It has a region of homology with CD24 and bioinformatics analysis predicts a long and quite conserved cytoplasmic tail, suggesting a possible role in as signal transduction/structural protein during the infection cycle. We characterized UL139 protein during the infection cycle in fibroblasts, using an engineered HCMV TR strain carrying a tagged version of the protein. We observed that the protein is a non-structural viral protein, it is expressed with a late kinetics and it co-localizes with early endosomes marker, enclosing the viral assembly complex. Data obtained by pull-down and MS experiments suggested the presence of cellular interactor(s) involved in the vesicular transport/cytoskeleton reorganization. Preliminary data strongly indicated Fascin, a cellular protein involved in actin rearrangement, as potential UL139 interacting protein. Further ongoing experiments aim to verify this hypothesis and uncover the biological significance of such interaction.