MS04-P12 Site-Specific Analysis of UL144 Protein from Human Cytomegalovirus Highlights its Role in Virus-Mediated Immune Evasion Ivana Nemčovičová (Department of Viral Immunology, Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovakia) Mário Benko (Department of Viral Immunology, Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovakia) Marek Nemčovič (Department of Glycobiology, Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia)email: viruivka@savba.sk
Human cytomegalovirus (HCMV, HHV-5) causes a spectrum of disease syndromes in children and adults. HCMV is a cause of mononucleosis in immunocompetent individuals and is a well-known cause of serious morbidity and sometimes fatal infections in immunocompromised patients, especially recipients of solid-organ or hematopoetic cell allografts and individuals with advanced AIDS. HCMV molds cell functions to support its replication and displays tropism for differentiated human cells that are critical for its life cycle. HCMV destroys infected cells by active lytic replication. The virus genes block apoptosis, interfere with the expression of immune recognition molecules on the surface of infected cells to avoid lysis by natural killer or cytotoxic T cells, and inhibit the antiviral effects [1]. The large size of the HCMV dsDNA genome, allows this virus to dedicate many genes to viral fitness; a number of these genes thwart the host inflammatory, innate, and adaptive immune responses. In clinical HCMV isolates, considerable genetic polymorphism exists and correlates with its clinical presentation. Of the viral genes, UL144 is particularly notable because of its role in modulating the host immune response. UL144 is found exclusively in clinical HCMV strains and encodes a structural homologue of the herpesvirus entry mediator. It has been proposed that UL144 plays a role in virus-mediated immune evasion by transmitting inhibitory signals to downregulate T-cell responses [2].
Sequence of HCMV UL144 shows 10 N-linked glycosylation sites located in extracellular part of the gene. Many of them are not present in other viral species that suggest that potential glycosylation is important only in humans and may play a role in ligand-binding recognition. Here, we present the characterization of such recombinant HCMV UL144 glycoprotein isolated from baculovirus-insect system. By liquid chromatography-tandem mass spectrometry (LC-MS/MS) we have identified up to 6 peptides of HCMV UL144 genes that have covered the most of the desired sequence. Intact protein analysis using a combination of LC and MS determined the accurate masses of these proteins (glycan-deficient mutein and native wild-type) and the relative abundance of their isoforms. We have also analyzed the glycan profiles and identified the most glycosylated UL144 species that showed mass of 20267,4 Da. As glycosylation plays an important role in receptor-ligand recognition we have performed the SPR binding studies to UL144 known cellular ligands that have show the importance of UL144 glycosylation in HCMV immune recognition.
References:

[1] Ploegh, H. L. (1998). Science, 280, 248-53.

[2] Benedict, C. A., Butrovich, K. D., Lurain, N. S., Corbei,l J., Rooney, I., Schneider, P., Tschopp, J., Ware, C. F. (1999). Journal of Immunology, 162, 6967–6970.

Keywords: immunomodulation, HCMV UL144, glycosylation