FellowDr. Ivana Nemčovičová
Project NameImmune modulation by HCMV and its immunotherapeutic potential.
Host organisationBiomedical Research Center
Duration of the project01.07.2015 - 30.06.2018

Abstract
Characterizing the structural and molecular basis of the host-virus protein interactions is critical to further our understanding of the immune equilibrium that is established during viral infection, and why disease can occur if this balance is disrupted. The ligands and receptors of Immunoglobulin (Ig) or Tumour Necrosis Factor (TNF) families are critical for host defence; therefore we have an interest in delineating evasion strategies used by virus to modulate immune recognition. Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that persistently infects the majority of the world’s population. HCMV is a paradigm for viral immune evasion that maintains a number of immunomodulatory genes that act to suppress Natural Killer (NK) cell function. In this regard, we are planning a preparation and molecular characterization of selected set of recombinant viral and human genes that are associated with cytotoxicity and viroprotectivity processes in the cell while these events will also be monitored. We expect by using this cross-disciplinary approach we will able to design and characterize suitable candidates with high immunomodulatory activity. In summary, project aims to examine the targets and mechanisms by which HCMV immunomodulation is achieved, and therefore discuss the development of immunomodulatory biologics for the treatment of HCMV infection. In addition, the understanding of mechanism by which HCMV modulates immune effector pathways highlights its possibility being used as a vaccine vector for the treatment of cancer or other autoimmune diseases.

Project Summary with Interim Results

As the largest human herpesvirus, HCMV is a paradigm of viral immune evasion and has evolved multiple mechanisms to evade immune detection and enable survival. The HCMV immunomodulatory genes studied here (Figure), promote virus persistence by their ability to target and downregulate various cell surface proteins. It was demonstrated HCMV systematically suppresses cell surface expression of death receptors and ligands for NK cell activation. We have discovered the HCMV UL141 gene plays a major role in such protection via direct novel and non-canonical interactions. In isolation, UL141 is capable of suppressing both CD155 and TRAIL-R2 cell surface expression, but an additional HCMV-encoded factor is known to be required for efficient down-regulation of cell surface CD112. Our recent data shown, UL141 and US2 couple together in the complex to facilitate CD112 binding and degradation. These viral proteins are therefore potential modulators of multiple immune-related pathways and could act as a multifunctional hub that inhibits the immune recognition and killing of HCMV-infected cells. Therefore, the molecular nature of HCMV UL141- and US2-mediated interactions with CD155, CD112 and additional TRAIL death receptors (TRAIL-R1, -R3, -R4) were successfully characterized. The overall glycosylation profiles were examined to dissect its relative importance. UL141 was found to maintains two N-linked glycans that are alternatively occupied within the active molecule. We have fully characterized the molecular complexes that have shown higher affinity and attempted crystallization for further structural analysis. For the TRAIL-R1–R4 the homology models were also prepared, analyzed and will be used for molecular replacement method. The binding requirements for HCMV UL141 signaling function were determined by generating mutants of UL141 that differentially bind to its multiple cognate ligands. These mutants (including dimerization-deficient mutant) allowed us to dissect the minimal binding requirements of UL141 in regulating immune signaling and NK inhibition. While canonical signaling by TNF receptors occurs upon their trimerization by cognate ligands, our preliminary data show that UL141 induces NFκB activation when added to TRAIL-DRs-expressing cells. However, the signaling function of UL141 was not confirmed or disproved by any other assay yet. In addition to work planned, we have determine the molecular basis of herpesvirus entry mediator (HVEM) interactions to CD160, a novel NK cell bidirectional ligand. Here, we present the molecular characterization and preliminary crystallographic analysis of CD160 and HVEM and therefore the complex formation. This is the first report on CD160 molecule studied at protein level that has demonstrated its active disulphide-linked multimeric form involved in bidirectional HVEM signaling pathways. Moreover, we have also participated on studies related to human hexosaminidase enzyme that play essential roles in cellular physiology and health being recently highlighted because of its high immunomodulatory effects on T lymphocytes while being used as a molecular marker of degranulation in mast cells. In collaboration, we have provided the first mechanistic studies on human immunomodulatory nucleoplasmic gene called HexD, defined the catalytic mechanism of this enzyme and established the identities of key enzymic residues.

The sophisticated cross-regulation of all studied proteins may represent a common mechanism of immune regulation targeted by multiple pathogens, which by extension are the potential targets for therapeutic manipulation and this has to be further investigated.