Characterizing Newly Identified Human Cytomegalovirus Entry Receptors

Human cytomegalovirus (HCMV) is a ubiquitous, usually benign virus. Nevertheless, HCMV frequently contributes to rejection of organs in immunosuppressed transplant patients and fetusinfections of the can causepost-encephalitic impairment of the infant brain. Among the important cells that HCMV infects in vivo are epithelial and endothelial cells. Three HCMV glycoproteins: trimer, gH/gL/gO, the pentamer gH/gL/UL128-131, and the viral fusion protein gB mediate entry of virus particles into biologically relevant epithelial and endothelial cells. Our model for how HCMV enters epithelial and endothelial cells suggets that HCMV trimers bind to cell surface receptors then viruses are internalized and pentamer acts in endosomes to trigger gB- mediated fusion of the virion envelope with cellular membranes. Given their importance in virus entry, trimer and pentamer are also important targets of antibodies and are considered key players in the design of HCMV vaccines. It was shown that trimer binds to platelet growth factor receptor-alpha (PGFR) to mediate entry into fibroblasts, but PDGFRa is not expressed in epitheial and endothelial cells. Thus, there are, as yet unidentfied, trimer receptors in these two cell types. It is also known that pentamer binds neuropilin-2 (NRP-2) , which is expressed in epithlial and endothelial cells, in a process that appears to allow HCMV to escape endosomes. However, pentamer binds NRP-2 with relatively low affinity, so that other pentamer receptors may exist. We utilized a novel receptor capture technology, which allowed covalent coupling of trimer or pentamer onto epithelial and endothelial cell surface proteins. Proteins bound by trimer and pentamer were identified by mass spectrometry. These studies identified a trimer-binding protein, Transforming Growth Factor  Receptor-3 (TGFR-3) and we showed that soluble form of TGFR-3 inhibited HCMV entry. Four interesting pentamer binding proteins were also identified and characterized including: thrombomodulin (TBHD) and a soluble form of TBHD blocked HCMV entry and calsyntensin-1, a protein that promotes entry of hepatitis C virus. Another pentamer-binding proteins calsyntensin-1 blocked HCMV cell-to-spread but not entry. We will further characterize these novel trimer and pentamer binding proteins in order to detemine if these proteins are important for HCMV entry into epithelial and endothelial cells. These studies will involve: producing soluble forms of these proteins, silencing the receptors, and characterizing anti-receptor antibodies that might block virus entry. High resolution deconvolution imaging, super resolution 3D imaging, and live cell imaging experiments, coupled with HCMV recombinants expressing fluorescent proteins, will be used characterize entry pathways. Whether these receptors act to sort virus particles during virus entry or whether the receptors signal into cells to alter cytoskeleton or activate signaling pathways will also be determined. These receptor candidates, combined with better characterized proteins such as NRP-2, will be extremely useful handles to better understand the cell biology of HCMV entry into biologically relevant epithelial and endothelial cells.