UL/b' = unique long b' region; BTLA = Band T-lymphocyte attenuator; CD155 = cluster of differentiation 155, poliovirus receptor/nectin-like molecule 5; CD160 = natural killer cell-activating receptor; CMV = cytomegalovirus; CXC = chemokine motif; DNAM-1 = DNAX accessory molecule 1, CD226; ER = endoplasmic reticulum; gH = glycoprotein H (gB, gM, gN, gL, gO); HVEM = herpes virus entry mediator; NK = natural killer; NKG2D = NK group 2D; RANTES = regulated on activation, normal T cell expressed and secreted; TB40E = bacterial artificial chromosome (BAC) clones of the HCMV; TIGIT = T cell immunoreceptor with Ig and ITIM domains; TNF = tumor necrosis factor; TRAIL-Rs (1 and 2) = TNF-related apoptosis inducing ligand receptors 1 and 2; vCXCL1 and 2 = viral chemokine homolog 1 and 2; VRPs = viral replicon particles

To establish infection, a wide variety of pathogens, including viruses, have evolved a number of strategies to avoid immune elimination. Viruses have acquired and optimized molecules that interact with the host chemokine network in order to disrupt immune surveillance and defense of vertebrates, helping to promote cell entry, facilitating dissemination of infected cells, and evasion the immune response. Viral immunomodulators include ligands, chemokine receptors and chemokinebinding proteins (vCKBPs) functioning as either cell surface receptor mimics, ligand mimics, or secreted chemokine-binding proteins. vCKBPs specifically modulate chemokine gradient formation and ligandreceptor recognition when they have a potential to even completely block chemokine-mediated responses to viral infection. Members of only two virus families (Herpesviridae and Poxviridae) encode vCKBPs capable of sequestering host chemokines through either the chemokine receptor, GAG-binding pocket, or both, which may result in the inhibition of chemotaxis in vivo. Here, we focused on vCKBPs encoded by α-, β-, and γ-herpesviruses, of which several have been experimentally used as anti-inflammatory or anti-immune reagents in animal models. Current results suggest that vCKBPs could be used to regulate the activity of both chemokines and chemokine receptors for the treatment of infections such as AIDS, diseases such as arthritis, neurotrauma, inflammatory CNS disorders, atherosclerosis, transplant rejection, and metastatic spread and angiogenesis. Better understanding of vCKBPs biology will help evaluate, which human diseases related to chemokine network dysregulation might be effectively treated with these novel promising immunomodulatory drugs to enable the manipulation of chemokine functions and leukocyte trafficking.

An increasing number of surface-exposed ligands and receptors acting on immune cells are being considered as a starting point in drug development applications. As they are dedicated to manipulating a wide range of immune responses, accurately predicting their molecular interactions will be necessary for the development of safe and effective therapeutics to enhance immune responses and vaccination. Here, we focused on the characterization of human CD160 and HVEM immune receptors, whose mutual engagement leads to bidirectional signaling (e.g., T cell inhibition, natural killer cell activation or mucosal immunity). In particular, our study reports on the molecule preparation, characterization and initial crystallographic analysis of the CD160–HVEM complex and both HVEM and CD160 in the absence of their binding partner. Despite the importance of the CD160–HVEM immune signaling and its therapeutic relevance, the structural and mechanistic basis underlying CD160–HVEM engagement has some controversial evidence. On one hand, there are studies reporting on the CD160 molecule in monomeric form that was produced by refolding from bacterial cells, or as a covalently linked single-chain complex with its ligand HVEM in insect cells. On the other hand, there are older reports providing evidence on the multimeric form of CD160 that acts directly on immune cells. In our study, the native non-linked CD160–HVEM complex was co-expressed in the baculovirus insect host, purified to homogeneity by anion-exchange chromatography to provide missing evidence of the trimeric form in solution. Its trimeric existence was also confirmed by the initial crystallographic analysis. The native CD160–HVEM complex crystallized in the orthorhombic space group with unit cell parameters that could accommodate one trimeric complex (3:3) in an asymmetric unit, thus providing ample space for the multimeric form. Crystals of the CD160–HVEM complex, CD160 trimer and HVEM monomer (reported in two space groups) diffracted to a minimum Bragg spacing of 2.8, 3.1 and 1.9/2.1 Å resolution, respectively. The obtained data will lead to elucidating the native structure of the complex.

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