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Background Transmission of latent human cytomegalovirus (HCMV) via organ transplantation with post-transplant viral reactivation is extremely prevalent and results in substantial adverse impact on outcomes. Therapies targeting the latent reservoir within the allograft to mitigate viral transmission would represent a major advance. Here, we delivered an immunotoxin (F49A-FTP) that targets and kills latent HCMV aiming at reducing the HCMV reservoir from donor lungs using ex-vivo lung perfusion (EVLP). Methods HCMV seropositive human lungs were placed on EVLP alone or EVLP+1mg/L of F49A-FTP for 6 hours (n=6 , each). CD14+ monocytes isolated from biopsies pre and post EVLP underwent HCMV reactivation assay designed to evaluate viral reactivation capacity. Off-target effects of F49A-FTP were studied evaluating cell death markers of CD34+ and CD14+ cells using flow cytometry. Lung function on EVLP and inflammatory cytokine production were evaluated as safety endpoints. Results We demonstrate that lungs treated ex-vivo with F49A-FTP had a significant reduction in HCMV reactivation compared to controls, suggesting successful targeting of latent virus (76% median reduction in F49A-FTP vs. 15% increase in controls, p=0.0087). Furthermore, there was comparable cell death rates of the targeted cells between both groups, suggesting no off-target effects. Ex-vivo lung function was stable over 6 hours and no differences in key inflammatory cytokines were observed demonstrating safety of this novel treatment. Conclusions Ex-vivo F49A-FTP treatment of human lungs targets and kills latent HCMV, markedly attenuating HCMV reactivation. This approach demonstrates the first experiments targeting latent HCMV in a donor organ with promising results towards clinical translation.

G protein-coupled receptors constitute the largest family of membrane proteins. As targets of >30% of the FDA-approved drugs, they are valuable for drug discovery. The receptor is composed of seven membrane-spanning helices and intracellular and extracellular domains. BILF1 is a receptor encoded by Epstein-Barr virus (EBV), which evades the host immune system by various strategies. BILF1 facilitates the virus immune evasion by downregulating MHC class I and is capable of inducing signaling-mediated tumorigenesis. BILF1 homologs from primate viruses show highly conserved extracellular domains. Here, we show that conserved residues in the extracellular domains of EBV-BILF1 are important for downregulating MHC class I and that the receptor signaling and immune evasion can be inhibited by drug-like small molecules. This suggests that BILF1 could be a target to inhibit the signaling-mediated tumorigenesis and interfere with the MHC class I downregulation, thereby facilitating virus recognition by the immune system., The Epstein-Barr virus (EBV) BILF1 gene encodes a constitutively active G protein-coupled receptor (GPCR) that downregulates major histocompatibility complex (MHC) class I and induces signaling-dependent tumorigenesis. Different BILF1 homologs display highly conserved extracellular loops (ECLs) including the conserved cysteine residues involved in disulfide bridges present in class A GPCRs (GPCR bridge between transmembrane helix 3 [TM-3] and ECL-2) and in chemokine receptors (CKR bridge between the N terminus and ECL-3). In order to investigate the roles of the conserved residues in the receptor functions, 25 mutations were created in the extracellular domains. Luciferase reporter assays and flow cytometry were used to investigate the G protein signaling and MHC class I downregulation in HEK293 cells. We find that the cysteine residues involved in the GPCR bridge are important for both signaling and MHC class I downregulation, whereas the cysteine residues in the N terminus and ECL-3 are dispensable for signaling but important for MHC class I downregulation. Multiple conserved residues in the extracellular regions are important for the receptor-induced MHC class I downregulation, but not for signaling, indicating distinct structural requirements for these two functions. In an engineered receptor containing a binding site for Zn+2 ions in a complex with an aromatic chelator (phenanthroline or bipyridine), a ligand-driven inhibition of both the receptor signaling and MHC class I downregulation was observed. Taken together, this suggests that distinct regions in EBV-BILF1 can be pharmacologically targeted to inhibit the signaling-mediated tumorigenesis and interfere with the MHC class I downregulation.

Purpose Post-transplant CMV reactivation from latently-infected lung allografts is extremely prevalent and results in inferior long-term outcomes. CMV latently infected cells ubiquitously express a viral surface protein, US28. We aimed to evaluate the therapeutic effects of a novel immunotoxin (F49A-FTP) that targets US28 in human lungs during EVLP, with the ultimate goal of minimizing or preventing CMV transmission and reactivation post-transplantation. Methods Human lungs rejected for transplantation were randomly placed on EVLP alone or EVLP+1mg/L of F49A-FTP (n=6 each) for 6 hours. Biopsies were collected pre and post EVLP and subjected to an in vitro assay designed to evaluate viral reactivation capacity (Fig 1A). Based on US28’s homology to the human CX3CR1 chemokine receptor, potential off-target effects of the toxin were studied evaluating cell death markers of CD34+ and CD14+ using flow cytometry, and the ratio of cells post:pre perfusion were calculated. Lung function on EVLP and inflammatory cytokine production were evaluated as safety endpoints. Results Delivery of F49A-FTP during EVLP showed no acute toxic events demonstrated by no significant differences in lung physiology and levels of IL-8, IL1-b and IFN-γ between the groups. Furthermore, while control lung tissue demonstrated a median increase from baseline of 32% (Range -16% to +112.5%) in viral reactivation, treated lungs demonstrated a significant median reduction in CMV reactivation of 76% (Range -15% to -99.9%) (Fig 1B, p=0.0087). No off-target effects were noted upon F49A-FTP delivery, demonstrated by similar amounts of cell death between the groups (Fig 1C). Conclusion We demonstrate that ex vivo F49A-FTP treatment of human lungs on EVLP markedly attenuates CMV reactivation and could potentially be used to treat donor organs prior to transplant. Ongoing in vivo reactivation experiments using the EVLP treated samples in a humanized mouse will further validate these findings.

The use of receptor-ligand interactions to direct toxins to kill diseased cells selectively has shown considerable promise for treatment of a number of cancers and, more recently, autoimmune disease. Here we move the fusion toxin protein (FTP) technology beyond cancer/autoimmune therapeutics to target the human viral pathogen, human cytomegalovirus (HCMV), on the basis of its expression of the 7TM G protein-coupled chemokine receptor US28. The virus origin of US28 provides an exceptional chemokine-binding profile with high selectivity and improved binding for the CX3C chemokine, CX3CL1. Moreover, US28 is constitutively internalizing by nature, providing highly effective FTP delivery. We designed a synthetic CX3CL1 variant engineered to have ultra-high affinity for US28 and greater specificity for US28 than the natural sole receptor for CX3CL1, CX3CR1, and we fused the synthetic variant with the cytotoxic domain of Pseudomonas Exotoxin A. This novel strategy of a rationally designed FTP provided unparalleled anti-HCMV efficacy and potency in vitro and in vivo.

Immunotoxins as antiviral therapeutics are largely unexplored but have promising prospective due to their high selectivity potential and their unparalleled efficiency. One recent example targeted the virus-encoded G protein-coupled receptor US28 as a strategy for specific and efficient treatment of human cytomegalovirus (HCMV) infections. US28 is expressed on virus-infected cells and scavenge chemokines by rapid internalization. The chemokine-based fusion-toxin protein (FTP) consisted of a variant (F49A) of CX3CL1 specifically targeting US28 linked to the catalytic domain ofPseudomonas exotoxinA (PE). Here, we systematically seek to improve F49A-FTP by modifications in its three structural domains; we generated variants with (1) altered chemokine sequence (K14A, F49L, and F49E), (2) shortened and elongated linker region, and (3) modified toxin domain. Only F49L-FTP displayed higher selectivity in its binding to US28 versus CX3CR1, the endogenous receptor for CX3CL1, but this was not matched by a more selective killing of US28-expressing cells. A longer linker and different toxin variants decreased US28 affinity and selective killing. Thereby, F49A-FTP represents the best candidate for HCMV treatment. Many viruses encode internalizing receptors suggesting that not only HCMV but also, for instance, Epstein-Barr virus and Kaposi’s sarcoma-associated herpesvirus may be targeted by FTPs.

Human cytomegalovirus (HCMV) is the causative agent of life-threatening systemic diseases in immunocompromised patients as well as a risk factor for vascular pathologies, like atherosclerosis, in immunocompetent individuals. HCMV encodes a G-protein-coupled receptor (GPCR), referred to as US28, that displays homology to the human chemokine receptor CCR1 and binds several chemokines of the CC family as well as the CX3C chemokine fractalkine with high affinity. Most importantly, following HCMV infection, US28 activates several intracellular pathways, either constitutively or in a chemokine-dependent manner. In this study, our goal was to understand the molecular interactions between chemokines and the HCMV-encoded US28 receptor. To achieve this goal, a double approach has been used, consisting in the analysis of both receptor and ligand mutants. This approach has led us to identify several amino acids located in the N terminus of US28 that differentially contribute to the high affinity binding of CC versus CX3C chemokines. Additionally, our results highlight the importance of secondary modifications occurring at US28, such as sulfation, for ligand recognition. Finally, the effects of chemokine dimerization and interaction with glycosaminoglycans (GAGs) on chemokine binding and activation of US28 were investigated as well using CCL4 as model ligand. In line with the two-state model describing chemokine/receptor interaction, we show that an aromatic residue in the N-loop region of CCL4 promotes tight binding to US28, whereas receptor activation depends on the presence of the N terminus of CCL4, as shown previously for CCR5.

Both beta- and gammaherpesviruses encode G protein-coupled receptors (GPCRs) with unique pharmacological phenotypes and important biological functions. An example is ORF74, the γ2-herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded GPCR, which is highly constitutively active and considered the key oncogene in Kaposi's sarcoma pathogenesis. In contrast, the current annotation of the Epstein-Barr virus (EBV) genome does not reveal any GPCR homolog encoded by this human oncogenic γ1-herpesvirus. However, by employing bioinformatics, we recognized that the previously established EBV open reading frame BILF1 indeed encodes a GPCR. Additionally, BILF1 is a member of a new family of related GPCRs exclusively encoded by γ1-herpesviruses. Expression of hemagglutinin-tagged BILF1 in the HEK293 epithelial cell line revealed that BILF1 is expressed as an approximately 50-kDa glycosylated protein. Immunocytochemistry and confocal microscopy revealed that BILF1 localizes predominantly to the plasma membrane, similar to the localization of KSHV ORF74. Using chimeric G proteins, we found that human and rhesus EBV-encoded BILF1 are highly potent constitutively active receptors, activating Gα i . Furthermore, BILF1 is able to inhibit forskolin-triggered CREB activation via stimulation of endogenous G proteins in a pertussis toxin-sensitive manner, verifying that BILF1 signals constitutively through Gα i . We suggest that EBV may use BILF1 to regulate Gα i -activated pathways during viral lytic replication, thereby affecting disease progression.

Open reading frame 74 (ORF74) encoded by human herpesvirus 8 is a highly constitutively active seven transmembrane (7TM) receptor stimulated by angiogenic chemokines, e.g. growth-related oncogene-alpha, and inhibited by angiostatic chemokines e.g. interferon-gamma-inducible protein. Transgenic mice expressing ORF74 under control of the CD2 promoter develop highly vascularized Kaposi's sarcoma-like tumors. Through targeted mutagenesis we here create three distinct phenotypes of ORF74: a receptor with normal, high constitutive signaling through the phospholipase C pathway but deprived of binding and action of chemokines obtained through deletion of 22 amino acids from the N-terminal extension; an ORF74 with high constitutive activity but with selective elimination of stimulatory regulation by angiogenic chemokines obtained through substitution of basic residues at the extracellular ends of TM-V or TM-VI; and an ORF74 lacking constitutive activity but with preserved ability to be stimulated by agonist chemokines obtained through introduction of an Asp residue on the hydrophobic, presumed membrane-exposed face of TM-II. It is concluded that careful molecular dissection can selectively eliminate either agonist or inverse agonist modulation as well as high constitutive activity of the virally encoded oncogene ORF74 and that these mutant forms presumably can be used in transgenic animals to identify the molecular mechanism of its transforming activity.

Human cytomegalovirus (HCMV)-encoded G protein-coupled-receptor US28 is believed to participate in virus dissemination through modulation of cell migration and immune evasion. US28 binds different CC chemokines and the CX3C chemokine CX3CL1. Membrane-anchored CX3CL1 is expressed by immune-activated endothelial cells, causing redirection of CX3CR1-expressing leukocytes in the blood to sites of infection. Here, we used stable transfected cell lines to examine how US28 expression affects cell migration on immobilized full-length CX3CL1, to model how HCMV-infected leukocytes interact with inflamed endothelium. We observed that US28-expressing cells migrated more than CX3CR1-expressing cells when adhering to immobilized CX3CL1. US28-induced migration was G protein-signalling dependent and was blocked by the phospholipase Cβ inhibitor U73122 and the intracellular calcium chelator BAPTA-AM. In addition, migration was inhibited in a dose-dependent manner by competition from CCL2 and CCL5, whereas CCL3 had little effect. Instead of migrating, CX3CR1-expressing cells performed ‘dancing-on-the-spot’ movements, demonstrating that anchored CX3CL1 acts as a strong tether for these cells. At low receptor expression levels, however, no significant difference in migration potential was observed when comparing the migration of CX3CR1- and US28-expressing cells. Thus, these data showed that, in contrast to CX3CR1, which promotes efficient cell capture upon binding to anchored CX3CL1, US28 acts to increase the migration of cells upon binding to the same ligand. Overall, this indicates that infected cells probably move more than uninfected cells in inflamed tissues with high CX3CL1 expression, with soluble chemokines affecting the final migration.

Viral infections depend on an intimate relationship between the infectious agent and the host cells. Viruses need the host cells for replication, while the innate- and adaptive- immunesystem of the host is fighting to kill the infected cell in order to clear out the pathogen and survive the infection. However, since both virus and host exist, the organisms struggle must reach an ecological equilibrium. Among the best-studied interactions between viruses and the host immune system are those between herpesviruses and their hosts. Herpesviruses are known to devote a significant part of their large genomes on immuno-modulatory genes, some encoding chemokines or chemokine receptors. These genes, which may be dispensable for viral replication in vitro, are highly important for viral growth in vivo, for viral dissemination and disease progression. Indeed, all beta and gamma-herpesviruses have acquired homologs of both chemokines and chemokine receptors belonging to the 7 transmembrane (7TM) spanning, G protein-coupled receptor family. 7TM receptors are very efficient drug targets and are currently the most popular class of investigational drug targets. A notable trait for the virus encoded chemokine receptors seems to be their constitutive activity. The biological function of the constitutive activity is still unclear, but it has become clear that the receptors are involved in important parts of the viral lifecycle in vivo, and that the receptor signaling is involved in gamma-herpesvirus mediated cell transformation. Therefore, blocking the signaling of these receptors will provide an efficient and highly specific way to inhibit viral replication in vivo and disease progression in the hosts.

The highly conserved Arg in the so-called DRY motif (Asp-Arg-Tyr) at the intracellular end of transmembrane helix 3 is in general considered as an essential residue for G protein coupling in rhodopsin-like seven transmembrane (7TM) receptors. In the open reading frame 74 (ORF74) receptor encoded by equine herpesvirus 2 (EHV2), the DRY motif is substituted with a DTW motif. Nevertheless, this receptor signaled with high constitutive activity through Gi as determined by a receptor-mediated inhibition of forskolin-induced cAMP-production and by an induction of the serum response element-driven transcriptional activity through a pertussis toxin-sensitive manner. Gs and Gq were not activated constitutively as determined by the lack of inositol phosphate turnover and activities of the three transcription factors: cAMP response element-binding protein (CREB), nuclear factor-kappaB, and nuclear factor of activated T cells. Coexpression of the ORF74-EHV2 receptor with the promiscuous G protein Gqi4myr supported the constitutive Gi activation as determined by inositol phosphate turnover and CREB activation. The constitutive activity was inhibited by nonpeptide inverse agonists with micromolar potencies, and the chemokine CXCL6 acted as a high-affinity agonist. It is noteworthy that reconstitution of the DRY motif resulted in a 4- to 5-fold decrease of the constitutive activity. Both the wild type and the receptor with the reconstituted DRY motif were expressed at the cell surface as indicated by immunohistochemistry and enzyme-linked immunosorbent assay analysis. It is concluded that the Arg of the DRY motif in transmembrane helix 3 is not essential for G protein coupling based on the constitutive as well as the ligand-mediated activity observed for ORF74-EHV2.

The 7TM receptor, US28, encoded by human cytomegalovirus binds a broad spectrum of endogenous CC chemokines with sub-nanomolar affinity as determined in homologous competition binding assays. We here find that US28 also recognizes the membrane-associated CX3C chemokine, fractalkine, with sub-nanomolar affinity (IC50=0.42±0.09 nM). Importantly, although fractalkine could compete with high affinity against the binding of CC chemokines, the secreted CC chemokines were only able to compete for binding against radioactive fractalkine with very low affinity. It is concluded that US28, which is known to enhance cell-cell fusion processes through interaction with an as yet unidentified, human cell-specific factor, has been optimized by cytomegalovirus to selectively recognize the membrane-associated fractalkine. It is suggested that US28 expressed on the surface of infected cells and possibly on the envelope of the virion is involved in transfer of the virus from cell to cell.

Cellular infection by cytomegalovirus (CMV) is associated with very early G-protein-mediated signal transduction and reprogramming of gene expression. Here we investigated the involvement of human CMV (HCMV)-encoded US27, US28, and UL33 receptors as well as murine CMV-encoded M33 transmembrane (7TM) receptors in host cell signaling mechanisms. HCMV-encoded US27 did not show any constitutive activity in any of the studied signaling pathways; in contrast, US28 and M33 displayed ligand-independent, constitutive signaling through the G protein q (Gq)/phospholipase C pathway. In addition, M33 and US28 also activated the transcription factor NF-κB as well as the cyclic AMP response element binding protein (CREB) in a ligand-independent, constitutive manner. The use of specific inhibitors indicated that the p38 mitogen-activated protein (MAP) kinase but not the extracellular signal-regulated kinase 1/2-MAP kinase pathway is involved in M33- and US28-mediated CREB activation but not NF-κB activation. Interestingly, UL33—the HCMV-encoded structural homologue of M33—was only marginally constitutively active in the Gq/phospholipase C turnover and CREB activation assays and did not show any constitutive activity in the NF-κB pathway, where M33 and US28 were highly active. Hence, CMVs appear to have conserved mechanisms for regulating host gene transcription, i.e., constitutive activation of certain kinases and transcription factors through the constitutive activities of 7TM proteins. These data, together with the previous identification of the incorporation of such proteins in the viral envelope, suggest that these proteins could be involved in the very early reprogramming of the host cell during viral infection.