The Glycoprotein B Disintegrin-Like Domain Binds Beta 1 Integrin To Mediate Cytomegalovirus Entry

Cellular integrins were identified as human cytomegalovirus (HCMV) entry receptors and signaling mediators in both fibroblasts and endothelial cells. The goal of these studies was to determine the mechanism by which HCMV binds to cellular integrins to mediate virus entry. HCMV envelope glycoprotein B (gB) has sequence similarity to the integrin-binding disintegrin-like domain found in the ADAM (a disintegrin and metalloprotease) family of proteins. To test the ability of this region to bind to cellular integrins, we generated a recombinant soluble version of the gB disintegrin-like domain (gB-DLD). The gB-DLD protein bound to human fibroblasts in a specific, dose-dependent and saturable manner that required the expression of an intact β1 integrin ectodomain. Furthermore, a physical association between gB-DLD and β1 integrin was demonstrated through in vitro pull-down assays. The function of this interaction was shown by the ability of cell-bound gB-DLD to efficiently block HCMV entry and the infectivity of multiple in vivo target cells. Additionally, rabbit polyclonal antibodies raised against gB-DLD neutralized HCMV infection. Mimicry of the ADAM family disintegrin-like domain by HCMV gB represents a novel mechanism for integrin engagement by a virus and reveals a unique therapeutic target for HCMV neutralization. The strong conservation of the DLD across beta- and gammaherpesviruses suggests that integrin recognition and utilization may be a more broadly conserved feature throughout the Herpesviridae.Like many other herpesviruses, human cytomegalovirus (HCMV) is an opportunistic pathogen that is able to asymptomatically infect the human population with high incidence throughout the world. Primary infection is followed by a life-long latent phase that may reactivate and cause disease during the immunosuppression experienced by AIDS patients and organ transplant recipients (14, 52). HCMV disease is also a cause of significant morbidity and mortality during primary congenital infections (66). Currently there is no effective HCMV vaccine, and HCMV antiviral therapies, such as ganciclovir, are highly toxic and unsuitable for treating pregnant women in the congenital setting (92).HCMV disease can manifest itself in most organ systems and tissue types. Pathology from HCMV-infected individuals reveals that HCMV can infect most cell types, including fibroblasts, endothelial cells, epithelial cells, smooth muscle cells, stromal cells, monocytes/macrophages, neutrophils, neuronal cells, and hepatocytes (20, 25, 77, 83, 87). The broad intrahost organ and tissue tropism of HCMV is paralleled in vitro with the virus'' ability to bind and fuse with nearly every vertebrate cell type tested (40, 62, 78). However, full productive infection is limited to secondary strains of fibroblasts and endothelial cells. The ability of HCMV to enter such a diverse range of cell types is indicative of multiple cell-specific receptors, broadly expressed receptors, or a complex entry pathway in which a combination of both cell-specific and broadly expressed cellular receptors are utilized.The genes that encode envelope glycoprotein B (gB) and gH are essential (37), play several key roles during virus entry and egress, and are conserved throughout the Herpesviridae (reviewed in reference 80). A soluble form of gB truncated at the transmembrane domain (gBs) binds to permissive cells specifically, blocks virus entry, and is sufficient to trigger signal transduction events that result in the activation of an interferon-responsive pathway that is also activated by HCMV virions (10, 12, 13).HCMV entry requires initial tethering of virions to cell surface heparan sulfate proteoglycans (HSPGs) (22, 80). The HCMV envelope contains at least two separate glycoprotein complexes with affinities for heparan sulfate: gB (22) and the gM/gN complex (48). The gM/gN complex is more abundant than gB within the envelope (88) and binds heparin with higher affinity (49). Thus, the gM/gN complex is thought to be the primary heparin-binding component of the HCMV envelope.Virus-cell tethering via HSPGs is followed by a more stable interaction and subsequent signal transduction cascades. This interaction was proposed to be mediated via cell surface epidermal growth factor receptor (EGFR) (17, 95). These data, however, conflicted with more recent reports that demonstrate EGFR is not explicitly required for infection (21, 42). Platelet-derived growth factor receptor (PDGFR) has also been reported to function as an attachment receptor that functions to activate signaling cascades required for infection (79). The relative contribution of signaling and virus-host cell attachment for each of these growth factor receptors remains to be further characterized. The possibility also exists that additional attachment receptors still remain unidentified.Integrins are expressed on the cell surfaces of all vertebrate cells, a characteristic that parallels the promiscuity of HCMV entry. Additionally, β1 integrins are capable of mediating many of the same signal transduction pathways that are triggered during HCMV entry into host cells. Upon binding and fusing with host cell surfaces, HCMV triggers changes in Ca²⁺ homeostasis (36) and the activation of phospholipases C and A2, as well as an increased release of arachidonic acid and its metabolites (2). Additionally, mitogen-activated protein kinase (MAPK) (44, 45), phosphatidylinositol-3-OH kinase (PI3-K) (46), and G proteins are activated (73). Indeed, it was shown that HCMV entry led to an activation of integrin signaling pathways that reorganized the actin cytoskeleton (31) and phosphorylated β1 and β3 integrin cytoplasmic domains (31), focal adhesion kinase (FAK) (31), and Src (94). Integrin antibody blocking studies in combination with HCMV infectivity assays in β1 integrin-null GD25 cells identified α2β1, α6β1, and αVβ3 integrins as HCMV “postattachment” entry receptors (31). Certain integrin signaling events could be triggered by both HCMV and a soluble version of gB and require the expression of β1 integrin, identifying this specific viral ligand in integrin engagement (31).ADAM family members are multifunctional proteins that contain a metalloproteinase domain involved in ectodomain shedding and a disintegrin module of approximately 90 amino acids that confers RGD-independent integrin binding (43, 81, 99). The minimum component of the disintegrin module required for integrin engagement is the 12- to 13-amino-acid disintegrin loop, for which a consensus sequence has been described: RX₆DLXXF (29). The 20-amino-acid stretch encompassing the gB disintegrin-like domain is highly conserved, with greater than 98% amino acid identity among HCMV clinical isolates. Additionally, this domain is present in most gammaherpesviruses and all betaherpesviruses, suggesting that integrin engagement may be a conserved feature for most of the Herpesviridae. Synthetic peptides of the gB disintegrin loop block virus fusion (tegument delivery) but not virus attachment (31). This fact suggests a disintegrin-mediated molecular mechanism of herpesvirus-integrin engagement. Glycoprotein H (gH) has also been identified as an αVβ3 integrin ligand (94). However, gH contains no previously identified integrin recognition motifs, and the αVβ3 integrin heterodimer does not typically engage ADAM family proteins.Herein, we explore the molecular mechanism of integrin engagement by HCMV envelope gB. We provide multiple lines of evidence that demonstrate a physical interaction between the gB disintegrin module with β1 integrin. Furthermore, this interaction has significant consequences to the viral life cycle, since a soluble version of the gB disintegrin module efficiently blocks HCMV infection at a postattachment step during entry into multiple in vivo cell targets. Similarly, polyclonal antibodies directed against the gB disintegrin-like domain neutralize HCMV infectivity. These data identify the molecular mechanism of an HCMV ligand-receptor interaction required for virus-host fusion.