Disruption of CCL21-induced chemotaxis in vitro and in vivo by M3, a chemokine-binding protein encoded by murine gammaherpesvirus 68

Chemokine-binding proteins represent a novel class of antichemokine agents encoded by poxviruses and herpesviruses. One such protein is encoded by the M3 gene present in the murine gammaherpesvirus 68 (MHV-68) genome. The M3 gene encodes a secreted 44-kDa protein that binds with high affinity to certain murine and human chemokines and has been shown to block chemokine signaling in vitro. However, there has been no direct evidence that M3 blocks chemokine activity in vivo, nor has the nature of M3-chemokine interaction been defined. To better understand the ability of M3 to block chemokine activity in vivo, we examined its interaction with a specific subset of chemokines expressed in lymphoid tissues, areas where gammaherpesviruses characteristically establish latency. Here we show that M3 blocks in vitro chemotaxis induced by CCL19 and CCL21, chemokines expressed constitutively in secondary lymphoid tissues. Moreover, we provide evidence that chemokine M3 binding exhibits positive cooperativity. In vivo, the expression of M3 in the pancreas of transgenic mice inhibits recruitment of lymphocytes induced by transgenic expression of CCL21 in this organ. The ability of M3 to block the biological activity of chemokines may represent an important strategy used by MHV-68 to evade immune detection and favor viral replication in the infected host.     

Felid herpesvirus 1 glycoprotein G is a structural protein that mediates the binding of chemokines on the viral envelope

Glycoprotein G (gG) orthologues have been described in several alphaherpesviruses. gG is expressed both as a membrane-anchored form on infected cells and as a secreted form. Recently, we reported that both forms of gG encoded by alphaherpesviruses infecting large herbivores and by Felid herpesvirus 1 (FeHV-1) bind with high affinity to a broad range of CXC, CC and C-chemokines. Based on the viral species, gG has been reported either as a structural or a non-structural protein. To date, the incorporation of FeHV-1 gG into virions has never been tested, nor the property of alphaherpesvirus structural gG to bind chemokines on the virion surface. In the present study, to address these questions, various FeHV-1 gG recombinant strains were produced using an original technique based on an infectious FeHV-1 BAC clone and restriction endonuclease mediated recombination. Using the recombinants produced, we were able to determine that FeHV-1 gG is a structural protein that acts as a chemokine-binding protein on the virion surface. In the light of these results, putative roles of gG in alphaherpesvirus infections are discussed, and an evolutionary scenario is proposed to explain the structural versus non-structural property of gG amongst alphaherpesviruses.     

The Viral KSHV Chemokine vMIP-II Inhibits the Migration of Naive and Activated Human NK Cells by Antagonizing Two Distinct Chemokine Receptors

Natural killer (NK) cells are innate immune cells able to rapidly kill virus-infected and tumor cells. Two NK cell populations are found in the blood; the majority (90%) expresses the CD16 receptor and also express the CD56 protein in intermediate levels (CD56^Dim CD16^Pos) while the remaining 10% are CD16 negative and express CD56 in high levels (CD56^Bright CD16^Neg). NK cells also reside in some tissues and traffic to various infected organs through the usage of different chemokines and chemokine receptors. Kaposi''s sarcoma-associated herpesvirus (KSHV) is a human virus that has developed numerous sophisticated and versatile strategies to escape the attack of immune cells such as NK cells. Here, we investigate whether the KSHV derived cytokine (vIL-6) and chemokines (vMIP-I, vMIP-II, vMIP-III) affect NK cell activity. Using transwell migration assays, KSHV infected cells, as well as fusion and recombinant proteins, we show that out of the four cytokine/chemokines encoded by KSHV, vMIP-II is the only one that binds to the majority of NK cells, affecting their migration. We demonstrate that vMIP-II binds to two different receptors, CX3CR1 and CCR5, expressed by naïve CD56^Dim CD16^Pos NK cells and activated NK cells, respectively. Furthermore, we show that the binding of vMIP-II to CX3CR1 and CCR5 blocks the binding of the natural ligands of these receptors, Fractalkine (Fck) and RANTES, respectively. Finally, we show that vMIP-II inhibits the migration of naïve and activated NK cells towards Fck and RANTES. Thus, we present here a novel mechanism in which KSHV uses a unique protein that antagonizes the activity of two distinct chemokine receptors to inhibit the migration of naïve and activated NK cells.     

Herpesvirus chemokine-binding glycoprotein G (gG) efficiently inhibits neutrophil chemotaxis in vitro and in vivo

Glycoprotein G (gG) of alphaherpesviruses has been described to function as a viral chemokine-binding protein (vCKBP). More recently, mutant viruses devoid of gG have been shown to result in increased virulence, but it remained unclear whether the potential of gG to serve as a vCKBP is responsible for this observation. In this study, we used equine herpesvirus type 1 (EHV-1) as a model to study the pathophysiological importance of vCKBP activity. First, in vitro chemotaxis assays studying migration of immune cells, an important function of chemokines, were established. In such assays, supernatants of EHV-1-infected cells significantly inhibited IL-8-induced chemotaxis of equine neutrophils. Identification of gG as the responsible vCKBP was achieved by repeating similar experiments with supernatants from cells infected with a gG-negative mutant, which were unable to alter IL-8-induced equine neutrophil migration. Furthermore, rEHV-1 gG was able to significantly reduce neutrophil migration, establishing gG as a bona fide vCKBP. Second, and importantly, in vivo analyses in a murine model of EHV-1 infection showed that neutrophil migration in the target organ lung was significantly reduced in the presence of gG. In summary, we demonstrate for the first time that EHV-1 gG not only binds to chemokines but is also capable of inhibiting their chemotactic function both in vitro and in vivo, thereby contributing to viral pathogenesis and virulence.     

Relationships between glycosaminoglycan and receptor binding sites in chemokines-the CXCL12 example

Chemokines are small proteins, promoting directional migration and activation of different cells through binding to specific receptors. Most chemokines also bind to heparan sulfate (HS), a family of complex and highly sulfated glycosaminoglycan (GAG) found at the cell surface and in the extracellular matrix. This class of molecules has recently emerged as critical regulators of many events involving cell response to the external environment. Binding to HS is thought to be functionally important. Current models suggested that HS ensures the correct positioning of chemokines within tissues and maintains haptotactic gradients of the proteins along cell surfaces, thus providing directional cues for migrating cells. On the chemokine surface, the GAG binding epitopes can be displayed on different areas, some of which overlap the receptor binding domain, while others are clearly separated. We review here some structural aspects of the interaction between GAGs or receptors and chemokines. In particular, we will address the case of CXCL12, a chemokine whose receptor binding site is distinct from the GAG binding site and whose different isoforms display different GAG binding abilities. This chemokine system thus offers an unprecedented opportunity to ascertain the importance of chemokine/GAG interaction in the regulation of cell migration.     

Enhancement of Chemokine Function as an Immunomodulatory Strategy Employed by Human Herpesviruses

Herpes simplex virus (HSV) types 1 and 2 are highly prevalent human neurotropic pathogens that cause a variety of diseases, including lethal encephalitis. The relationship between HSV and the host immune system is one of the main determinants of the infection outcome. Chemokines play relevant roles in antiviral response and immunopathology, but the modulation of chemokine function by HSV is not well understood. We have addressed the modulation of chemokine function mediated by HSV. By using surface plasmon resonance and crosslinking assays we show that secreted glycoprotein G (SgG) from both HSV-1 and HSV-2 binds chemokines with high affinity. Chemokine binding activity was also observed in the supernatant of HSV-2 infected cells and in the plasma membrane of cells infected with HSV-1 wild type but not with a gG deficient HSV-1 mutant. Cell-binding and competition experiments indicate that the interaction takes place through the glycosaminoglycan-binding domain of the chemokine. The functional relevance of the interaction was determined both in vitro, by performing transwell assays, time-lapse microscopy, and signal transduction experiments; and in vivo, using the air pouch model of inflammation. Interestingly, and in contrast to what has been observed for previously described viral chemokine binding proteins, HSV SgGs do not inhibit chemokine function. On the contrary, HSV SgGs enhance chemotaxis both in vitro and in vivo through increasing directionality, potency and receptor signaling. This is the first report, to our knowledge, of a viral chemokine binding protein from a human pathogen that increases chemokine function and points towards a previously undescribed strategy of immune modulation mediated by viruses.     

Structural basis of chemokine sequestration by CrmD, a poxvirus-encoded tumor necrosis factor receptor

Pathogens have evolved sophisticated mechanisms to evade detection and destruction by the host immune system. Large DNA viruses encode homologues of chemokines and their receptors, as well as chemokine-binding proteins (CKBPs) to modulate the chemokine network in host response. The SECRET domain (smallpox virus-encoded chemokine receptor) represents a new family of viral CKBPs that binds a subset of chemokines from different classes to inhibit their activities, either independently or fused with viral tumor necrosis factor receptors (vTNFRs). Here we present the crystal structures of the SECRET domain of vTNFR CrmD encoded by ectromelia virus and its complex with chemokine CX3CL1. The SECRET domain adopts a β-sandwich fold and utilizes its β-sheet I surface to interact with CX3CL1, representing a new chemokine-binding manner of viral CKBPs. Structure-based mutagenesis and biochemical analysis identified important basic residues in the 40s loop of CX3CL1 for the interaction. Mutation of corresponding acidic residues in the SECRET domain also affected the binding for other chemokines, indicating that the SECRET domain binds different chemokines in a similar manner. We further showed that heparin inhibited the binding of CX3CL1 by the SECRET domain and the SECRET domain inhibited RAW264.7 cell migration induced by CX3CL1. These results together shed light on the structural basis for the SECRET domain to inhibit chemokine activities by interfering with both chemokine-GAG and chemokine-receptor interactions.     

HHV8-encoded vMIP-I selectively engages chemokine receptor CCR8. Agonist and antagonist profiles of viral chemokines.

Uncertainty regarding viral chemokine function is mirrored by an incomplete knowledge of host chemokine receptor usage by the virally encoded proteins. One such molecule is vMIP-I, a C-C type chemokine of undefined function and binding specificity, encoded by the Kaposi's sarcoma herpesvirus HHV-8. We report here that vMIP-I binds to and induces cytosolic [Ca(2+)] signals in human T cells selectively through CCR8, a CC chemokine receptor associated with Th2 lymphocytes. Furthermore, using a panel of 65 different human, viral, and rodent chemokines, we have established a comprehensive ligand binding "fingerprint" for CCR8. The receptor exhibits marked "high" affinity (K(d) < 15 nM) only for four chemokines, three of them of viral origin: vMIP-I, vMIP-II, vMCC-I, and human I-309. A previously unreported second class of lower affinity ligands includes MCP-3 and possibly two other viral chemokines. vMIP-I and I-309 appear to act as CCR8 agonists: binding to and inducing cytosolic [Ca(2+)] elevation through the receptor. By contrast, vMIP-II and vMCC-I act as potent antagonists: binding without inducing signaling, and blocking the effects of I-309 and vMIP-I. These results suggest a ligand hierarchy for CCR8, identifying vMIP-I as a selective viral chemokine agonist. CCR8 may thus engage a specific subset of chemokines with the potential to regulate each other during viral infection and immune regulation.     

Intracellular signalling pathways induced by chemokines in natural killer cells.

Chemokines are small peptides involved in the recruitment of various cell types into inflammatory sites. They are divided into four sub-families depending on the presence of amino acids separating the cysteine residues in their N-terminal region. These are the alpha (CXC), beta (CC), gamma (C) and delta (CX)C) chemokines. In addition, five CXC chemokine (CXCR1-5), nine CC chemokine (CCR1-9), one C chemokine (XCR1) and one C-X3C chemokine (CX3CR1) receptors have been identified. These receptors belong to the seven transmembrane spanning domain family, and are coupled to the heterotrimeric guanine nucleotide binding (G) proteins. Chemokines activate various immune cells, and in particular the anti-viral/anti-tumour effectors, the natural killer (NK) cells by activating members of the heterotrimeric G proteins. The importance of the family of chemokines is highlighted by the ability of its members to inhibit the replication of HIV-1 strains in CD4+ cells, where chemokine receptors act as HIV-1 co-receptors. This review discusses the intracellular signalling pathways induced by chemokines in NK and other cell types, and the relationships to HIV-1 signalling in these cells.     

The Multiple Chemokine–Binding Bovine Herpesvirus 1 Glycoprotein G (BHV1gG) Inhibits Polymorphonuclear Cell but Not Monocyte Migration into Inflammatory Sites

Chemokines facilitate the recruitment of inflammatory cells into tissues, contributing to target organ injury in a wide range of inflammatory and autoimmune diseases. Targeting either single chemokines or chemokine receptors alters the progression of disease in animal models of rheumatoid arthritis and lupus with varying degrees of efficacy, but clinical trials in humans have been less successful. Given the redundancy of chemokine–chemokine receptor interactions, targeting of more than one chemokine may be required to inhibit active inflammatory disease. To test the effects of multiple chemokine blockade in inflammation, we generated an adenovirus expressing bovine herpesvirus 1 glycoprotein G (BHV1gG), a viral chemokine antagonist that binds to a wide spectrum of murine and human chemokines, fused to the fragment crystallizable (Fc) portion of murine immunoglobulin (IgG)2a. Administration of the adenovirus significantly inhibited thioglycollate-induced migration of polymorphonuclear leukocytes into the peritoneal cavity of BALB/c mice and reduced both clinical severity and articular damage in K/BxN serum transfer-induced arthritis. However, treatment with BHV1gG-Ig fusion protein did not prevent monocyte infiltration into the peritoneum in the thioglycollate model and did not prevent renal monocyte infiltration or nephritis in lupus-prone NZB/W mice. These observations suggest that the simultaneous inhibition of multiple chemokines by BHV1gG has the potential to interfere with acute inflammatory responses mediated by polymorphonuclear leukocytes, but is less effective in chronic inflammatory disease mediated by macrophages.     

Human herpesvirus 7 open reading frame U12 encodes a functional beta-chemokine receptor

Human herpesvirus 7 (HHV-7), which belongs to the betaherpesvirus subfamily, infects mainly CD4+ T cells in vitro and infects children during infancy. After the primary infection, HHV-7 becomes latent. HHV-7 contains two genes (U12 and U51) that encode putative homologs of cellular G-protein-coupled receptors. To analyze the biological function of the U12 gene, we cloned the gene and expressed the U12 protein in cells. The U12 gene encoded a calcium-mobilizing receptor for the EBI1 ligand chemokine-macrophage inflammatory protein 3beta (ELC/MIP-3beta) but not for other chemokines, suggesting that the chemokine selectivity of the U12 gene product is distinct from that of the known mammalian chemokine receptors. These studies revealed that U12 activates distinct transmembrane signaling pathways that may mediate biological functions by binding with a beta-chemokine, ELC/MIP-3beta.     

Human herpesvirus-8-encoded signalling ligands and receptors

Analysis of the genome of human herpesvirus 8 (HHV-8) led to the discovery of several novel genes, unique among the characterized gammaherpesviruses. These include cytokines (interleukin-6 and chemokine homologues), two putative signal-transducing transmembrane proteins encoded by genes K1 and K15 at the genome termini, and an OX-2 (CD200) receptor homologue that had not previously been identified in a gammaherpesvirus. HHV-8 also specifies a diverged version of the gammaherpesvirus-conserved G protein-coupled chemokine receptor (vGCR) and a latently expressed protein unique to HHV-8 specified by open reading frame (ORF) K12. These cytokine and receptor homologues mediate signal transduction or modulate the activities of other endogenous cytokines and receptors to enhance viral productive replication, regulate latent-lytic switching, evade host attack, or mediate cell survival. The viral signalling ligands and receptors are also potential contributors to virus-associated diseases, Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease, and so represent potentially important targets for therapeutic and antiviral drugs. Understanding these proteins' modes of action and functions in viral biology and disease is therefore of considerable importance, and the subject of this review.     

Structural determinants of chemokine binding by an Ectromelia virus-encoded decoy receptor

The EVM1 protein encoded by Ectromelia virus is a member of a highly conserved family of poxvirus chemokine binding proteins that interfere with host immune surveillance processes. EVM1 is abundantly expressed early during mousepox infection and is able to selectively bind CC chemokines and inhibit their interactions with host receptors. Here, we characterize the interaction between EVM1 and the human and murine chemokines CCL3 (MIP-1alpha), CCL2 (MCP-1), and CCL5 (RANTES). Each of these CC chemokines binds EVM1 with 1:1 stoichiometry and equilibrium dissociation constants ranging from 29 pM to 20 nM. The interactions are characterized by rapid-association kinetics between acidic EVM1 and generally basic chemokines with half-lives enduring up to 30 min. The 2.6-A crystal structure of EVM1 reveals a globular beta sandwich with a large, sequence-conserved surface patch encircled by acidic residues on one face of the protein. To determine whether this conserved cluster of residues is involved in chemokine engagement, a structure-based mutational analysis of EVM1 was employed. Mapping of the mutational results onto the surface of EVM1 reveals that a cluster of five residues (I173, S171, S134, N136, and Y69) emanating from one beta sheet is critical for CCL2 and CCL3 sequestration. Additionally, we find that the extended beta2-beta4 loop flanking this conserved cluster is also essential for high-affinity, lasting interactions with chemokines. This analysis provides insight into the mechanism of CC-chemokine inhibition employed by the poxvirus family of chemokine decoy receptors.     

Structural basis of chemokine sequestration by a herpesvirus decoy receptor

The M3 protein encoded by murine gamma herpesvirus68 (gamma HV68) functions as an immune system saboteur by the engagement of chemoattractant cytokines, thereby altering host antiviral inflammatory responses. Here we report the crystal structures of M3 both alone and in complex with the CC chemokine MCP-1. M3 is a two-domain beta sandwich protein with a unique sequence and topology, forming a tightly packed anti-parallel dimer. The stoichiometry of the MCP-1:M3 complex is 2:2, with two monomeric chemokines embedded at distal ends of the preassociated M3 dimer. Conformational flexibility and electrostatic complementation are both used by M3 to achieve high-affinity and broad-spectrum chemokine engagement. M3 also employs structural mimicry to promiscuously sequester chemokines, engaging conservative structural elements associated with both chemokine homodimerization and binding to G protein-coupled receptors.     

Structural Basis of Chemokine Sequestration by a Tick Chemokine Binding Protein: The Crystal Structure of the Complex between Evasin-1 and CCL3

Background

Chemokines are a subset of cytokines responsible for controlling the cellular migration of inflammatory cells through interaction with seven transmembrane G protein-coupled receptors. The blocking of a chemokine-receptor interaction results in a reduced inflammatory response, and represents a possible anti-inflammatory strategy, a strategy that is already employed by some virus and parasites. Anti-chemokine activity has been described in the extracts of tick salivary glands, and we have recently described the cloning and characterization of such chemokine binding proteins from the salivary glands, which we have named Evasins.

Methodology/Principal Findings

We have solved the structure of Evasin-1, a very small and highly selective chemokine-binding protein, by x-ray crystallography and report that the structure is novel, with no obvious similarity to the previously described structures of viral chemokine binding proteins. Moreover it does not possess a known fold. We have also solved the structure of the complex of Evasin-1 and its high affinity ligand, CCL3. The complex is a 1∶1 heterodimer in which the N-terminal region of CCL3 forms numerous contacts with Evasin-1, including prominent π-π interactions between residues Trp89 and Phe14 of the binding protein and Phe29 and Phe13 of the chemokine.

Conclusions/Significance

However, these interactions do not appear to be crucial for the selectivity of the binding protein, since these residues are found in CCL5, which is not a ligand for Evasin-1. The selectivity of the interaction would appear to lie in the N-terminal residues of the chemokine, which form the “address” whereas the hydrophobic interactions in the rest of the complex would serve primarily to stabilize the complex. A thorough understanding of the binding mode of this small protein, and its other family members, could be very informative in the design of potent neutralizing molecules of pro-inflammatory mediators of the immune system, such as chemokines.     

Recombinant Herpesvirus Glycoprotein G Improves the Protective Immune Response to Helicobacter pylori Vaccination in a Mouse Model of Disease

Alphaherpesviruses, which have co-evolved with their hosts for more than 200 million years, evade and subvert host immune responses, in part, by expression of immuno-modulatory molecules. Alphaherpesviruses express a single, broadly conserved chemokine decoy receptor, glycoprotein G (gG), which can bind multiple chemokine classes from multiple species, including human and mouse. Previously, we demonstrated that infection of chickens with an infectious laryngotracheitis virus (ILTV) mutant deficient in gG resulted in altered host immune responses compared to infection with wild-type virus. The ability of gG to disrupt the chemokine network has the potential to be used therapeutically. Here we investigated whether gG from ILTV or equine herpesvirus 1 (EHV-1) could modulate the protective immune response induced by the Helicobacter pylori vaccine antigen, catalase (KatA). Subcutaneous immunisation of mice with KatA together with EHV-1 gG, but not ILTV gG, induced significantly higher anti-KatA IgG than KatA alone. Importantly, subcutaneous or intranasal immunisation with KatA and EHV-1 gG both resulted in significantly lower colonization levels of H. pylori colonization following challenge, compared to mice vaccinated with KatA alone. Indeed, the lowest colonization levels were observed in mice vaccinated with KatA and EHV-1 gG, subcutaneously. In contrast, formulations containing ILTV gG did not affect H. pylori colonisation levels. The difference in efficacy between EHV-1 gG and ILTV gG may reflect the different spectrum of chemokines bound by the two proteins. Together, these data indicate that the immuno-modulatory properties of viral gGs could be harnessed for improving immune responses to vaccine antigens. Future studies should focus on the mechanism of action and whether gG may have other therapeutic applications.     

Identification of a gammaherpesvirus selective chemokine binding protein that inhibits chemokine action

Chemokines are involved in recruitment and activation of hematopoietic cells at sites of infection and inflammation. The M3 gene of gammaHV68, a gamma-2 herpesvirus that infects and establishes a lifelong latent infection and chronic vasculitis in mice, encodes an abundant secreted protein during productive infection. The M3 gene is located in a region of the genome that is transcribed during latency. We report here that the M3 protein is a high-affinity broad-spectrum chemokine scavenger. The M3 protein bound the CC chemokines human regulated upon activation of normal T-cell expressed and secreted (RANTES), murine macrophage inflammatory protein 1alpha (MIP-1alpha), and murine monocyte chemoattractant protein 1 (MCP-1), as well as the human CXC chemokine interleukin-8, the murine C chemokine lymphotactin, and the murine CX(3)C chemokine fractalkine with high affinity (K(d) = 1. 6 to 18.7 nM). M3 protein chemokine binding was selective, since the protein did not bind seven other CXC chemokines (K(d) > 1 microM). Furthermore, the M3 protein abolished calcium signaling in response to murine MIP-1alpha and murine MCP-1 and not to murine KC or human stromal cell-derived factor 1 (SDF-1), consistent with the binding data. The M3 protein was also capable of blocking the function of human CC and CXC chemokines, indicating the potential for therapeutic applications. Since the M3 protein lacks homology to known chemokines, chemokine receptors, or chemokine binding proteins, these studies suggest a novel herpesvirus mechanism of immune evasion.     

Examination of the function of RANTES, MIP-1alpha, and MIP-1beta following interaction with heparin-like glycosaminoglycans

Chemokines are a group of small proteins that have a variety of functions, including the activation and recruitment of immune cells during episodes of inflammation. In common with many cytokines, it has been observed that chemokines have the potential to bind heparin-like glycosaminoglycan molecules, which are normally expressed on proteoglycan components of the cell surface and extracellular matrix. The significance of this interaction for chemokine activity remains a subject of debate. In this study, Chinese hamster ovary cells were transfected separately with the human chemokine receptors CCR1 and CCR5, and these receptors were shown to induce an intracytoplasmic Ca(2+) flux and cellular chemotaxis following stimulation with the natural CC chemokine ligands (MIP-1alpha, RANTES (regulated on activation normal T cell expressed), and MIP-1beta). In further experiments, mutant CHO cells, with a defect in normal glycosaminoglycan (GAG) expression, were also transfected with, and shown to express similar levels of, CCR1 and CCR5. Although these receptors were functional, it was found that the mutant cells required exposure to higher concentrations of ligands than the wild-type cells in order to produce the same intracytoplasmic Ca(2+) flux. Radioligand binding experiments demonstrated that specific chemokine receptors expressed by wild-type cells had a significantly greater affinity for MIP-1alpha than similar receptors expressed by GAG-deficient mutants. However, there was no significant difference between these cells in their affinity for RANTES or MIP-1beta. In conclusion, it has been demonstrated clearly that GAG expression is not necessary for the biological activity of the chemokines MIP-1alpha, RANTES, or MIP-1beta. However, the presence of cell surface GAGs does enhance the activity of low concentrations of these chemokines by a mechanism that appears to involve sequestration onto the cell surface.     

The bovine herpesvirus 4 Bo10 gene encodes a nonessential viral envelope protein that regulates viral tropism through both positive and negative effects

All gammaherpesviruses encode a glycoprotein positionally homologous to the Epstein-Barr virus gp350 and the Kaposi's sarcoma-associated herpesvirus (KSHV) K8.1. In this study, we characterized the positional homologous glycoprotein of bovine herpesvirus 4 (BoHV-4), encoded by the Bo10 gene. We identified a 180-kDa gene product, gp180, that was incorporated into the virion envelope. A Bo10 deletion virus was viable but showed a growth deficit associated with reduced binding to epithelial cells. This seemed to reflect an interaction of gp180 with glycosaminoglycans (GAGs), since compared to the wild-type virus, the Bo10 mutant virus was both less infectious for GAG-positive (GAG(+)) cells and more infectious for GAG-negative (GAG(-)) cells. However, we could not identify a direct interaction between gp180 and GAGs, implying that any direct interaction must be of low affinity. This function of gp180 was very similar to that previously identified for the murid herpesvirus 4 gp150 and also to that of the Epstein-Barr virus gp350 that promotes CD21(+) cell infection and inhibits CD21(-) cell infection. We propose that such proteins generally regulate virion attachment both by binding to cells and by covering another receptor-binding protein until they are displaced. Thus, they regulate viral tropism both positively and negatively depending upon the presence or absence of their receptor.