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(Journal of Leukocyte Biology. 2002;72:24-34.)
© 2002 by Society for Leukocyte Biology

Viral chemokine-binding proteins

Bruce T. Seet and Grant McFadden

Department of Microbiology and Immunology, The University of Western Ontario, London, Canada; and Viral Immunology and Pathogenesis Laboratories, The John P. Robarts Research Institute, London, Ontario, Canada

Correspondence: Grant McFadden, The John P. Robarts Research Institute, 1400 Western Road, SDRI Room 133, London, Ontario N6G 2V4 Canada. E-mail: mcfadden{at}rri.on.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 POXVIRUS LOW AFFINITY CKBP...
 POXVIRUS HIGH AFFINITY CKBP-II
 HERPESVIRUS HIGH AFFINITY CKBP...
 VIRAL CKBPs AS THERAPEUTIC...
 CONCLUSION
 REFERENCES
 
The chemokines are a large family of small signaling proteins that bind to G-protein-coupled receptors (GPCRs) on target cells and mediate the directional migration of immune cells into sites of infection or inflammation. The large DNA viruses, particularly the poxviruses and herpesviruses, have evolved several mechanisms to corrupt the normal functioning of the chemokine network. Two strategies rely on mimicking chemokines or chemokine receptors. A third strategy involves the production of secreted chemokine-binding proteins (CKBPs) that exhibit no sequence similarity to any known host proteins, yet function to competitively bind and inhibit the interactions of chemokines with cognate receptors. Each strategy has provided unique insights into the elusively complex world of the chemokines. Here, we focus on recent advances made in the understanding of secreted CKBPs encoded by poxviruses and herpesviruses. A better understanding of how viral CKBPs function to manipulate the immune response may provide further clues as to how to develop specific therapeutic agents to abrogate chemokine-mediated disease conditions.

Key Words: poxvirus • herpesvirus • chemokine receptor • viral immune evasion • viral chemokine inhibitor • chemokine antagonist • vCCI • CKBP


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
POXVIRUS LOW AFFINITY CKBP...
 POXVIRUS HIGH AFFINITY CKBP-II
 HERPESVIRUS HIGH AFFINITY CKBP...
 VIRAL CKBPs AS THERAPEUTIC...
 CONCLUSION
 REFERENCES
 
The successful propagation of viruses within the mammalian host requires the skillful evasion or manipulation of the host immune arsenal [1 2 3 4 ]. Large DNA viruses, particularly the poxviruses and herpesviruses, provide some of the most extensive inventories of gene products that serve to defend these viruses against the aggressive assault executed by the host inflammatory response. In addition to producing proteins that systematically undermine the interferon (IFN) system, the tumor necrosis factors (TNF), various interleukins (ILs), complement and major histocompatibility complex presentation [1 , 5 6 7 8 9 10 ], the chemokine network represents a significant target for disruption or exploitation by these viruses [3 , 4 , 11 , 12 ].

Chemokines comprise a large family of small, 6–14 kDa proteins that function to directionally attract and activate leukocytes during processes of inflammation and infection [13 14 15 16 17 ]. Based on the presence and relative positioning of the N-terminal cysteine residue(s), chemokines are divided into four classes: CXC ({alpha}), CC (ß), CX3C ({gamma}), and C ({delta}) (where X is any residue). Chemokine receptors are expressed differentially on leukocyte subsets as G-protein-coupled receptors (GPCRs), which generally only recognize a small group of chemokines restricted to a given class [17 ]. Thus, CXC-chemokines tend to bind to CXC-chemokine receptors (CXCRs) and function to attract neutrophils, whereas CC-chemokines generally bind to CC-chemokine receptors (CCRs) and tend to attract monocytes, lymphocytes, eosinophils, and basophils. The C-chemokine, lymphotactin (XCL1), binds to XCR1 and is shown to induce neutrophil and T and B cell migration [18 19 20 ]. Finally, the CX3C-chemokine, fractalkine (CX3CL), is capable of functioning as a membrane-anchored form or as a shed ligand and binds CX3CR1 on blood-derived neutrophils, monocytes, natural killer cells, and T lymphocytes [21 , 22 ].

Chemokines are also believed to form solid-phase gradients through interactions with endothelial or matrix glycosaminoglycan (GAG) molecules, which are found as the post-translational modifications of proteoglycans [23 24 25 26 ]. Such chemokine gradients are thought to facilitate the luring of leukocytes along and into sites of infection or inflammation through a complex process of rolling, adhesion, and extravasation [26 27 28 29 ]. Moreover, cell surface GAG interactions may regulate the activity of chemokines by sequestering chemokines to sites of inflammation or facilitating the presentation of chemokines to their receptors [30 31 32 33 ]. Given the importance of chemokines in coordinating the proper maneuvering of immune cells to sites of infection, the high affinity interactions of chemokines with chemokine receptors and the low affinity interactions with GAGs offer two attractive targets for disruption by viruses [34 ].

Since the first identification of chemokine receptor homologs in herpesviruses over a decade ago [35 ], members of the herpesvirus and poxvirus families continue to furnish an impressive and expanding number of proteins that modulate the chemokines (Fig. 1 ). These virus-encoded proteins commonly conform to one of three general strategies that include the production of proteins with homology to chemokines, proteins that resemble chemokine GPCRs, or secreted chemokine-binding proteins (CKBPs) that competitively interact with chemokines and prevent chemokine interactions with cellular host GPCRs or GAGs (Fig. 2 and Table 1 ) [3 , 4 , 11 , 35 , 40 , 42 , 47 , 48 , 56 , 57 ]. The observation that viruses produce and deploy proteins that function to modulate or mitigate the normal functioning of the complex network of chemokines underscores the significant influence of the chemokines in coordinating the host immune response during viral infection [4 , 11 , 61 ]. Here, we will focus on the secreted CKBPs encoded by the poxviruses and the herpesviruses and the insights these proteins have provided in understanding the chemokines.



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  		Figure 1. Virus modulation of the chemokine network. Depicted are the viral gene products (inner circle) that are chemokine receptor homologs (CXCRs or CCRs), chemokine homologs (CC or CXC), or secreted chemokine-binding proteins (CKBPs) that bear no homology to any known proteins. Host or viral ligands that bind viral chemokine receptors or viral CKBPs are illustrated on the outer circle. Similarly, host or viral receptors that are engaged by viral chemokines are also depicted on the outer circle. Ligands and receptors (outer circle) that are affected agonistically by the viral gene products are shown in blue; those affected antagonistically by viral gene products are shown in red. Those labeled in black text bind the viral gene product, but the interaction is uncharacterized. Note that some chemokines may activate a viral receptor positively but may be sequestered (e.g., hCMV US28). Inverse agonists are not shown. Virus abbreviations: CapV, capripoxvirus; FPV, fowlpox virus; EHV, HHV, and MHV, equine, human, and mouse herpesvirus, respectively; HSV, herpes saimiri virus; hCMV and mCMV, human and mouse cytomegalovirus, respectively; LSDV, lumpy skin disease virus; MDV, Marek’s disease virus; MC, molluscum contagiosum; SPV, swinepox virus; YLDV, Yaba-like disease virus. For a full review of the information depicted here, see refs [3 , 4 , 10 , 12 , 36 37 38 39 ].

 


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  		Figure 2. Schematic of the strategies used by poxviruses or herpesviruses to disrupt the chemokines. (A) Viral chemokine homologs can function as agonists to promote the influx of immune cells into sites of infection aiding in virus dissemination or as antagonists to competitively inhibit the binding of host chemokines to cognate receptors (reviewed in refs [3 , 4 ]). (B) Viral chemokine receptor homologs can function as antagonists to bind and sequester chemokines or as agonists that are constitutively active or activated inducibly by chemokines (reviewed in refs [4 , 11 ]). (C) Secreted viral CKBPs can competitively block the binding of chemokines to cellular chemokine receptors (as shown) or can block GAG-bound chemokine gradients (not shown).

 

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  		Table 1. Secreted Viral Chemokine-Binding Proteins

 

   POXVIRUS LOW AFFINITY CKBP TYPE I (CKBP-I)
TOP
 ABSTRACT
 INTRODUCTION
 POXVIRUS LOW AFFINITY CKBP...
POXVIRUS HIGH AFFINITY CKBP-II
 HERPESVIRUS HIGH AFFINITY CKBP...
 VIRAL CKBPs AS THERAPEUTIC...
 CONCLUSION
 REFERENCES
 
The myxoma virus is a pathogenic poxvirus that causes a highly lethal disease in European rabbits called myxomatosis [62 ]. Like other poxviruses, myxoma virus encodes a secreted IFN-{gamma} receptor (IFN-{gamma}R) homolog, termed M-T7, which binds IFN-{gamma} with high affinity and inhibits binding of IFN-{gamma} to cellular IFN-{gamma}Rs [63 , 64 ]. However, unlike other poxvirus IFN-{gamma}R homologs, M-T7 was uniquely shown to bind CXC, CC, and C-chemokines with no apparent species restriction and was subsequently given a second designation as CKBP-I [40 ].

The binding of chemokines by CKBP-I was shown to be a low affinity interaction and could be blocked by heparin, suggesting that the heparin-binding domain of chemokines mediates the interaction [40 ]. Furthermore, the N-terminal region of IL-8, which is involved in the high affinity interaction of IL-8 with its cellular receptor, was dispensable for the interaction with CKBP-I [40 ]. Conversely, the C-terminal region of IL-8, a region that constitutes the heparin-binding domain of IL-8, was shown necessary for the interaction with CKBP-I [25 , 40 ]. Rabbit IFN-{gamma} could compete for the chemokine interaction with CKBP-I, suggesting that the chemokine-binding domain and the IFN-{gamma}-binding domain on M-T7 may overlap.

It has been proposed that binding the heparin-binding domain of chemokines by CKBP-I may disrupt GAG-dependent cell-surface gradients formed by chemokines [40 ]. In vivo evidence suggesting that a haptotactic chemokine gradient may be impaired by CKBP-I during viral infection is the observation that infection of rabbits with a recombinant myxoma virus with a disruption of the CKBP-I gene dramatically increases the focalized migration of leukocytes into sites of infection [65 ]. The extent to which the inhibition of IFN-{gamma} or chemokines by CKBP-I plays in mediating this phenotype has yet to be determined. However, it has been shown that CKBP-I can mediate the inhibition of inflammatory cells in rodent model systems of inflammation in which CKBP-I cannot inhibit IFN-{gamma} and is free to exert its species nonspecific inhibition of chemokines (described below) [41 ].


   POXVIRUS HIGH AFFINITY CKBP-II
TOP
 ABSTRACT
 INTRODUCTION
 POXVIRUS LOW AFFINITY CKBP...
 POXVIRUS HIGH AFFINITY CKBP-II
HERPESVIRUS HIGH AFFINITY CKBP...
 VIRAL CKBPs AS THERAPEUTIC...
 CONCLUSION
 REFERENCES
 
Many poxviruses produce a secreted CKBP that binds to many CC-chemokines with high affinity and competitively inhibit CC-chemokines from binding their cognate high affinity cellular receptors [42 , 47 , 48 ]. Members of this family of related proteins have been called type II CKBPs (CKBP-II), viral CC-chemokine inhibitors (vCCI), or the T1/35 kDa family of CC-CKBPs, reflecting the nomenclature of the T1 genes from leporipoxviruses and the 35 kDa genes from the orthopoxviruses. Binding and inhibition studies of chemokines by CKBP-II have demonstrated a specificity for CC-chemokines, but not C, CXC, and CX3C chemokines [42 , 47 , 48 , 54 , 55 ]. Unlike cellular chemokine receptors that typically only recognize restricted subgroups of chemokines, these poxvirus CKBPs are capable of interacting with many CC chemokines, frequently with picomolar- to nanomolar-affinity constants, and can effectively block the binding of these chemokines to their receptors [42 , 47 , 48 , 55 ]. Remarkably, the amino acid sequences of CKBP-II proteins bear no resemblance to any known chemokine receptor, GPCR, or mammalian protein [42 , 47 , 48 , 55 ].

Kinetic analyses of the binding of the vaccinia virus (VV) CKBP-II (also known as VV-35 kDa, B29R, or vCCI) with the human monocyte chemoattractant protein-1 (MCP-1) determined that the high affinity interaction is a result of very rapid association rates (kon=>106 M-1s-1) and slow dissociation rates (koff=<10-2s-1), providing very favorable kinetic parameters for the competitive inhibition of CC-chemokines [52 , 53 ]. A functional comparison of two orthopoxvirus CC CKBPs, the VV-CKBP-II protein and the rabbitpox virus 35 kDa protein (RPV-CKBP-II), with the myxoma virus (MV) CKBP-II (also known as M-T1), demonstrated that despite possessing only 40% identity, these CKBPs have very similar in vitro capabilities of inhibiting CC-chemokine-induced signaling and chemotaxis [43 ].

Disruption of the M-T1 gene from myxoma virus or the disruption of the RPV-CKBP-II in rabbitpox virus resulted in an increase in early leukocyte infiltration into tissue sites of virus infection, although there was little or no effect on overall virulence or lethality of either deletion virus [42 , 44 , 46 ]. However, the observation that these poxvirus CKBP-II proteins can potently inhibit the in vivo accumulation of inflammatory leukocytes in the context of virus infection [42 , 44 ] and as purified proteins [48 , 50 ] underscores the promise of these proteins as potential anti-inflammatory therapeutic agents.

Structural aspects of the interaction of poxvirus CKBP-II with CC-chemokines
The unique, conserved sequences of the poxvirus CKBP-II proteins and their capacity to interact with many CC-chemokines from various species have generated much interest in defining the structural basis for how this family of proteins broadly recognizes and inhibits these ligands. The X-ray crystal structure of the cowpox virus (CPV) CKBP-II protein confirmed that the structure is indeed unlike any GPCR or known mammalian receptor, although it possesses a structural fold that distantly resembles the collagen-binding domain of Staphylococcus aureus adhesin [49 ]. The CPV-CKBP-II structure possesses two short {alpha}-helices and two parallel ß-sheets (ß-sheets I and II) that form a ß-sandwich of novel topology [49 ]. Importantly, the structure revealed a negatively charged surface within ß-sheet II of the CPV-CKBP-II that contains a region of relatively conserved residues found among other members of the poxvirus CKBP-II proteins [49 ]. It was predicted that this region might be a potential binding site for positively charged chemokines, although this has yet to be confirmed experimentally [49 ].

The ability of CKBP-II to prevent CC-chemokines from binding to cellular receptors suggested that cellular CCRs and CKBP-II likely recognize overlapping surfaces on CC chemokines [48 , 49 , 52 , 53 ]. Early characterization demonstrated that the CKBP-II binding site on CC-chemokines likely does not involve the heparin-binding domain of the chemokine, as soluble heparin could not inhibit the interaction competitively [48 ]. To define the precise binding site on CC-chemokines used by the poxvirus CKBP-II proteins, two recent studies used surface plasmon resonance analysis to identify the CKBP-II binding site on a CC-chemokine [52 , 53 ].

Both studies used a panel of independently constructed, site-directed mutants of the human CC-chemokine MCP-1 whose surface-exposed residues were altered individually to alanine in order to evaluate the effect of individual residues on the binding of MCP-1 to a model CKBP-II [52 , 53 ]. To this end, both studies also used the VV-CKBP-II to screen for binding against the panel of MCP-1 mutants. However, Beck et al. [53 ] used an Fc fusion of the VV-CKBP-II protein immobilized onto an anti-human Fc-antibody surface, whereas Seet et al. [52 ] used a VV-CBKP-II Fc-fusion protein captured on an immobilized protein A surface as well as a purified baculovirus-expressed VV-CKBP-II protein that was immobilized directly to a Biacore chip using amine-coupling chemistry. Despite using different coupling strategies and forms of the CKBP-II, most of the affinity constants derived for each mutant were remarkably similar, although some of the minor decreases in affinity were not completely in agreement (Table 2 ). Nonetheless, the general conclusions were consistent between the two studies.


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  		Table 2. Affinity Constants Generated by Two Independent Biacore Studies with VV-CKBP-II-Binding MCP-1 Mutants

 
Seet et al. [52 ] determined that the interaction affinity between VV-CKBP-II and wild-type (wt) human MCP-1 possessed a KD of 340 pM (kon=5.6x107 M-1s-1; koff=1.9x10-2s-1), while Beck et al. [53 ] determined that the VV-CKBP-II Fc fusion interacted with wt MCP-1 with a KD of 294 pM (kon=2.47x106 M-1s-1; koff=6.79x10-4s-1). The observation that the affinity constants are within the same range for human MCP-1 binding to its receptor, CCR2b (KD=35–440 pM; refs [66 67 68 ]), suggests that VV-CKBP-II, at relatively low concentrations, can efficiently compete and inhibit MCP-1 binding to its receptor. Both studies arrived at the conclusion that the surface residues on MCP-1 required for the interaction with VV-35 kDa define an overlapping region on MCP-1 that is also used by CCR2b [52 , 53 , 66 ]. Both studies identified Y13, R18, and R24 as major MCP-1-binding determinants for VV-CKBP-II and also identified two other residues (K19 and K38) that led to modest two- and fivefold decreases in the affinity of MCP-1 for VV-CKBP-II. Importantly, these residues correspond to findings that CCR2b uses residues Y13, R24, K35, K38, K49, and to a lesser extent, R18 and K19 on MCP-1 [66 ] and indicate that CKBP-II proteins function by occluding the CCR2b-binding site on MCP-1 [52 , 53 ]. Moreover, it was noted that MCP-1 residues important for binding GAGs [69 ] were not involved in the MCP-1 interaction with VV-CKBP-II [53 ], confirming observations that heparin does not compete for the binding of chemokines to VV-CKBP-II [48 ].

Several MCP-1 surface residues when changed to alanine actually increased the affinity of the interaction with VV-CKBP-II [52 , 53 ]. For example, both studies observed that the K49A (defined as lysine replaced with alanine) mutant increased the affinity by approximately two- to threefold. Beck et al. [53 ] also observed a 2.5-fold increase in affinity for the V9A mutant and proposed that the removal of the valine side chain may result in a small conformational change leading to better access to the critical Y13 residue. Similarly, it was argued that the removal of K49, which is positioned close to the important R24 residue, might facilitate the interaction of MCP-1 with VV-CKBP-II [53 ]. The exact reason why these MCP-1 mutations result in increased affinities for VV-CKBP-II remain unknown; however, these residue positions might be important for determining chemokine specificity for CKBP-II proteins or CCRs.

A recent, comprehensive analysis of the binding profile of the VV-CKBP-II with an extensive panel of more than 80 chemokines has provided further insights into the specificity of CKBP-II interactions for chemokines [54 ]. Using this binding profile and inspecting the conservation of the corresponding residues on MCP-1 involved in the interaction with VV-CKBP-II, it is possible to better predict the possible physicochemical rules that govern the specificity of these interactions. For instance, among those chemokines that bind with high affinity to VV-CKBP-II, the residue corresponding to Y13 of MCP-1 is conserved primarily as a hydrophobic residue that can be aromatic (Tyr, Phe) or not (Leu or Ile), and R18 is usually conserved as a basic residue (Fig. 3 ). Residues that correspond to R24 of MCP-1 appear as basic residues (Arg, Lys) or hydrophobic residues (Phe, Leu, Val). Importantly, those chemokines that had low or no affinity to VV-CKBP-II [54 ] often lacked one or more of these residues, although MCP-2 is one exception (Fig. 3) . It should also be noted that the absence of lysine at position 49 or valine at position 9 among CC chemokines might compensate for the absence of those residues that are required for increased affinity. The balance between the presence of residues that increase affinity and those that decrease affinity likely plays a role in determining specificity and affinity.



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  		Figure 3. Alignment of CC-chemokines that bind VV-CKBP-II. Alignment is based on an analysis of the ability of more than 80 chemokines to inhibit the binding of a signature CC-chemokine to VV-CKBP-II Fc-fusion protein [54 ]. Shown are CC-chemokines that are either considered high affinity interacting CC-chemokines (upper set) or those that are considered to have lower to no affinity (lower set). Notably, CC-chemokines that are considered to have low-to-no affinity exhibit a lack of conserved residues at key residues (shown in color) that mediate VV-CKBP-II interaction (see text). Sequences shown are truncated at the N-terminus (before the dicysteine motif) and at the C-terminus (three residues after the third conserved cysteine).

 
It has been noted that the MCP-1-binding face used by CKBP-II corresponds to a common binding region for recognition by CCRs on other CC chemokines [70 71 72 ]. Together, the conserved interaction epitopes among CC chemokines for binding to CKBP-II and the general use of this same molecular face by CCRs help define how CKBP-II may be able to achieve promiscuous interaction and inhibition specificity toward many CC-chemokines [52 , 53 ]. Overall, these findings are consistent with the notion that a relatively small number of conserved surface residues on CC-chemokines, which overlap with CCR-binding regions, dictate the binding affinity and recognition specificity required for the inhibitory interaction with CKBP-II.

Stoichiometry of the CKBP-II interaction with MCP-1
Although the interaction stoichiometry of CKBP-II with CC-chemokines was demonstrated to be 1:2 originally [48 ], the interaction was detected recently as a 1:1 complex [52 ]. The different conclusions were based on observations derived from two different techniques that examined the interactions of different chemokines with the VV-CKBP-II Fc-fusion protein. The former deduction was based on observations that demonstrated two chemokines binding the viral CKBP-II in an electrophoretic mobility shift assay after chemical cross-linking in addition to calculations based on Scatchard analysis of data derived from a scintillation proximity assay [48 ].

The latter deduction was based on observations using surface plasmon resonance technology, which can monitor the precise amount of protein bound on the chip surface from which mass ratios, molecular mass ratios, and binding at saturation can be used effectively to calculate valency—in this case, two MCP-1 for each dimer of VV-CKBP-II Fc [52 ]. To assess if MCP-1 bound as a dimer or monomer to the VV-CKBP-II Fc, a monomeric mutant of MCP-1 (proline-altered to alanine at position 8, P8A), was shown to have identical saturation-binding profiles on a defined surface of noncovalently captured VV-CKBP-II Fc-fusion protein (Fig. 4 ). These saturation levels were observed even when the concentration of wt MCP-1 was in excess of the KD for dimerization of MCP-1 (not shown). Importantly, these observations demonstrate that one VV-CKBP-II interacts with one MCP-1 [52 ].



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  		Figure 4. Sensorgrams of wt MCP-1 and the monomeric mutant of MCP-1, P8A, binding VV-CKBP-II Fc fusion. VV-CKBP Fc fusion was captured onto a protein A surface immobilized covalently onto a Biacore CM5 chip. Chemokines were serially diluted in running buffer and injected over the VV-CKBP Fc surface (black curves: 0.93, 2.8, 8.3, and 25 nM, from bottom to top) at a fast flow rate (100 µl/min) [52 ]. Double-referenced [73 ] curves show the mass of the chemokine (in response units, RU) binding the VV-CKBP-II Fc-fusion protein as a function of time(s). Data were globally fit (red line) to a 1:1 mass transport model using BIAevaluation 3.0 software. Both plots demonstrate that the binding responses saturate at equivalent Rmax values, supporting a model that VV-CKBP-II binds the monomeric form of MCP-1. Calculations based on the molecular mass of the dimeric VV-CKBP Fc-fusion protein and the molecular mass of the chemokine also support a 1:1 interaction stoichiometry (see text) [52 ]

 
Further evidence that the interaction occurs as a 1:1 complex was the finding that Y13 is involved intimately in mediating the interaction of MCP-1 with VV-35 kDa [52 ]. As Y13 is fully sequestered in the MCP-1 homodimer interface [74 , 75 ], the possibility that MCP-1 might interact as a dimer with CKBP-II was ruled out [52 ]. As other CC-chemokines form very similar quaternary structures, it is predicted that CKBP-II proteins also interact with the monomeric form of other CC-chemokines by occluding their homodimer interface. An expanded analysis of other CC-chemokines using a range of biophysical tools will be necessary to determine if this model of stoichiometry is consistent among of other CC-chemokines. Nonetheless, the notion that a distinct site on CKBP-II interacts specifically with the monomeric form of CC-chemokines is consistent with the observation that chemokines are predominantly monomeric at physiological concentrations and can function effectively as monomers with cellular receptors [74 , 76 77 78 ].

GAG binding by a member of the CKBP-II family
Several immunomodulatory poxvirus proteins have been shown to interact with GAG molecules, such as the complement-control protein from vaccinia virus and the molluscum contagiosum virus chemokine homolog, MC148 [79 80 81 ]. Recently, the CC-CKBP from the myxoma virus, termed M-T1 (MV-CKBP-II), was shown to possess the ability to interact with GAGs, such as heparin, heparan sulfate, and chondroitin sulfate, in addition to its documented ability to bind CC-chemokines [45 ]. Whereas the interaction with CC-chemokines is of a high affinity nature (KD=pM to low nM), the MV-CKBP-II interaction affinity with heparin is of a lower affinity nature (KD=446 nM) [45 ].

The MV-CKBP-II interaction with GAGs and with CC-chemokines was not mutually exclusive, suggesting that the binding sites for CC-chemokines and GAGs are spatially distinct [45 ]. Using structural folding models and deletion analysis of MV-CKBP-II, the heparin-binding domain was localized to where ß-sheet I would be predicted to form on M-T1. This region of MV-CKBP-II contains a high abundance of positively charged amino acids as well as two predicted Cardin and Weintraub heparin-binding consensus motifs [45 ]. It is interesting that the homologous VV-CKBP, which was shown not to bind heparin, had neither this clustered basic charge domain nor any obvious heparin-binding consensus motifs within this region [45 ]. Both proteins, however, share a negatively charged ß-sheet II, supporting the idea that the ß-sheet II region contains the putative CC-chemokine-binding domain [45 , 49 ]. The distinct surfaces of positive and negative charges on MV-CKBP-II appear to correspond to the heparin-binding and CC-chemokine-binding regions of MV-CKBP-II, respectively, and support the prediction that the binding sites for heparin and CC chemokine constitute functionally different sites on MV-CKBP-II [45 ].

Precedence for a cell-surface localization strategy has been observed in other poxvirus immunomodulatory factors. For instance, the poxvirus TNF-R and IFN-{alpha}/ßR viroceptors localize to cell surfaces through an undefined mechanism, yet still bind to their respective ligands while bound to the cell surface [82 83 84 ]. The complement control protein from vaccinia virus (VCP) has also been shown to be capable of binding complement and heparin simultaneously [80 , 81 , 85 ]. GAG binding by VCP imparts several functions that include the ability to displace GAG-bound chemokines, facilitate uptake by mast cells, and mediate attachment of VCP to endothelial cells [80 , 81 , 85 ]. The finding that MV-CKBP-II, but not VV-CKBP-II, can simultaneously bind GAGs and CC-chemokines may bestow several distinct functions in vivo not shared by the VV-CKBP-II protein. For instance, MV-CKBP-II may loosely tether to cell surfaces through GAG interactions at the site of infection in order to bind and neutralize CC-chemokines that are produced locally as a result of virus infection. Furthermore, GAG-binding by MV-CKBP-II may prevent diffusion effects in vivo and enable MV-CKBP-II to increase its local concentration at sites of infection. Finally, like other heparin-binding proteins, GAG binding by MV-CKBP-II may protect against protease degradation. Further study into the role of GAG-binding by MV-CKBP-II during viral infection is required. Nonetheless, cell-surface localization by MV-CKBP-II through GAG interactions provides another example of how viruses might have evolved to control the local microenvironments of infected tissues more efficiently.


   HERPESVIRUS HIGH AFFINITY CKBP-III
TOP
 ABSTRACT
 INTRODUCTION
 POXVIRUS LOW AFFINITY CKBP...
 POXVIRUS HIGH AFFINITY CKBP-II
 HERPESVIRUS HIGH AFFINITY CKBP...
VIRAL CKBPs AS THERAPEUTIC...
 CONCLUSION
 REFERENCES
 
Herpesvirurses encode a number of proteins that are produced to subvert the chemokines at various levels [3 , 4 , 10 ]. Murine {gamma}-herpesvirus-68 (MHV-68) is a natural pathogen of rodents that is closely related to human herpesvirus 8 (HHV8) and Epstein-Barr virus [86 87 88 ]. MHV-68 was recently shown to encode an abundantly secreted 44 kDa protein, termed M3 (CKBP-III), which is translated from a 1.4 kb early-late lytic transcript [58 ]. Similar to the CKBP-II family, the M3 protein can competitively inhibit the binding of many chemokines from their receptors and is able to prevent chemokine-induced signaling through chemokine receptors [56 , 57 ]. M3 possesses no known mammalian homologs but exhibits 25% identity and 45% similarity to the MHV-68 M1 gene product, which is not known to bind chemokines [58 ]. Although M1 possesses homology to the SPI-1 poxvirus serpin [89 ], M3 does not share this similarity [58 ].

Unlike most cellular chemokine receptors and the CC chemokine class-specific CKBP-II family, M3 can broadly bind multiple chemokine classes, including the CC, CXC, C, and CX3C chemokines, although some of the CXC chemokines tested were shown to have low or no affinity for M3, indicating some level of specificity for chemokine interactions [56 , 57 ]. This unique broad-spectrum binding profile suggests that the structural mechanism by which M3 functions likely differs from that of the CKBP-II family, although both families mediate the occlusion of the receptor-binding site on the chemokine. Similar to the CKBP-II family, the M3 interaction with chemokines is not dependent on the heparin-binding domain of the chemokines because heparin is unable to compete for the interaction [56 ], suggesting that M3 occludes only the high affinity receptor binding site on chemokines.

Cross-competition analysis of chemokines binding to M3 suggests that M3 likely possesses a single binding region recognizing common structural features that are conserved among all chemokine families or distinct but overlapping regions on the chemokines [56 , 57 ]. The former case would, however, necessitate that M3 have greater recognition plasticity not shared by the CKBP-II family as conserved residues among CC-chemokines, but not CXC-chemokines, appear to mediate CKBP-II recognition [52 , 53 ]. Elucidating the molecular basis for this unique binding profile of M3 with chemokines should provide useful insights into the processes of molecular recognition of distinct, but related, protein structures as well as provide clues toward how class-unrestricted chemokine antagonists may be developed for therapeutic use.

Role of M3 in establishing latency
One of the characteristic features of MHV-68 is the initial replication in respiratory epithelial cells and the subsequent dissemination to lymphoid tissue where the virus establishes latency in B cells, dendritic cells, and macrophage [90 91 92 ]. M3 has been shown to be abundantly expressed during lytic infections in culture and in lungs [58 , 87 ] and when the latent virus first reaches the spleen [59 , 93 ], suggesting that chemokine inhibition could be necessary for some aspect of lytic infection, establishment of latency, or reactivation from latency.

To investigate the role of M3 on viral infection and latency in vitro and in vivo, a recent study constructed a recombinant MHV-68 mutant virus containing a disrupted M3 open reading frame using a LacZ espression cassette (M3LacZ) [60 ]. The recombinant M3LacZ virus abolished the production of the M3 protein and the chemokine-binding activity [56 ]. It was also demonstrated that the insert had no effect on the transcription of flanking M2 and M4 genes [60 ]. Growth curves demonstrated that the absence of M3 had no significant effect on lytic replication in vitro. Moreover, initial in vivo lytic replication after intranasal infection of mice was not significantly different from wt MHV-68 virus, although an enhanced clearance of virus was observed at late times of lytic replication after infection [60 ]. Based on these observations, it was concluded that M3 is not required to protect MHV-68 from immune surveillance during the acute phase of lytic replication [60 ].

It has been suggested that MHV-68 lytic and latent virus amplification are distinct processes [60 , 94 ], and therefore we evaluated the role of M3 during the establishment of latency. The absence of M3 in the M3LacZ virus had little effect compared with wt MHV-68 on the establishment of mediastinal lymph node (MLN) latency and virus-driven B cell activation 7 days post-infection (p.i.). This contrasted with the severely impaired amplification of M3LacZ latent virus in the spleen and MLN [60 ]. Moreover, although splenic B cell activation was only slightly impaired at day 7 p.i. compared with wt virus, there was a significant defect in B cell activation at 13 day p.i. [60 ]. Importantly, a revertant virus (M3R) containing restored M3 function could reverse these M3LacZ phenotypes to that of wt.

To determine the immunological mechanism that might be mediating the lower levels of latent M3LacZ virus despite normal seeding, CD8+ T cells were depleted before intranasal challenge with M3LacZ [60 ]. The ability of M3LacZ to establish latency in the spleen was restored partially in CD8+ T cell-depleted mice, suggesting that the clearance of the M3LacZ virus by cytotoxic T cells is impaired [60 ]. Given that CD8+ T cell depletion resulted in only a partial restoration of M3LacZ latency, it is probable that other immune clearance mechanisms are inhibited by M3. Further study to determine the cell subsets that are modulated by M3 is warranted.

A full appreciation of how M3 modulates virus infection has yet to be determined. However, the MHV-68 model of pathogenesis provides a unique window into how this virus manipulates the chemokines to provide a favorable environment for viral infection. Furthermore, the broad spectrum and high affinity inhibition of chemokines by the M3 protein make M3 an attractive candidate to exploit as an antichemokine therapeutic. Future studies that compare and contrast the use of this protein with the poxvirus CKBP proteins as therapeutic agents should aid in understanding the role of different chemokine subsets in various disease indications.


   VIRAL CKBPs AS THERAPEUTIC AGENTS
TOP
 ABSTRACT
 INTRODUCTION
 POXVIRUS LOW AFFINITY CKBP...
 POXVIRUS HIGH AFFINITY CKBP-II
 HERPESVIRUS HIGH AFFINITY CKBP...
 VIRAL CKBPs AS THERAPEUTIC...
CONCLUSION
 REFERENCES
 
Chemokines regulate, and sometimes exacerbate, the progression of various disease pathologies such as multiple sclerosis, diabetes, arthritis, or allergy-induced inflammation [95 96 97 98 99 100 ]. Preventing the recruitment of leukocytes into sites of inflammation using chemokine antagonists provides an upstream point of intervention prior to subsequent tissue damage [95 ]. However, the complexity and redundancy of the chemokine network imply that specific chemokine inhibitors may not be as effective as broad-spectrum chemokine antagonists. Thus, viral CKBPs are attractive as potential anti-inflammatory molecules because of their inherent capacity as broad-spectrum inhibitors of inflammatory chemokines [34 , 101 ].

The potential of CKBPs as therapeutic agents has been explored through their use as stand-alone treatments of diseases based on acute inflammation. One of the first examples demonstrating the potency of CKBPs in a model of inflammation was a study that used a purified recombinant VV-CKBP-II His-fusion protein in a guinea pig model of allergic inflammation [48 ]. Importantly, intradermal injections of the VV-CKBP-II His-fusion protein effectively inhibited eosinophil accumulation induced by eotaxin, but not C5a or leukotriene B4 [48 ]. More recently, the CPV-CKBP-II fused to the Fc region of human immunoglobulin G1 was used as a therapeutic agent to block airway inflammation in a murine model of allergen-induced perivascular and peribronchial inflammation [50 ]. Single doses of CPV-CKBP-II Fc administered intranasally were sufficient to prevent recruitment of inflammatory leukocytes into the lung while sparing the inflammatory responses in the peritoneum. Importantly, systemic immune responses were not altered by the use of CPV-CKBP-II Fc [50 ]. These encouraging results suggest that CKBP-II proteins may be amenable to treat other forms of CC chemokine-mediated inflammatory conditions.

We also explored the application of the general CKBP, CKBP-I protein (M-T7), to disrupt chemokine functions. A single intravenous injection of CKBP-I protein administered to rats or rabbits immediately after balloon angioplasty attenuated intimal hyperplasia dramatically after angioplasty injury, including reduction in neointima formation and macrophage infiltration [41 ]. The inhibitory activity of CKBP-I was attributed to the chemokine binding activity of CKBP-I and not its IFN-{gamma} binding properties, as CKBP-I inhibition is highly specific for rabbit IFN-{gamma} [41 , 102 ]. The inhibition of inflammation in both animal models is consistent with the broad species nonspecific interaction of CKBP-I with chemokines [40 ]. The low effective doses and absence of any toxicity indicate that CKBP-I offers another viral candidate for therapeutic use in inflammatory-based diseases.

Collectively, these studies are beginning to provide validation to the assertion that viral CKBPs may be highly amenable as therapeutic agents [101 ]. It has been noted that timing of administration, bioavailability, pharmacokinetics, and antigenicity represents a hurdle for the application of these proteins in the clinical setting [101 ]. Continued investigations to evaluate the efficacy of these proteins as anti-inflammatory therapeutics will permit a better grasp of the full promise of these exciting, new immunomodulatory compounds.


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 POXVIRUS LOW AFFINITY CKBP...
 POXVIRUS HIGH AFFINITY CKBP-II
 HERPESVIRUS HIGH AFFINITY CKBP...
 VIRAL CKBPs AS THERAPEUTIC...
 CONCLUSION
REFERENCES
 
The observation that the poxvirus and herpesvirus CKBPs discovered to date have no obvious resemblance to known chemokine receptors or any other proteins within the mammalian genome suggests that the chemokines exerted significant selection pressures that necessitated the evolution of distinctive antichemokine strategies. The discovery of new viral CKBPs and the elucidation of their mechanisms are providing insights into the diversity of strategies that are used by viruses to modulate the chemokines. Knowledge of how viruses function to oppose chemokines is providing important insights into how the immune system combats viral infection and contributes a better understanding of how we may better control unwanted chemokine-mediated inflammation.


   ACKNOWLEDGEMENTS
 
B. T. S. is supported by an Ontario Graduate Scholarship, and G. M. holds a Canada Research Chair in Molecular Virology. This work has been supported by the Canadian Institutes for Health Research and the National Cancer Institute of Canada. We thank John Barrett for useful comments and discussion.

Received December 3, 2001; revised February 3, 2002; accepted February 5, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 POXVIRUS LOW AFFINITY CKBP...
 POXVIRUS HIGH AFFINITY CKBP-II
 HERPESVIRUS HIGH AFFINITY CKBP...
 VIRAL CKBPs AS THERAPEUTIC...
 CONCLUSION
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