Vol. 65, No. 6JOURNAL OF VIROLOGY, June 1991, p. 3411-34150022-538X/91/063411-05$02.00/0Copyright C) 1991, American Society for Microbiology

An Fc Receptor for Human Immunoglobulin G Is Located withinthe Tegument of Human Cytomegalovirus

LINDA M. STANNARD* AND DIANA R. HARDIE

Department of Medical Microbiology, University of Cape Town Medical School, Observatory, 7925, South Africa

Received 12 October 1990/Accepted 22 February 1991

Immunogold electron microscopy has demonstrated that human immunoglobulin G (IgG) can bind to thetegument of human cytomegalovirus virions by the Fc portion of the molecule. This binding was inhibited bypreincubation of the Fc probes with protein A. Treatment of AD169 virions with Triton X-100 allowed releaseof the Fc-binding proteins, which were precipitated and characterized by polyacrylamide gel electorphoresis(PAGE). Polypeptides of approximately 69 and 33 kDa were recovered and shown by immunoblotting to retaintheir capacity to bind Fc-gold after separation under both reducing and nonreducing conditions. The combinedresults of blocking experiments, PAGE of precipitates, and Western blots (immunoblots) indicate that thetegument proteins which bind IgG-Fc are identical to those which bind 12 microglobulin.

Although human cytomegalovirus (HCMV) has long beenrecognized as an important pathogen, especially in immuno-compromised individuals, an understanding of the pathogen-esis at a molecular level is still relatively incomplete. Cellsinfected with HCMV are able to bind the Fc portion ofimmunoglobulin G (IgG) (6)-a property also manifested byother herpesviruses such as herpes simplex virus (HSV) (24)and varicella-zoster virus (14)-but neither the functionalrole nor the chemical nature of these HCMV-induced Fcreceptors has been elucidated. We report here the detectionof a non-membrane-bound Fc receptor which resides withinthe tegument of the HCMV virion itself and suggest that thepresence of an Fc-binding protein in this unexpected loca-tion may assist in maintaining the infectious potential ofthose virus particles which have lost their envelopes.

Electron-dense probes of colloidal gold coupled to specificproteins have been valuable in the identification of preciseultrastructural locations of viral proteins (20, 21) or specificsites of viral protein interactions. Thus, the ability ofHCMVto bind beta-2-microglobulin (P2m) (7, 8) has previously beenshown to exist in the tegument (19). While the interactionbetween purified HCMV virions and human IgG was beingstudied by immunogold electron microscopy, it was notedthat IgG purified from sera that assayed as seronegative forantibodies to HCMV by enzyme-linked immunosorbent as-say consistently bound to the tegument protein of the virusparticles. IgG from seropositive individuals bound, as ex-pected, to all components of the virion (envelope, tegument,and capsid), while monoclonal antibody (MAb) F5 (12),which is specific for the HCMV gp52 envelope glycoproteinknown as gB (21), bound only to the envelope (Fig. lc). Fivedifferent MAbs specific for HSV glycoprotein gB, gC, or gD(16) did not bind to any component of the HCMV virions(Table 1). In order to establish the nature of the interactionbetween nonimmune-human IgG and the HCMV tegument,the binding properties of separated Fc and Fab fragments ofIgG were examined.

Standard methods were used to separate IgG fromHCMV-seronegative human serum by DEAE-cellulose an-ion-exchange chromatography. Concentrated IgG sus-pended in 0.01 M phosphate buffer was subjected to diges-

* Corresponding author.

tion with papain (Sigma) for 8 h at 37°C, using 1 mg of papainper 100 mg of IgG. Incubation was followed by dialysisagainst 0.01 M phosphate buffer and ion-exchange chroma-tography on Sephadex C50 as described by Franklin andPrelli (5) to separate the Fab and Fc components. Purepreparations of Fab were obtained by gel filtration, and Fcfractions were purified by repeated crystallization (17). Ho-mogeneity of the final fragments was confirmed by both gelimmunoprecipitation and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Each of thepurified fractions was coupled to colloidal gold as previouslydescribed (20). Briefly, protein in 0.2 mM borax buffer (pH9.0) was rapidly mixed with 10 times the volume of colloidalgold, which had been adjusted to a pH close to the isoelectricpoint of the protein. The mixture contained the minimalconcentration of protein required to stabilize the gold colloidand prevent its flocculation in the presence of 1% NaCl.Gold probes were separated from free protein by two cyclesof centrifugation and finally suspended in 20 mM Tris buffer(pH 8.2) containing 0.5% bovine serum albumin and 0.02 Msodium azide (TBSA). For microscopy, HCMV AD169virions were concentrated from cell culture fluids of infectedhuman embryonic fibroblasts, placed on Formvar-coatedgrids, and floated on drops of the respective probe (diluted inTBSA) for 2 h at 37°C. Grids were then washed with distilledwater, stained with 1% phosphotungistic acid, and examinedin a Hitachi 600 electron microscope.

Fc-gold probes showed a strong affinity for the tegument(Fig. la). The degree of binding of the purified Fc fragmentswas equal to or greater than that observed with the wholeIgG molecules. In contrast, the reaction between virions andthe Fab portion of IgG was greatly diminished in comparisonwith that seen with either the Fc portion or the wholemolecule. Nevertheless, we consistently observed a lowlevel of binding to the tegument (Fig. lb), an interestingphenomenon which remains to be explained.

Protein A from Staphylococcus aureus is known to bind tothe Fc portion of human IgGl, IgG2, and IgG4 (11), and thebinding site is said to be situated between IgG constantdomains 2 and 3. When Fc probes were preincubated withprotein A before exposure to HCMV virions, their attach-ment to the tegument was almost totally blocked (Fig. 2c).This inhibition of Fc binding suggests that the site (on the Fcportion of IgG) which binds to the tegument protein of

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a

V

FIG. 1. HCMV virions after reaction with electron-dense probes consisting of colloidal gold coupled to specific proteins. (a) Purified Fcportion of seronegative human IgG binds strongly to the virion tegument. (b) Purified Fab portion of seronegative human IgG shows reducedbinding to the tegument. (c) MAb F5, specific for HCMV gp52, binds only to the virion envelope.

HCMV is in close proximity to the attachment site forprotein A. The low level of residual binding observed inthese blocking experiments may be explained by the pres-ence of IgG3-Fc fragments, which do not bind protein A.

In view of the previous finding (19) that r2m binds to theHCMV virion tegument at locations similar to those whichwere shown to bind human IgG-Fc (Fig. 2a and b), additionalblocking experiments were performed whereby the virusparticles were preexposed to either unlabeled P2m or unla-beled Fc preparations before being allowed to react with theheterologous protein-gold probes. The binding of IgG-Fcwas indeed inhibited by P2m (Fig. 2d) and vice versa, which

TABLE 1. Murine MAbs tested by immunogold electronmicroscopy on HCMV AD169 virions

Binding visualized byMurine IgG immunogold electronMAbW Specificity subclass microscopyb

Envelope Tegument

F5 HCMV gp52 (gB) 2a Pos Neg1-59-2 HSV gB 3 Neg Neg111-114 HSV gD 2b Neg Neg1-99-1 HSV gD 2a Neg Neg11-512-3 HSV gC 3 Neg Neg11-481-B HSV gE 2b Neg Neg

a HSV MAbs are described in reference 16.b Pos, binding occurred; Neg, no binding occurred.

a C

FIG 2 Effect of blocking Fc binding with protein A or v2MHCMV virions reacted with Fc-gold (a) or P2m-gold (b) showbinding to similar locations on the virion tegument Fc-gold preincubated with protein A retains only a low level of binding (c) andpreincubation of virus with unlabeled 32m totally blocks binding ofFc-gold (d).

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implied that both proteins bind to the same or closely relatedsites on the virion tegument.Immunogold probes were found to be of value not only for

electron microscopy but also as "immunosorbents" for theseparation of the reactive tegument proteins. Purified virionpreparations were agitated in k'uffer containing 1% TritonX-100, 10 mM Tris-HCl, 1 mM CaCl2, 0.15 M NaCl. 2 mMphenylmethylsulfonyl fluoride, and 1% ethanol (pH 7.3).This caused disintegration of the viral envelopes, allowingrelease of the Fc-binding tegument proteins into the Triton-soluble phase. Whereas the insoluble components (mainlynucleocapsids) were shown by electron microscopy to havelost their ability to bind IgG-Fc, the Triton-dispersed pro-teins retained their Fc-binding capacity and could be precip-itated with Fc-specific colloidal-gold probes. Precipitateswere concentrated by centrifugation, washed twice in 0.01 Mphosphate buffer, and subjected to SDS-PAGE (10) with 10or 12% polyacrylamide gels. Polypeptides of approximately69 and 33 kDa were resolved by staining with Coomassieblue (Fig. 3A). The mobilities of these proteins remainedunchanged after incubation for 18 h at 37°C with N-glycosi-dase F (Boehringer Mannheim), indicating the absence ofN-linked oligosaccharides. In parallel studies, the sameenzyme was shown to reduce the molecular weight ofHCMV envelope glycoprotein gB (precipitated from Tritonsuspensions with specific MAb-gold probes) by approxi-mately 9 kDa (data not shown). Tegument proteins arepresumably acquired in the nucleus of the infected cell priorto the budding of the virion through the nuclear envelope,and the presence of N-linked oligosaccharides in the tegu-ment would therefore be unexpected. Nevertheless, since ithas been reported that the major component of HCMVteguments, a 149-kDa phosphoprotein, contains residues ofN-acetylglucosamine attached by an unusual 0 linkage (3),the presence of similar 0-linked modifications of the Fc-binding tegument proteins cannot be totally excluded.

In order to test the evidence obtained in electron micros-copy blocking experiments, which suggested that the sametegument proteins might be involved in both Fc and P2mbinding, SDS-PAGE analysis was performed on gold-proteinprecipitates obtained with P2m-gold. Proteins precipitatedwith P2m were the same size as those obtained with IgG-Fc(Fig. 3A). Grundy and co-workers (7) used polyclonal rabbitserum specific for P2m to immunoprecipitate Triton-solubleHCMV proteins which had been radiolabeled with 125I. Theyrecovered polypeptides with comparable molecular weightsof 65 and 36 kDa; the differences from our calculated sizespossibly reflect differences in methods.PAGE-separated proteins from both Fc- and P2m-gold

precipitates were electrophoretically transferred to nitrocel-lulose, treated with 1% bovine serum albumin to blocknonspecific binding, and exposed to fresh preparations of theoriginal Fc- or 32m-gold probes for 16 h at room tempera-ture. After a thorough washing with distilled water, adsorbedgold particles were visualized by enhancement with silverions (Intense-M; Janssen). While only weak reactions wereobserved at the 69-kDa position, the 33-kDa proteins fromeither the Fc-gold or the P2m-gold precipitates retained theircapacity to combine strongly with homologous gold probes.Furthermore, they bound equally well to the heterologousgold probe, thus strengthening the evidence that the Fc-binding tegument proteins are the same as those which bind132m. The immunoblotting experiments were repeated onelectrotransfers of proteins, from the original Triton ex-tracts, after subjection to PAGE under either reducing ornonreducing conditions. Both P2m-gold and Fc-gold bound

A 2 3 4

-69-55

4.ip

-33

B NR R

-69

V -33

1 2 3 4

FIG. 3. Comparison of proteins with affinity for human IgG-Fcor 02m. (A) Triton-soluble proteins of purified HCMV virions wereprecipitated by using colloidal gold coupled either to the Fc frag-ment of human IgG or to P2m. Precipitates were analyzed bySDS-PAGE on 10% polyacrylamide gels, and protein bands werevisualized by staining with Coomassie blue. Polypeptides of similarmolecular masses were precipitated by human IgG-Fc (lane 2) andP2m (lane 3). Precipitates obtained with MAb F5, which is specificfor the major envelope glycoprotein of HCMV (gB), are shown inlane 4. Molecular weight markers are shown in lane 1. (B) HCMVvirion proteins extracted with Triton X-100 were separated bySDS-PAGE under nonreducing (NR; lanes 1 and 2) or reducing (R;lanes 3 and 4) conditions and then electrotransferred to nitrocellu-lose. Separate strips were allowed to react with either Fc-gold (lanes1 and 3) or ,32m-gold (lanes 2 and 4). Bound gold probes werevisualized by enhancement with silver. Both Fc and 12m probesbound strongly to a 33-kDa protein and less strongly to a 69-kDaprotein (visible in lane 4).

to transfers of both reduced and nonreduced proteins atpositions equivalent to that of the 33-kDa protein (Fig. 3B).Weak reactions were occasionally also visible at the 69-kDaposition (Fig. 3B, lane 4). It is possible that the receptor is anoncovalently linked heterodimer with the binding epitopesresiding predominantly in the 33-kDa subunit. An interactionof two viral glycoproteins is known to occur in the case ofthe HSV Fc-receptor, where gE and gI together form acomplex (9) which binds IgG-Fc more effectively than doesgE alone (2).The location of an Fc-binding protein within the tegument

of the HCMV virus particle is intriguing, since most Fcreceptors described to date either are membrane-anchoredproteins or, as in the case of the type III Fcy receptor ofnatural killer cells (18), are attached to the lipid bilayer bymeans of a phosphatidyl-inositol glycan tail. Although thefunctional role of this unconventional non-membrane-boundFc receptor of HCMV can be discussed only in the broadest

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speculative terms, it is not unlikely that its presence helpspreserve the infectivity of uncoated virions. Previous studieswith Allerton bovine herpesvirus have shown that removalof virion envelopes by sonication does not result in loss ofinfectivity (unpublished observations). In vivo, the infectiv-ity of unenveloped HCMV particles may well be protectedby their abilities to bind nonimmune host globulin, thusshielding the particles from immune recognition. However,the manner in which particles coated with Fc-anchored IgGmight attach to target cells and initiate infection is not clear.Unlike specific immune complexes of viruses, they would beunable to utilize host cell surface Fc receptors and enter thecell by phagocytosis. Of relevant interest is the recent reportby Lenert et al. (13) that the CD4 molecule of T lymphocytescan bind to the Fab portion of human IgG. This raises thequestion of whether CD4, or indeed other unknown butcomparable receptors for Fab constant domains, could serveas cell surface ligands for IgG-coated HCMV nucleocapsids.Alternatively, the tegument Fc receptor may itself representa cell attachment protein. Of relevance are our findings thatthe tegument proteins which bind IgG-Fc also bind P2m andthat interaction with either of these two ligands (both mem-bers of the immunoglobulin superfamily) can block bindingof the other. The constant domains of IgG share homologywith 12m (15), a factor which might be responsible for theirshared affinity for the HCMV tegument. Consequently it ispossible that the respective tegument protein has equalaffinity for yet another, as-yet-unidentified homologous pro-tein, conceivably also of the immunoglobulin superfamily,which could represent the principal ligand for unenvelopedHCMV virions. Cell surface molecules with homology toIgG (26) have been shown to serve as receptors for virusessuch as rhinoviruses, poliovirus, and the human immunode-ficiency virus (25). Intact enveloped HCMV virions un-doubtedly represent the principal infectious unit, and cellsurface receptors for envelope proteins probably differ fromthose for nonenveloped virus particles. Taylor and Cooper(22, 23) have shown that the major receptor for HCMV onhuman fibroblasts is a glycoprotein of approximately 30 kDain molecular mass, but some binding to both 28- and 92-kDamembrane constituents was also observed. Although it haspreviously been postulated that P2m-coated virions attach totarget cells by displacing the P2m on class I human leukocyteantigen molecules on the cell surface (8), the demonstrationof HCMV receptors unrelated in size to class I humanleukocyte antigen (23) and our finding of (at least) a dualaffinity for the P2m-binding protein of the virion suggest thatother modes of virus-cell attachment should be considered.DNA sequence analysis has shown that the HCMV genomeencodes a molecule with homology to human and murinemajor histocompatibility complex class I antigens (1). Sub-sequent studies (4) have made use of vaccinia virus recom-binants constructed to contain either the class I homologousgene, termed UL18, or a 32m-coding gene sequence. WhenBHK cells were coinfected with both recombinant viruses, a67-kDa protein could be coprecipitated with 132m from celllysates by using a MAb to 132m, and cell surface expressionof P2m (measured by immunofluorescence) was increased10-fold over that found with the 132m-vaccinia virus recom-binant alone. They conclude that the HCMV class I homologis able to bind to P2m and facilitate its transfer to the cellsurface. The relationship between the UL18 gene product,presumed to behave as a conventional transmembrane pro-tein in the recombinant studies, and the non-membrane-bound P2m-binding component of extracellular HCMV iden-tified in this study is not known, but an investigation of the

Fc-binding properties of the UL18 gene product may beenlightening.

We thank Patricia Spear for providing the MAbs to HSV glyco-proteins and Janet Rider for the MAb to HCMV gp52. We aregrateful to Keith Dumbell and John Moodie for critical reading of themanuscript.

This work was supported in part by the South African Poliomy-elitis Research Foundation.

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