key: cord-270814-krw8zmr5
authors: Rao, Pasupuleti V.; Kumari, Suman; Gallagher, Thomas M.
title: Identification of a Contiguous 6-Residue Determinant in the MHV Receptor That Controls the Level of Virion Binding to Cells
date: 1997-03-17
journal: Virology
DOI: 10.1006/viro.1997.8446
sha: 
doc_id: 270814
cord_uid: krw8zmr5

Abstract Murine carcinoembryonic antigens serve as receptors for the binding and entry of the enveloped coronavirus mouse hepatitis virus (MHV) into cells. Numerous receptor isoforms are now known, and each has extensive differences in its amino terminal immunoglobulin-like domain (NTD) to which MHV binds via its protruding spike proteins. Some of these receptor alterations may affect the ability to bind viral spikes. To identify individual residues controlling virus binding differences, we have used plasmid and vaccinia virus vectors to express two forms of MHV receptor differing only in their NTD. The two receptors, designated biliary glycoproteins (Bgp) 1aand 1b NTD, varied by 29 residues in the 107 amino acid NTD. When expressed from cDNAs in receptor-negative HeLa cells, these two Bgp molecules were displayed on cell surfaces to equivalent levels, as both were equally modified by a membrane-impermeant biotinylation reagent. Infectious center assays revealed that the 1aisoform was 10 to 100 times more effective than 1b NTDin its ability to confer sensitivity to MHV (strain A59) infection. Bgp1awas also more effective than Bgp1b NTDin comparative virus adsorption assays, binding 6 times more MHV (strain A59) and 2.5 times more MHV (strain JHMX). Bgp1awas similarly more effective in promoting the capacity of viral spikes to mediate intercellular membrane fusion as judged by quantitation of syncytia following cocultivation of spike and receptor-bearing cells. To identify residues influencing these differences, we inserted varying numbers of 1bresidues into the Bgp1abackground via restriction fragment exchange and site-directed mutagenesis. Analysis of the resulting chimeric receptors showed that residues 38 to 43 of the NTD were key determinants of the binding and fusion differences between the two receptors. These residues map to an exposed loop (C-C′ loop) in a structural model of the closely related human carcinoembryonic antigen.

nized MHV strains (Barthold et al., 1986; Barthold, 1987) . These receptors are type I single-pass transmembrane Coronaviruses are attractive objects for studies of aniglycoproteins containing an ectodomain structure made mal virus entry into cells because there exists a remarkup of four immunoglobulin-like domains (Dveksler et al. , able level of variation in the ligands that engage virions 1991). The variability between 1 a and 1 b lies primarily in with cells. Several strains of MHV are now recognized the most membrane-distal domain (Yokomori et al., (Siddell, 1995) and several receptors are also known 1992b, Dveksler et al., 1993a) . This amino terminal do- (Dveksler et al., 1991; Yokomori and Lai, 1992b; Nedellec main (NTD) is essential for virus binding in vitro (Dveksler et al., 1994; Chen et al., 1995) . This abundant natural et al., 1993b) and is similarly required to render cells variation provides the opportunity to compare different susceptible to infection by MHV (Dveksler et al., 1995) . receptors for their ability to interact with different virus Initial studies on the coronavirus-binding functions of strains. Such comparative analyses will allow one to Bgp1 a and 1 b indicated that they differ substantially in identify the determinants responsible for differences in virion adsorptive capacity -when the two receptors virus:cell interactions.

were immobilized on nitrocellulose filters, only the 1 a In this study, we have focussed attention on two allelic isoform supported detectable virus-binding (Dveksler et isoforms of the MHV receptor, biliary glycoprotein (Bgp) al., 1993a) . This finding suggested that one or more of isoforms 1 a (Dveksler et al., 1991; the 29 amino acid differences between the 1 a and 1 b 1992a) and 1 b (Yokomori and Lai, 1992b; NTDs (Yokomori and Lai, 1992b ) are responsible for 1993a; McCuaig et al., 1993) , and we have examined controlling virion adsorptive capacity, either because their capacity to interact with purified enveloped corothey provide direct receptor:virion contact points or benavirions. The 1 a isoform is expressed in MHV suscepticause they control the overall conformation of the recepble mice, while mouse strains homozygous for the 1 b tors. If the NTD differences were indeed critical, then isoform are generally resistant to many currently recogidentification of the relevant residues could be achieved by constructing 1 a /1 b hybrid cDNAs and then measuring virus binding to the protein products. We have used duce chimeras that have been useful in identifying resi-changed. 1 b 84-122 and 1 b 70-122 were made by exchange of AccI -PstI and BamHI-PstI fragments, respec-dues controlling the virus-adsorptive capacity of the MHV receptor.

tively (see Fig. 7 ). Additional exchanges were performed after creation of a NdeI site at codon 53 using the megaprimer mutagenesis procedure (Aiyar and Leis, 1993) .

NdeI site creation altered codon 53 (N53H) in Bgp1 a and Cells and viruses codons 53 and 54 in Bgp1 b NTD (N53H, K54M). 1 b 54-122 and 1 b 10-54 were made by exchange of NdeI -NdeI Baby hamster kidney (BHK), rabbit kidney (RK13), and and NcoI -NdeI fragments, respectively (see Fig. 7 ). Rehuman carcinoma (HeLa) cells were grown in Dulbecco combinant 1 b 38-43 was made by oligonucleotide-dimodified Eagle medium (DMEM) supplemented with 10% rected PCR mutagenesis using a primer spanning coheat-inactivated fetal bovine serum (DFBS). Murine 17 dons 38-43 and the NdeI site (Scharf, 1990) . Nucleotide cl 1 cells (Sturman and Takemoto, 1972) were grown in sequencing methods (Sanger et al., 1977) were employed DMEM containing 5% tryptose phosphate broth (TPB;

to confirm that Bgp1 a and 1 b clones matched those re-Difco Laboratories, Detroit, MI) and 5% DFBS. Murine ported for these genes (McCuaig et al., 1993) and to astrocytoma (DBT) cells (Hirano et al., 1978) were grown verify the isolation of all of the mutant receptors dein minimal essential medium (MEM) containing 10% TPB scribed herein. and 5% DFBS; these cells were used to measure MHV infectivities by plaque assay. Murine coronavirus strains Generation of vaccinia virus recombinants. A59 and JHMX (Makino et al., 1984) (Felgner et al., (ICN Radiochemicals, Irvine, CA) . Clarified supernatants 1987) . At 3 days postinfection, cultures were frozen at were overlaid above 30% (w/w) sucrose cushions in PBS 080Њ. After three freeze -thaw cycles, debris was recontaining 0.01% BSA and subjected to ultracentrifugamoved by centrifugation and virus in the clarified matetion (Beckman-Spinco SW28 rotor for 3 hr at 28,000 rpm).

rial was plated on RK13 cells. Plaques arising from spe-Virion-containing pellets were resuspended in ice-cold cific amplification of recombinant virus were revealed PBS containing 0.01% BSA and stored in silanized glass by overlay of cells with mycophenolic acid-containing vials at 080Њ. medium, as described by Falkner and Moss (1988) . Virus from well isolated plaques was grown in RK13 cells Synthesis and cloning of MHV receptor cDNA to generate stocks of recombinant virus (vBgp1 a , constructs vBgp1D, vBgp1 b NTD , vBgp1 a Q10G,V123G , and additional 1 a /1 b Methods for construction of vaccinia virus insertion/ chimeras). expression plasmids (pTM3; Moss et al., 1990) encoding MHV receptor isoform Bgp1 a or Bgp1 a lacking codons Immunoblot analysis of Bgp proteins 10-122 (Bgp1D) have been described previously (Gal-Cell monolayers were infected with the respective lagher, 1996). These plasmid vectors were further modi-vBgp recombinants (m.o.i. Å 10) and coinfected with fied by reinsertion of Bgp NTD sequences into Bgp1D.

vTF7.3 (m.o.i. Å 1). At 20 hr postinfection, lysis buffer (10 To this end, the Bgp1 a and 1 b NTD sequences were mM Tris -HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 0.5% amplified from outbred CD1 mouse liver RNA by RT-PCR NP-40, 0.1 mM PMSF, and 0.3 U/ml aprotinin) was added, (Kawasaki, 1990) using primers based on the reported and 10-ml aliquots (equivalent to 2 1 10 4 cells) were Bgp1 a sequence (McCuaig et al., 1993) and products subjected to Western immunoblotting. Receptor was encoding for the 1 a and 1 b NTD were identified by identified using antireceptor antibody 874 (a gift of Dr. restriction mapping and by sequencing (Sambrook et M. J. Buchmeier, Scripps Research Institute, La Jolla, CA) al., 1989) . 1 b NTD sequences were cloned in-frame into which was directed against conserved carboxy-terminal the unique XmaI site of Bgp1D to generate the chimeric residues 443-458. Bgp1 b NTD . Two undesired codon alterations that resulted at the junctions of the insertion (Q10G and V123G)

Quantitation of relative Bgp cell surface levels were introduced into a control Bgp1 a construct by performing a parallel insertion of the Bgp1 a NTD into pTM3-At 20 hr postvaccinia infection, HeLa cells expressing the Bgp molecules were washed three times with ice-Bgp1D. The control recombinant was designated as Bgp1 a Q10G,V123G . cold PBS and incubated for 1 hr at 4Њ with PBS containing 1 mg/ml NHS-LC biotin (sulfosuccinimidyl-6-(biotina-To construct additional Bgp chimeras for use in identifying virus binding determinant(s), restriction fragments mido) hexanoate; Pierce Co., Rockford, IL). Unreacted reagent was then quenched by the addition of 20 mM between pTM3-Bgp1 a and pTM3-Bgp1 b NTD were ex-Tris-HCl (pH 7.5) for 15 min at 4Њ. Monolayers were Virus binding assays washed thoroughly with PBS and lysed (2 1 10 6 cells/ HeLa cells overexpressing Bgp receptors from vacml in lysis buffer). Clarified cytoplasmic extracts (250 ml) cinia vectors were washed twice with ice-cold SFM at were then mixed with antipeptide antibody 874 (12.5 ml) 20 hpi, then radiolabeled MHV particles were diluted in and left overnight at 4Њ. Twenty microliters of Gammabind SFM and added at varying multiplicities. After incuba-G-Sepharose (Pharmacia Biotech) was then added for 1 tions at 4Њ, unadsorbed virions were removed and cells hr at 4Њ. Sepharose beads were collected by centrifugawere rinsed five times with ice-cold PBS containing 0.5% tion, washed twice in RIPA buffer (10 mM Tris-HCl (pH BSA and 0.05% Tween 20. RIPA buffer was added, and 7.2) 150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, radioactivities associated with the cell lysates and superand 1% Triton X-100), then once in 10 mM Tris -HCl (pH natant media were quantified by scintillation counting. 7.2) containing 0.1% v/v Nonidet P-40. Immunoprecipitated receptors were then subjected to Western blotting, Intercellular fusion assay and the biotinylated fraction of each sample was identi-

The cell fusion-dependent reporter gene (b-galactosified by incubation of blots for 1 hr with 0.1% v/v streptavidase) activation assay of Nussbaum et al. (1994) was din-horseradish peroxidase (HRP) conjugate (Pierce adapted for studies of MHV fusion as described pre-Co.) in PBS/3% BSA. Immobilized HRP was detected by viously (Gallagher, 1996) , with minor modifications. In incubation in PBS containing 0.5 mg/ml 4-chloro-1-naphbrief, stably transfected HeLa-Bgp1 a cells were infected thol, 17% v/v methanol, and 0.015% hydrogen peroxide.

with MHV-A59 (m.o.i. Å 10) and with vaccinia virus strain Blots were photographed (Polaroid 665 film) and signal WR (m.o.i. Å 10). After 1 hr at 37Њ, these cells were further intensities were quantified with an LKB ultrascan XL dentransfected by lipofection with the reporter gene consitometer.

struct pG1NT7b-gal (kindly provided by Dr. Richard A. Morgan, National Center for Human Genome Research,

Bethesda, MD). Five hours later (before the onset of MHV Vaccinia-infected BHK cells were challenged with A59-induced syncytium formation yet after pG1NT7b-gal MHV-A59 (m.o.i. Å 10) at 10 hr postvaccinia infection.

DNA amplification via vaccinia-encoded polymerases) Fourteen hours later, cells were washed with PBS, fixed the cells were trypsinized, suspended in DMEM 10% in acetone, and incubated for 1 hr at 22Њ with a 1:250 DFBS, and 0.6-ml (3 1 10 5 cell) aliquots were overlaid dilution of antispike Mab 4B11.6 (Collins et al., 1982) on to confluent 5-cm 2 monolayers of HeLa cells that were in PBS/2% BSA. Bound Mab was detected with FITCcoinfected 20 hr earlier with vTF7.3 and vBgp recombiconjugated goat Ig directed against mouse Ig (Cappell, nants. After 3 hr at 37Њ, the mixed monolayers were fixed Durham, NC) and cells were photographed using a Leitz and stained with x-gal for in situ localization of b-gal fluorescence microscope.

activity (MacGregor et al., 1989) . Alternatively, the monolayers were lysed by addition of 0.5% NP-40 in PBS and Infectious center assays the quantity of b-galactosidase in each lysate was calculated using a colorimetric enzyme assay involving hydro-cDNA constructs encoding Bgp1 a and Bgp1 b NTD were lysis of chlorophenyl red b-galactopyranoside (Nussinserted into plasmid expression vector pUHD-10-3 baum et al., 1994). OD 590 values were normalized by (Gossen and Bujard, 1992) and the plasmids (0.5 mg) comparing the hydrolysis rates for each sample with that were transfected by lipofection into aliquots of 3 1 10 5 obtained for a standard preparation of E. coli b-galactosi-HeLa-tTA cells (Gossen and Bujard, 1992) . To determine dase (Calbiochem, La Jolla, CA) and were expressed as transfection efficiencies, parallel cultures were cotransnanograms/well. fected with each pUHD-Bgp construct in conjunction with 0.05 mg of the b-galactosidase-encoding pCMV-b (Clon-RESULTS tech Labs, Inc.). In situ x-gal staining (MacGregor et al., 1989) at 36 hr posttransfection revealed 11.8 and 12.5%

Synthesis of chimeric MHV receptors from vaccinia b-gal-positive cells for the 1 a and 1 b NTD transfectants, revirus vectors spectively. To assess susceptibilities to MHV infection, cells were washed at 36 hr posttransfection with cold A method for receptor overexpression on cell surfaces was necessary to reproducibly measure the binding of serum-free DMEM (SFM) and inoculated with MHV-A59 at 4Њ for 1 hr. Unbound virus was removed by washing MHV particles to a series of different receptors. HeLa cell lines in which the MHV receptor gene was stably and with PBS containing 0.5% BSA and 0.05% Tween 20 and cells were then incubated in DMEM 10% DFBS for 2 hr constitutively expressed (HeLa-Bgp1 a cells) have been developed (Gallagher, 1996) ; however, we found that at 37Њ. Cells were then trypsinized, washed twice with DMEM 10% DFBS, and serial dilutions were plated on clones varied dramatically in receptor levels (data not shown). Therefore we anticipated difficulty in identifying DBT indicator monolayers. Plaques were visualized after a 2-day incubation period. a series of stable HeLa cell transfectants in which differ- The carboxy-terminal 16 amino acids are identical for to increase the likelihood of equivalent receptor producall four receptors; and an antipeptide antibody (874) tion, different receptor cDNAs were expressed from vacraised against these cytoplasmic residues was available cinia virus vectors. Such vectors have historically produced to us for use in monitoring receptor levels. Initial tests high levels of surface glycoprotein (Broder et al., 1994; Nus- using this anti-C-terminal antibody were performed by sbaum et al., 1995) and their preparation is known to be immunoblotting BHK and HeLa cell lysates collected 20 relatively straightforward (Falkner and Moss, 1988) .

hr after coinfection with vTF7.3 and vTM3-Bgp. The re-A set of cDNAs capable of encoding four distinct sults (Fig. 2 ) revealed identical patterns of immunoreacforms of MHV receptor were each recombined into tive protein in lysates expected to contain Bgp1 b NTD , the vaccinia virus genome using insertion vector pTM3 1 a Q10G,V123G , and 1 a (lanes 1-3, respectively). A sharp band (Elroy-Stein and Moss et al., 1990) and at c. 55 kDa was detected, which likely represent underrecombinant viruses (designated vTM3-Bgp) were seglycosylated form(s) of the 424 amino acid proteins. The lected as described under Materials and Methods. The series of bands ranging from 80 to 120 kDa indicated use of the pTM3 vector places the cDNAs under the that equivalent levels of the various glycoforms of receptranscriptional control of a bacteriophage T7 promoter; tor were present in each infected culture. Lysates exthus expression of MHV receptors required coinfection pected to contain Bgp1D had a similar, slightly more of HeLa cells with vTM3-Bgp and vTF7.3, which encodes bacteriophage T7 RNA polymerase (Fuerst et al., 1986) .

The four translation products predicted from this expression scheme are depicted in Fig. 1 . The mature Bgp1 a , after removal of its signal peptide, has an ectodomain composed of four immunoglobulin-like domains, designated D1 to D4 (Dveksler et al., 1991) . A deletion mutant of Bgp1 a (Bgp1D) was prepared by excision of nucleotides encoding the bulk of the virus-binding D1 domain as well as 15 amino acids of the D2 domain. Corresponding sequences from the allelic Bgp1 b (McCuaig et al., 1993) were then placed back into the truncated cDNA to generate a hybrid capable of encoding the chi- To test the contribution that these two changes might an antipeptide antibody directed against conserved carboxy-terminal have on interaction with virions, cDNA for a Bgp1 a con- (4) taining the mutations (designated Bgp1 a Q10G,V123G ) was Bgp1D, (5) vTF7.3 alone, (6) molecular weight markers, kilodaltons.

prepared by ligating the NTD sequences of Bgp1 a into Identical profiles of immunoreactive protein were observed in similarly infected HeLa cell lysates.

the deletion mutant. sion vector pUHD-10-3 and transfected into HeLa-tTA cells (Gossen and Bujard, 1992) tantly, the results of Fig. 4 showed that Bgp1 only 1 to 2 log 10 higher. Moreover, these differences in receptor effectiveness could not be eliminated by increasing input multiplicities during virus challenge. Fiintense immunoreactive protein profile, with the apparent nally, we observed a straightforward correlation between molecular weights of the bands lower by approximately infectious centers and virus yields; 24 hr after challenge 20 kDa (lane 4).

of the Bgp transfectants with MHV A59 at 10 PFU per To determine whether the four different MHV receptors cell, yields were 69000, 3000, and 200 PFU/ml for the 1 a , were displayed on the cell surface at similar levels, we 1 b NTD , and control transfectants, respectively. Thus, when first made the assumption that each receptor contains expressed in the HeLa-tTA cells, Bgp1 a was utilized as similar numbers of exposed amines available for conjuan MHV-A59 receptor much more effectively than gation with the membrane-impermeant biotinylation re-Bgp1 b NTD . agent sulfo-NHS-LC biotin. With this in mind, we derivatized the proteins on intact cells at 20 hr postvaccinia infection with the reagent and then we lysed cells and

Virus binding capacities of the chimeric MHV immunoprecipitated the receptor proteins. The relative receptors proportion of biotinylated receptor among the immuno-With the HeLa cell monolayers each expressing a difprecipitates was then assessed by Western blotting usferent MHV receptor on the cell surface, straightforward ing streptavidin-horseradish peroxidase as a detection virus adsorption assays became feasible. Thus we colreagent. By this method, the relative abundance of biotinlected particles from supernatants of MHV-infected 17cl1 ylated receptors were similar in all cultures; densitometcells that had been incubated for 4 hr in the presence ric scans of the blots revealed less than 10% variation of trans[ 35 S] label, and we concentrated the radiolabeled in signal intensities ( percentage of added radioactivity remaining bound to the monolayers was determined. The results (Fig. 5) re-spike antigen (Fig. 3, panels 1, 2, and 3) while the truncated receptor failed to support MHV-A59 infection vealed a gradual increase in the levels of nonspecific adsorption of virions to cells displaying Bgp1D, from 0.8% (panel 4), in concert with the results of Dveksler et al. (1993b) .

after 15 min, to 1.6% by 1 hr, and 3.5% by 8 hr. MHV-A59 bound far more avidly to cells with Bgp1 a or 1 b NTD ; 9.5 Additional examination of the various receptors for their ability to confer susceptibility to infection involved and 3%, respectively, after 1 hr. Subtraction of nonspecific adsorption values revealed that the Bgp1 b NTD was 6-fold infectious center assays. To this end, cDNAs encoding Bgp1 a or Bgp1 b NTD were introduced into plasmid expres-less effective than Bgp1 a in virion adsorptive capacity. This difference in efficiency of binding was maintained identify the residues involved in virus binding by systematically exchanging 1 a and 1 b residues. This was accomthroughout the 8-hr, 4Њ incubation period. Moreover, this plished through a series of restriction fragment ex-pattern whereby Bgp1 a bound more virus than 1 b NTD rechanges to produce recombinant receptors containing mained constant over a 10-fold range of virus input multivarying numbers of 1 b residues within a Bgp1 a backplicities (data not shown). ground (Fig. 7) . These receptors were expressed in In all subsequent assays, we chose to interact [ 35 S] HeLa cells from vaccinia vectors and then tested for virions with receptor-bearing cells for 1 hr at 4Њ, as bindtheir ability to bind [ 35 S] labeled MHV particles. Chimeric ing differences between the two receptor isoforms were receptors containing 1 b residues from positions 54 to 122 readily discernable under these conditions. Our next were as effective as 1 a in binding, while the reciprocal assay involved testing the related strain JHMX for bindchimeras containing 1 b residues 10 to 54 were ineffective ing. For JHMX, a similar pattern of virus adsorption (Table 2) . emerged (Fig. 6) . The amount of virion adsorption, how-To further narrow down residues controlling receptor ever, was lower for JHMX and the difference between binding efficiency, we focused on codons 38 to 43, as Bgp1 a and Bgp1 b NTD in JHMX adsorptive capacity was only this region is the most variable among the presently se-2.5-fold, comparatively lower than the 6-fold difference quenced Bgps (see Fig. 8 ; boxed region). Additionally observed when strain A59 was used.

this stretch of six residues is predicted to form a protruding loop connecting two b strands that form the frame-Mapping the MHV receptor determinants that control work of the immunoglobulin-like CEA domain (Bates et virus adsorption al., 1992) . Thus the Bgp1 a residues 38 to 43 were The two receptors contain a limited number of amino changed to 1 b residues by oligonucleotide-directed mutaacid differences and show measurable differences in genesis. The resulting chimera (1 b 38-43) exhibited a weak binding efficiency that was equivalent to that of virus binding capacity. This provided an opportunity to (Table 2) . Thus a key determinant of binding differences between the isoforms was present within enhance intercellular fusion with S-expressing cells. To these six residues. do this, we employed a cell fusion-dependent reporter gene activation assay (Nussbaum et al., 1994) . In brief,

In performing these experiments comparing the efficiency of Bgp1 a and Bgp1 b NTD receptors, we readily identified a correlation between virus binding capacity and syncytium formation. Upon infection by MHV-A59, stable HeLa-Bgp1 a transfectants fused rapidly into polykaryons, while the corresponding HeLa-Bgp1 b NTD cells were much less susceptible. This was readily evident by simple microscopic examination of infected cell monolayers anywhere from 10 to 30 hr postinfection. However, previously published information suggested that measurements of additional receptor mutants for virus binding and fusion promotion capacities might not reveal a direct relationship between these two properties. First, evidence for the uncoupling of virus binding and fusion activation functions has been demonstrated in studies of mutant retrovirus receptors (James et al., 1996; Lifson et al., 1988; Truneh et al., 1991) . Second, two reports focusing on the 

The results obtained from a subset of these tests is shown in Fig. 9 . When cells were fixed and stained with transfectants with vaccinia virus (strain WR) and MHV strain A59 and then loading a fraction of the cells with

x-gal (MacGregor et al., 1989) , intense blue clusters were abundantly evident in monolayers expressing Bgp1 a (Fig.  a transcriptionally silent b-galactosidase gene under the control of the phage T7 promoter. These cells, which 9a, panel A) and the Bgp1 a Q10G,V123G mutant. Fewer blue syncytia were seen in the Bgp1 b NTD -expressing mono-bear S proteins on their surface, were then overlaid at subconfluent densities onto monolayers of HeLa cells layers (Fig. 9a, panel C) , while blue cells were rare in the cultures lacking the Bgp NTD altogether (Fig. 9a , coinfected with vTF7.3 and the respective vTM1-Bgp recombinants. Transfer of the T7 polymerase to the S-ex-panel B). Quantitation of the b-galactosidase activity in detergent lysates prepared from each monolayer at vari-pressing cells causes b-galactosidase expression, which can be measured and taken as an indicator of the ous times after cell mixing revealed the potency of each receptor in promoting fusion (Fig. 9b) . From 2 to 4 hr after amount of fusion between receptor and spike-bearing cells.

cocultivation, specific b-galactosidase expression in cultures harboring Bgp1 a was 5-6 times the level found in Bgp1 b NTD -containing cultures, in concert with the sixfold Previous qualitative assays in which virions were al-bound to Bgp1 b NTD . When identical assays were performed using [ 35 S] MHV strain JHMX, 2.8% bound specifi-lowed to interact with denatured and immobilized MHV receptors showed that allelic receptor isoforms termed cally to Bgp1 a , while 0.8% bound Bgp1 b NTD (Fig. 6 ). These differences between the two receptors in virus binding Bgp1 a and 1 b do indeed differ in virion adsorptive capacity (Boyle et al., 1987; Dveksler et al., 1993a) , thereby capacity impact the outcome of MHV infection. We found that transfection of either Bgp1 a or Bgp1 b NTD genes into providing the opportunity for determining which nonhomologous region(s) control the differences. Our initial HeLa cells conferred sensitivity to MHV A59 infection, but Bgp1 b NTD was inefficiently utilized as revealed by goal was to establish assays in which we could accurately measure the relative virus binding capacities of quantitation of infectious centers (Fig. 4) . This latter finding was generally consistent with a variety of previous these two receptors. We anticipated that the success of these assays might require abundant receptor levels observations. Chen and Baric (1996) reported that challenge of stable BHK-Bgp1 a transfectants with MHV A59 because previous attempts to measure virion binding to murine cells endogenously expressing Bgps were not led to titers of 10 8 PFU/ml after 1 day, while parallel challenge of BHK-mmCGM2 (Bgp1 b ) transfectants sensitive enough to reveal any specific adsorption (Yokomori et al., 1993; Wilson and Dales, 1988) . Our binding yielded only 10 4 PFU/ml. In similar tests, Compton (1994) found analagous (but less dramatic) differences between assays therefore involved interaction of radiolabeled virions to native receptors that were overexpressed on the the two stably transfected BHK cell lines in their support of MHV A59 infection. Finally, Yokomori and Lai (1992b) surface of HeLa cells from vaccinia vectors. By this method, specific binding measurements were obtained, found that Cos7 cells transiently transfected with Bgp1 a or 1 b supported very low levels of MHV A59; the 1 a even though the proportions of cell-associated [ 35 S] virions were relatively low (Fig. 6) . Adsorption might be lim-transfectants were marginally superior as virus hosts.

We have exchanged portions of Bgp1 a cDNA with ited in part by slow diffusion to cell monolayers, as prolonged incubation periods gradually increased binding those from Bgp1 b and have assessed the resulting hybrid gene products for virus binding capacity. Our most infor- (Fig. 5) . Ligand densities also likely play a role-recombinant Bgp1 a that is immobilized onto Sepharose at ex-mative recombinant receptor was a Bgp1 a in which only residues 38 -43 were specific to 1 b . This chimera was traordinarily high densities will bind the majority of [ 35 S] virions in our recently developed in vitro receptor binding indistinguishable from the complete Bgp1 b NTD in its support of virus binding (Table 2 ; see data highlighted by assays (Gallagher, 1997) .

We established conditions in which parallel HeLa cell bold type), thus strongly suggesting that these six residues are the critical determinants controlling virus ad-monolayers displayed equivalent amounts of either Bgp1 a or a chimeric Bgp1 b NTD on the cell surface. These sorption levels. Despite this compelling data, it must be FIG. 9 . Quantitation of intercellular fusion between spike-and receptor-bearing HeLa cells. Stable HeLa-Bgp1 a transfectants were infected with MHV strain A59 (m.o.i. Å 2) and concomitantly transfected by lipofection with pG1NT7-bgal, which produces the b-gal product only in the presence of T7 RNA polymerase. 5 hr later, the spike-bearing cells were trypsinized and overlaid onto HeLa cell monolayers that had been infected 20 hr earlier with vTF7.3 and the indicated vTM3-Bgp recombinants. The resulting intercellular fusion levels were quantitated by measuring b-gal enzyme activities produced by the mixing of T7 RNA polymerase (in vTF7.3/vTM3-Bgp cytosol) with pG1NT7-bgal (in MHV-A59 cytosol). (a) Cocultivated cells were fixed after a 3-hr incubation and incubated with x-gal substrate to reveal the b-gal product in situ (A) Bgp1 a , (B) Bgp1D, (C) Bgp1 b NTD , (D) Bgp1 a Q10G,V123G . (b) At the indicated times after overlay, cell monolayers lysed with PBS containing 0.5% NP-40. b-gal activity in the lysates was measured by a colorimetric assay and the OD 590 nm values were normalized by comparison with OD590 nm produced by a preparation of purified bgalactosidase. remembered that a tremendous number of possible re-the binding capacities of the receptors. In fact we have come to similar conclusions using MHV JHM in place of ceptor chimeras can be generated, each with a unique combination of 1 a and 1 b residues. It therefore remains A59-while receptor independent fusion was observed with JHM, enhancement of this endogenous activity was possible that investigations of additional 1 a /1 b combinations will reveal a role for residues outside the 38-43 most pronounced in the presence of receptors with 1 a residues 38 -43. At present we have no evidence of sepa-stretch in binding, perhaps because some patterns will impact overall receptor conformation. rate domains on the Bgp molecule that are individually responsible for binding and induction of membrane fu-Exchange of Bgp1 a residues 38-43 with those from 1 b also eliminates a potential N-linked glycan addition site sion. However, such separate domains may eventually be identified, as studies of both the HIV and poliovirus at residue 37. However it has been established from thorough mutagenesis studies in the Holmes laboratory receptors have revealed specific regions that induce changes in virion conformation required for membrane that a carbohydrate at this position does not contribute to virus binding (Dveksler et al., 1995) . Identification of penetration (James et al., 1996; Morrison et al., 1994) . Finally, the results from these quantitative binding these critical amino acid residues led us to attempt blockade of binding with synthetic peptides. To date we assays may shed light on the determinants of murine coronavirus tropism in vivo. JHMX can infect murine brain have been unable to interfere with the virus:receptor interaction using synthetic Bgp1 a peptide 33-45, implying tissue more readily than A59 (Lavi et al., 1986; Robb and Bond, 1979) , and the overall abundance of Bgp1 a and 1 b a requirement for the immunoglobulin-like framework region in presenting a defined conformation of the resi-in brain is very low Lai, 1992a, 1992b) . Given that JHMX is actually less avid than A59 in the dues.

According to a predicted three-dimensional model for Bgp binding properties, it appears likely that the different in vivo tropisms of these strains are not dictated simply the corresponding human CEA NTD (Bates et al., 1992) , residues 38 to 43 would represent a ''loop'' that is held by their capacity to bind these Bgps but rather by utilization of alternative receptors or by additional events oc-in place by two of the beta strands (strands C and C) that comprise a portion of the framework region (Fig. 8) .

curring subsequent to binding. This C-C loop is further predicted to protrude from the internal framework and thus it is reasonable to infer its Choe and Sodroski, 1992; Harrison, 1994) . Po- Gossen and Herman Bujard for the HeLa-tTA cells. This work was liovirus binding is influenced by mutations in the C-CЉ, supported by NIH Grant R29-NS-31616 and by a grant from the

Cold Spring Harbor using recombinant vaccinia virus vectors

A pregnancy-specific glycoprotein is expressed in the brain and serves as a receptor for mouse hepatitis fusion mutants are attenuated and display altered hepatotropism

Utility of mouse cell line DBT for propagation and assay of mouse hepatitis virus persistence: Coevolution of increased host cell resistance and virus virulence

Contribution of charged amino acids in the CDR2 region of CD4 to HIV-1 gp120 binding

PCR Protocols'' (M. A. Monoclonal antibodies to murine hepatitis virus-4 (strain JHM) define the viral glycoprotein responsible for attachment and cell-cell fusion

The organ tropism of mouse hepatitis virus A59 in mice is dependent on dose in their use of murine carcinoembryonic antigen-related glycoprotein receptors

Synthetic CD4 peptide derivatives that inhibit HIV infection and cytopathicity. Science 241, 712-716. the mouse hepatitis virus (MHV) receptor: Expression in human and hamster cell lines confers susceptibility to MHV

Expression in Vivo

Analysis of genomic and intracellular viral RNAs of small plaque mutants of related glycoprotein family are functional receptors for the coronavirus mouse hepatitis virus-A59

virus strain A59 and blocking antireceptor monoclonal antibody bind to the N-terminal domain of cellular receptor

quantitative equilibrium and kinetic binding studies of mutants in conjunction with a high resolution CD4 atomic structure

Homolog-scanning mutagenesis reveals poliovirus receptor residues important for virus binding and replication

Escherichia coli gpt gene provides dominant selection for vaccinia virus open reading frame expression

Lipofection: A highly efficient, lipid-mediated DNA-transfection procedure. mouse hepatitis virus infection

Proc. Natl. Acad. Sci. USA

Bgp2, a new member of the carcinoembryonic antigen-related gene family, encodes an transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase

Fusogenic mechanisms of enveloped-virus glycoproteins analyzed by a novel recomindependent infection by a neurotropic murine coronavirus

Murine coronavirus membrane fusion is blocked by modification of thiols buried within the spike protein

A role for naturally occurring variation of the measles virus hemagglutinin and CD46

Pathogenic murine coronaviruses. murine coronavirus spike protein in stabilizing association with the cellular receptor

Characterization of biological behavior in vitro and virus-specific intracellular RNA of strongly neurotropic JHMV and weakly neuro-Gombold

Molecular Cloning: protein cleavage signal

Tight control of gene expression in A Laboratory Manual

In vivo and in vitro models of demyelinating disease: Efficiency of virus spread and formation of 5467

Cloning with PCR. In ''PCR Protocols'' (M. A. Innis, infectious centers among glial cells is genetically determined by the murine host

Mouse hepatitis virus utilizes two carcinoembryonic antigens as alternative receptors

tis virus in the resistant mouse strain SJL is functional: Implications for the requirement of a second factor for viral infection

5942-spike protein-dependent cellular factor other than the viral receptor is required for mouse hepatitis virus entry

CЉ-D, and D-E loops (Aoki et al., 1994; Morrison et al., Schweppe Foundation of Chicago. 1994 ). Additional comparisons of receptor structure and function will require definitive resolution of the Bgp1 a REFERENCES receptor through crystallographic methods. This is fast becoming a realistic possibility as a system for produc- Aiyar, A., and Leis, J. (1993) . Modification of the megaprimer method tion and purification of reasonably large quantities of of PCR mutagenesis: Improved amplification of the final product. 14, 366-368. that the relative fusion levels corresponded directly with