Type-C and type-D primate retrovirus envelope glycoproteins bind common cellular receptor sites

VIROLOGY

98,

373-384 (197%

Type-C and Type-D Primate Retrovirus Envelope Glycoproteins Common Cellular Receptor Sites CHARLES F. MOLDOW,” ROBERT S. KAUFFMAN,* SUSHILKUMAR DEVARE,t AND JOHN R. STEPHENSON-F,’

Bind G.

*Department of Medicine, University of Minnesota School of Medicine, Minneapolis, Minnesota 55455, and Wiml Genetics Section, Laboratory of Cellular and Molecular Biology, National Cancer Institute, Frederick, Maryland 21701 Accepted July 9, 1979 The major envelope glycoproteins (gp7Os) of representative type-C and type-D retroviruses have been purified to homogeneity under nondenaturing conditions, labeled to high specific activity with lz51,and analyzed for ability to specifically bind surface receptor sites on cultured mammalian cells. The specificity of such binding is indicated by its inhibition in either productively or nonproductively infected cell lines. In general, gp70 binding parallels host range although notable exceptions are seen. For instance, while MPMV exhibits a much narrower host range than SMRV, envelope glycoproteins isolated from the two viruses exhibit similar binding characteristics. Thus, the resistance to MPMV infection of most mammalian cell lines tested apparently involves replication steps subsequent to receptor binding. Interference studies reveal that gp7Os of type-C viruses of the highly related M7/RD114 primate group and representative type-D retroviruses, such as MPMV and SMRV, recognize the same or similar cellular receptors. Such receptors are strikingly different from those recognized by a murine amphotropic type-C virus isolate, 4070-A. These findings suggest either that type-D and primate-derived type-C retroviruses have acquired common env sequences involved in receptor site recognition by genetic recombination or alternatively, that such viruses share a common progenitor and that genetic sequences involved in receptor site recognition are evolutionarily conserved. INTRODUCTION

Increasing numbers of type-C and type-D primate retroviruses of Old and New World monkey origin are being described. Among type-C viruses, isolates of baboon origin represent true endogenous viruses of primates and have been shown to be present in a naturally integrated state within the primate genome for a prolonged evolutionary time (Benveniste and Todaro, 1976).A highly related group of endogenous feline viruses, of which RD114 is the prototype, is apparently of primate origin, having entered the germ line of an ancestral cat as a relatively recent evolutionary event (McAllister et al., 1972; Benveniste and Todaro, 1974). The type-D virus group consists of an endogenous langur virus (PO-l-Lu) (Todaroet al., 1978), * To whom reprint requests should be addressed. 373

squirrel monkey retroviruses (SMRV) (Herberling et aZ., 19’77),and Mason-Pfizer monkey virus (MPMV) (Chopra and Mason, 1970). While PO-l-Lu and SMRV are endogenous to the spectacled langur (Benveniste and Todaro, 1977) and squirrel monkey (Colcher et al., 1977) respectively, genetic sequences detected in the cellular DNA of rhesus monkeys exhibit only partial complementary to MPMV (Drohan et al., 1977). SMRV, PO-I-Lu, and the endogenous baboon viruses are transmitted vertically, in contrast to MPMV which appears to be horizontally transmitted (Drohan et al., 1977; Fine et al., 1978). Even when derived from evolutionarily distant species, type-C and type-D viruses exhibit considerable relatedness within their respective groups. For instance, functionally analogous structural proteins of diverse type-C viruses have been shown to possess 0042~6322/79/140373-12$02.00/O

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immunological cross reactivity (Geering et al., 1970; Stephenson et al., 1977), and in some instances, amino acid sequence homology (Oroszlan et al., 1975). While studies of type-D retroviruses have been less extensive, the major internal antigens of SMRV, MPMV, and PO-l-Lu are known to possess cross-reactive antigenic determinants (Hino et al., 1977; Colcher et al., 1977; Todaro et al., 1978; Devare et al., 1978a). The existence of common determinants shared by nonglycosylated internal antigens of type-C and type-D retroviruses has not as yet been demonstrated, although the envelope glycoproteins of primate type-C viruses of RD114/baboon group have been shown to share interspecies antigenic determinants with analogous type-D viral proteins (Stephenson et al., 1976; Devare et al., 197813). Viral envelope glycoproteins are involved in recognition of host cell membrane receptors during the process of infection and are important determinants of viral host range (Weiss, 1976). Studies of the binding of purified murine (De Larco and Todaro, 1976; Bishayee et al., 1978), and avian (Moldow et al., 1978) type-C viral glycoproteins to cells have established the kinetics and specificity of cellular receptor site binding and have provided preliminary estimates of the numbers of such receptors per cell. In view of the immunologic cross-reactivity between type-C and type-D viral envelope glycoproteins, it was of interest to establish whether the shared determinants, presumably acquired by genetic recombination, are involved in receptor site recognition. In the present study we have purified and performed binding studies utilizing envelope glycoproteins of diverse mammalian retroviruses to examine such a possibility. The results demonstrate that receptor site recognition is a requirement, but is not sufficient for virus infectivity, and suggest the possible existence of common or highly related receptor sites on the host cell membrane for envelope glycoproteins of type-C and type-D primate retroviruses. Such receptor sites appear distinct from those recognized by nonprimate retroviruses, such as the amphotropic murine leukemia virus isolate.

ET AL. MATERIALS

AND METHODS

Cells and viruses. Cells were grown in Dulbecco’s modification of Eagle’s medium (DMEM) supplemented with 10% calf serum. Cell lines used included canine embryo thymus (FCfZTh), bat lung (Tb-1-Lu), and fetal mink lung cells (CCL64) obtained from the American Type Culture Collection, Rockville, Maryland. Canine embryo thymus cells nonproductively infected with either of two nonconditional replication mutants of RD114, designated RD-52-1 and RD-54-5, or by M7-baboon virus mutants, M7-24-3 and M7-114-7, have been described elsewhere (Sacks et al., 1978). Other cell lines, including an NIH Swiss mouse embryo fibroblast line (NIH/3T3) (Jainchill et al., 1969) and a human rhabdomyosarcoma line (A673) (Giard et al., 1973) have been previously described. A kitten embryonic lung cell line (KELu) was developed in our laboratory. An endogenous type-C virus isolate of Papio cynocephalus baboon origin, (M7baboon virus), a highly related endogenous cat virus (RD114), a wild mouse amphotropic virus, 4070-A (Hartley and Rowe, 1976), as well as two type-D retroviruses, MPMV and SMRV, have also been described (Devare et al., 197813).Viruses were propagated and purified by sucrose density gradient centrifugation through the courtesy of R. V. Gilden, Frederick Cancer Research Center, Frederick, Maryland. PuriJication and lz51-labeling of viral envelope glycoproteins. Density gradient purified viruses (8-10 mg) were disrupted by sonication for 10 set in 0.05 M Tris-HCl, pH 8.5, buffer containing 1.0% Triton X-100 and 2.0 mM EDTA, clarified by centrifugation (100,000 g for 30 min), dialyzed overnight at 4” against 0.01 M sodium phosphate, pH 7.4, 0.14 M NaCl (PBS), and applied to agarose-Lens culinaris lectin (P-L Biochemicals, Milwaukee, Wis.) columns (1.5 x 5 cm). Columns were washed at 4” with 25 ml of PBS, and viral envelope glycoproteins eluted at room temperature with PBS containing 0.2 M a-methyl D-mannopyranoside. Glycoproteins were located by monitoring ultraviolet absorbance at 280 nm and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

PRIMATERETROVIRUSENVELOPEGLYCOPROTEINS (Laemmli, 1970). Peak &actions were pooled,

dialyzed at 4” overnight against TET (10 mM Tris-HCl, pH 7.8, 0.1 mM EDTA, 0.5% Triton X-100), and applied to diethylaminoethyl (DEAE)-cellulose (DE52 H. Reeve Angle, Clifton, N. J.) columns (1.5 x 5 cm) equilibrated with TET. Columns were washed with 25 ml of TET, and the glycoprotein was eluted with a loo-ml linear gradient of 0 to 0.5 M NaCl in TET. Purified glycoproteins were labeled with 1251(Amersham, Arlington Heights, Ill.) to specific activities of 1 x lo4 to 8 x lo4 cpm/ng protein by the solid phase iodogen (Pierce Biochemicals, Rockford, Ill.) procedure (Fraker and Speck, 19’78; Devare et al., 197813). Protein aliquots were maintained at -70” and used within 5-7 days after iodination. The radiochemical purity of 1251-labeledglycoproteins was established by SDS-PAGE and radioautography (Bonner and Laskey, 1974). Receptor site binding assay. Binding studies were performed using both suspensions of fibroblasts as described below and with monolayers of cells grown on 35-mm plastic dishes according to the method of De Larco and Todaro (1976). Results obtained using the two methods were in close agreement; nonspecific binding was lower but the binding took longer to reach saturation using the monolayer method. However, cell numbers were more accurately controlled and the results more reproducible using cell suspensions. For consistency, all of the results presented in the present studies were based on cell suspension assays and were obtained as follows. Cells were grown to confluence on petri dishes, scraped in the presence of trypsin (0.05%) and EDTA (0.0005%), suspended in DMEM containing 10% calf serum, washed in this medium, and transferred to 10 x 75mm plastic tubes previously washed with calf serum and air dried. The cells were washed once in DMEM and resuspended in binding buffer (DMEM containing 200 pg/ml ovalbumin and 8 mM Tris pH 7.8) at 1 x lo7 cells/ml and incubated at 37” with 1251-labeledgp70 for varying lengths of time. The binding reaction was terminated by the addition of 1 ml of ice-cold PBS; cells were sedimented at 1000 g for 5 min, washed twice with PBS, and the

375

amount of ‘251-labeledgp70 bound was measured in an LKB-1280 Ultrogamma counter. Specific binding values represent the means of triplicate determinations and have been corrected for background nonspecific binding by substrating appropriate blanks. RNA-chqxvdent DNA polyrmmse assays. Culture fluids (20 ml) were harvested, clarified by centrifugation at 10,000 rpm for 30 min, and virus was pelleted by centrifugation at 30,000 rpm for 60 min in a Beckman type 30 rotor and resuspended in 0.2 ml virus disruption buffer containing 50 mM Tris-HCl (pH 7.8), 100 mM KCl, 0.05% Triton X-100, and 2 mM DTT. For determination of vii-ion-associated RNA-dependent DNA polymerase enzymatic activity, 20 ~1 of the dissociated virus was incubated at 37” for 60 min in 0. l-ml reaction mixtures consisting of 50 mM Tris-HCl (pH 7.8), 90 mM KCl, 0.1 mM MnCl,, 0.05% Triton X-100, 2 mM DTT, 500ng poly(rA) ,375 ng oligo (dT) 12-18, (Collaborative Research, Inc., Waltham, Mass,) and 200 pmol [3H]TTP (55 Ci/ mmol, Schwartz/Mann, Orangeburg, N. Y.). The enzymatic activity of type D viruses was determined utilizing 7.5 mM MgCl, instead of MnCl, and in the presence of 16 mM KCl. DNA synthesis was measured according to previously described methods (Scolnick et al., 1970). Competition immummsuys. Cell extracts were prepared by sonication of cells for 20 seein an equal volume of 10 mM Tris-HCl buffer, pH 7.8, containing 100 mM NaCl, 0.5 mM EDTA, and 0.5% Triton X-100. Competition immunoassays were performed by testing unlabeled cell extracts at serial twofold dilutions for ability to compete with ‘251-labeledRD114 gp70 for binding limiting amounts of goat antiserum directed against detergent-disrupted M’I-baboon virus. Reaction mixtures contained 0.01 M Tris-HCl, pH 7.8, 1.0 mM EDTA, 0.4% Triton X-100, 1% bovine serum albumin, and 0.2 M NaCl in a total volume of 0.2 ml. Antiserum and unlabeled competing antigen were incubated at 37” for 1 hr followed by the addition of 10,000 cpm 1251-labeled antigen. After further incubation for 3 hr at 37” and 18 hr at 4”, antigen-antibody complexes were immunoprecipitated as previously described (Stephenson et cd., 1977).

376

MOLDOW ET AL. RESULTS

Isolation of Envelope Glycoproteins and Analysis of Their Interactions with Cell Receptors Envelope glycoproteins of type-D retroviruses including MPMV and SMRV, endogenous type-C viruses of the RDllWbaboon group, and a prototype amphotropic mouse type-C virus isolate, 4070-A, were purified and 1251-labeled at high specific activity as described under Materials and Methods. Each 12Y-labeled protein was shown to migrate as a single well-defined band by SDS-PAGE establishing a high degree of radiochemical purity (Fig. 1). Initial studies were undertaken to determine the influence of time and temperature on final extent of binding of each of the *251-labeledviral envelope glycoproteins to mink cells. The mink lung cell line, CCL64, A I

B I

C I

D I

E I

FIG. 1. SDS-PAGE analysis of purified viral envelope glycoproteins. Y-Labeled viral envelope glycoproteins (50,000cpm) isolated from (A) RD114; (B) M71 baboon virus; (C) SMRV; (D) MPMV; and (E) 4070-A were heated for 2 min at 90” in 0.65 M Tris-HCl, pH 6.7,1% SDS, 10%glycerol, 2.5% 2-mercaptoethanol and 0.1% bromophenol blue, and electrophoresed through a 5% acrylamide, 0.13% bisacrylamide stacking gel (pH 6.7) and a 5-208 linear polyacrylamide separation gel (pH 3.9) at 20 mA per gel (Laemmli, 1970).Molecular weight markers included “C-formalde hyde labeled phosphorylase B (98,000), bovine serum albumin (69,000), ovalbumin (SS,OOO),carbonic anhydrase (30,000), and cytochrome c (12,300). Radioactive proteins were visualized by autoradiography (Bonner and Laskey, 1974).

was arbitrarily chosen for this purpose. Binding was time and temperature dependent; at 37”, saturation was achieved within 90 min and the number of molecules bound remained relatively constant for an additional hour (Table 1). In contrast, at 4”, the extent of binding increased linearly with time up to 180min, and failed to reach saturation even when tested for periods as long as 4 hr (data not shown). Because binding was considerably more rapid at 37” and near saturation levels could be achieved, all further studies were performed at this temperature. In view of the fact that only between 1.0 and 8.0% of different input 1251-labeled gp7Os bound to CCL64 mink cells under conditions of receptor excess, it was important to rule out the possibility that the binding observed was due to minor contaminants in the labeled gp7Os. Therefore, cell bound gp7Os were analyzed by SDS-PAGE. The results of such an analysis establish that binding activity comigrates with input gp70, and thus confirms that the binding observed is gp70 specific (data not shown). Although the differences in molecular weight of the various gp7Osare minor, they are sufficiently large to rule out the possibility of a common contaminant of around 70,000 daltons in molecular weight. Moreover, in other studies we have shown that following receptor site binding by Prague Rous sarcoma virus gp85, the eluted protein has a molecular weight of 85,000 daltons. As a further test of the specificity of the gp70 binding observed, other viral structural proteins such as Rauscher MuLV ~12 and p30 were analyzed for receptor site binding capacity. As summarized in Table 2, neither exhibited significant levels of binding above background. Finally, by pretreatment of 4070-A envelope glycoprotein with high titer monospecific Rauscher-MuLV gp70 sera, it was possible to remove over 95% of the 1251-labeland simultaneously reduce receptor site binding to nondetectable levels. For quantitation of cell receptor sites for viral glycoproteins, it was first necessary to determine amounts of gp70 necessary to achieve saturation. The extent of binding to CCL64 cells was determined by varying input 1251-labeledRD114 gp70 over a range

PRIMATE RETROVIRUS

ENVELOPE

TABLE 1 DETERMINATION OF TIME AND GLY~OPROT~~~ CONCENTRATION REQUIRED FOR SATURATION OF CELLULAR RECEPTOR Sm3s

lzSI-Labeled gp70 (cpm x lo+) bound ata lz51-Labeled RD114 15 min 30 min 60 min 120 min gp70 (input) 1.0 1.8 NT NT NT NT NT NT

3.5 4.0 11.0 31 NT NT NT NT

4.0 5.0 13.5 NT 86 NT NT NT

377

GLYCOPROTEINS

isolates tested. It should be stressed, however, that these are only rough approximations and, because we have probably not reached complete saturation of binding sites, are likely to represent underestimations of the true values. Role ofReceptor Site Recognition in Establishment of Productive Virus Infection

5.0 7.2 14.8 78 115 150 143 lt54

To further examine specificity of receptor site binding by lz51-labeled gp7Os and to determine the extent to which receptor site recognition reflects host range, a broad range of mammali~ cell lines were analyzed for ability to bind the above described viral glycoproteins (Table 3). In parallel, cell lines were assayed for susceptibility to infection a Binding assays were performed using 1.0 X lo5 by the same viruses. The results indicate CCL64 mink cells per reaction mixture and 1251-labeled that susceptibility to infection requires, or RD114 gp’70 at a specific activity of 8.0 x lo4 cpmng. at least correlates positively, with gp70 reResults represent mean values of triplicate determiceptor site recognition. Receptor site bind0.2 x 0.6 x 1.0 x 4.0 x 12.0 x 18.0 x 20.0 x 24.0 x

lo6 lo@ 106 106 106 10” lo6 lo6

nations and have been corrected for nonspecific binding to reaction tubes in the absence of cells.

TABLE 2

from 0.2 x lo6 to 24 x lo6 cpm. The results (Table 1) indicate near saturation of binding using 18 x 10” cpm lz51-labeled gp70 when binding is allowed to proceed for at least 90 min. Moreover, as shown in Fig. 2, equilib~um was indeed achieved under such conditions, and an increase in cell number led to increased, although not absolutely proportional, binding. Thus, while it is not possible to fully saturate available gp70 binding sites under conditions of vast gp70 excess, the levels of binding seen at equilibrium in the presence of limited cell numbers, and limited but excess gp’70, reflect the product of binding site number times binding site affinity. This value may be used to eompare gp70 binding to various cell lines. By the same approach, we can estimate saturation binding levels of type-C and type-D retroviral gp?Os to CCL64 cells. Using the amount of gp70 bound by 1 x lo5 cells after 2 hr incubation, in combination with specific activities of the 1251-labeled gp?Os (2-8 x lo4 epmlng) and assuming one gp70 molecule binds to one receptor site, we estimate mink cells to possess between 1 x lo5 to 4 x lo5 gp70 binding sites for each of the type-C and type-D retrovirus

SPECIFIC INHIBITION OF lz51 gp70 RECEPTOR SITE BINDING BY A MONOSPECIFIC SERA DIRECTED

AGAINSTg~i’0’ ‘251-Labeled antigen bound (epm x IO-3 Control Sera Anti-p30 Anti-~70 Test antigen Total Bound Total Bound Total Bound gP76 P12 P30

9800 9400 9700

800 <2 <2

9300 7600 210

860 <2 <2

200 8900 9200

<2 <2 <2

” Monospecific sera used, prepared against highly purified Rauscher-MuLV gp70 and ~30, have been previously described (Reynolds et al., 1978). Antibody inhibition of binding was performed by addition of 10 ~1 undiluted sera to 0.5 ml 0.01 2M Tris-HCI, pH 7.8 reaction mixtures containing lO* cpm of the appropriate ‘251-Iabeledantigen. Following incubation for 2 hr at 37”, 0.1 ml of a 10% suspension of Sepharose-bound protein A (Pha~acia, Piscataway, N. J.) was added, incubation continued for 3 hr at 4”, and complexes were removed by ~ent~fugation at 2500rpm for 15 min. Results are expressed as cpm bound per 1@ cells after 2 hr incubation and represent mean values from triplicate determinations. Binding assays were performed using 1.0 x 10” uninfected NIH/3T3 mouse cells and LZ51-Iabeled4070-A gp70, ~30 or p12 as described under Materials and Methods.

378

MOLDOW ET AL.

binds readily to nonpermissive cells. RD114 gp’70, on the other hand, failed to bind nonpermissive NIH/3T3 cells as might be predicted based on a previously reported host range analysis of vesicular stomatitis virus pseudotypes of RD114 (Schnitzer et al., 1977). While these findings establish glycoprotein receptor site recognition as an important determinant of viral host range, other cellular controls must also influence host cell susceptibility to superinfection. Primate-Derived Type-C and Type-D Retroviruses Recognize Common Cell Receptors TIME(min)

FIG. 2. Binding of RD114 gp’70 to CCL64 mink cells under conditions of glycoprotein excess. Y-Labeled RD114 gp’70, 1.0 x lo6 cpm, was assayed for 2 hr at 37”forbindingl x 10”(O), 1 x 10’(A), 5 x 10’(O), and 1 x 107(0) mink ceils as described under Materials and Methods. Results have been corrected for nonspecific background binding and represent mean values from three separate determinations.

ing, however, clearly is not the sole determinant of infectivity. For instance, MPMV, which has an extremely narrow host range,

In view of the previously demonstrated immunologic cross-reactivity between envelope glycoproteins of type-C and type-D primate retroviruses (Stephenson et al., 1976;Devareet al., 1978b), it was of interest to examine whether viruses of these two distinct groups might recognize common cell surface receptors. Experiments were performed in which mink cells were infected with various viruses and binding of individual glycoproteins to infected cells was studied. As shown in Fig. 3, the extent to which lz51-labeled SMRV gp70 bound mink cells productively infected with SMRV was con-

TABLE 3 RETROVIRUSENVELOPE GLYCOPROTEINRECEPTORSITE RECOGNITIONAND VIRUS HOST RANGE ‘*Y-Labeled envelope glycoprotein bound (cpm x IO+) (supernatant reverse transcriptase activity (cpm X 10-3))a Host cell line

Species

MPMV

gp70

SMRV

gp70

RD114

gp70

A673 Tb-1-Lu FCf2Th KELu CCL64 NIH/3T3

Human Bat Dog Cat Mink Mouse

41.3

(33.1) (CO.2) (~0.2) (cO.2) (~0.2) (CO.2)

6 NT 16 7.3 9 (NT)

(430) (20.2) (210) (NT) (980) (NT)

16.5 10.9 20.0 13.0 15.2 1.0

(870) (130) (910) (205) (110) (CO.2)

3:: 37.2 16.5 9.0

M7/baboon gp70 6.1 NT 16.0 NT 9.0 13.0

(430)

(210) (450)

(230) (280) (10)

4070-A gp70 16.4 7.9 16.2 NT 14.1 NT

(450) (210) (760) (180) (810) (690)

a Binding assays were performed using 1.0 x 10Buninfected cells and L*51-labeledglycoproteins (1 x 10Bcpm) as described under Materials and Methods. Results are expressed as cpm bound per IO8cells after 2 hr incubation and represent mean values of triplicate determinations corrected for nonspecific background binding to tubes in the absence of cells. Viral host range was determined by pretreatment of designated cell lines with DMEM Polybrene (2 pg/ml) for 24 hr and subsequent infection with the appropriate virus. Ten days following infection, culture fluids were harvested and analyzed for virion-associated reverse transcriptase activity using poly (rA)oligo(dT),,-,, as template primer and either 0.1 mM MnZ+(type-C viruses) or 7.5 mAf Mg2+ (type-D viruses) as the divalent cation. Results obtained represent mean values from two independent determinations and are indicated in brackets. NT, not tested.

PRIMATE RETROVIRUS

ENVELOPE

siderably lower than to uninfected control cells. It should be noted, however, that productive virus infection did not entirely abolish binding. SMRV-infected cells still bound 1251-labeledSMRV to a final extent of around 25% of that seen with uninfected mink cells. Addition of excess cold virus prior to the labeled protein reduced the gp’70binding to uninfected cells to this same degree but did not further reduce the binding of gp70 to the productively infected cells (data not shown). The possibility that at least a portion of the residual binding to mink cells is nonspecific or due to phagocytosis of a fraction of the labeled antigen cannot be completely excluded. The binding of SMRV gp70 to RD114 or M7-baboon infected cells was also considerably reduced as compared to uninfected controls, but was consistently greater than exhibited by cells infected with SMRV (Fig. 3). Further evidence for cross-interference of binding between type-D and primatederived type-C retroviruses is indicated by the results summarized in Table 4. Reciprocal efficient cross-interference was observed between RD114 and M7 virus while cells infected with these viruses exhibited only partial interference with SMRV gp70 receptor site recognition. Neither SMRV nor type-C viruses of the M7/RD114 class competed significantly with binding by the wild mouse amphotropic virus isolate, 4070-A.

GLYCOPROTEINS

379

20-

60

120

IN

TlME(min)

FIG. 3. Binding of SMRV gp70 to productively infected mink lung cells. Binding assays were performed using 1.0 X lo6 cpm 1Z51-labeledSMRV gp70. Cell lines were used at a concentration of 1 x IO6cells per reaction and included uninfected mink cells (0) and mink cells productively infected with M’I-baboon virus (A), RD114 (Cl), or SMRV (0). The binding reaction was carried out for 2 hr at 37” as described under Materials and Methods. Results have been corrected for nonspecific background binding and represent mean values from three separate determinations.

These findings imply the existence of a common receptor site on mink cells shared by primate-derived type-C and type-D retroviruses and raise the possibility that the

TABLE 4

BINDINGOFVIRAL ENVELOPEGLYCOPROTEINS TOUNINFECTEDCONTROL ANDPRODUCTIVELY INFECTEDFETALMINK LUNGCELLS ‘*SI-Labeled gp70 bound (% of uninfected control)” Envelope glycoprotein (gP70) MWbaboon virus RD114 MPMV SMRV 4070-A

Infected with Uninfected control

M’llbaboon

RD114

SMRV

4070-A

100 100 100 100

22.9 24.3 50.1 53.1

21.6 24.0 46.6 49.3

19.0 16.0 36.0 34.3

100

92

98.0

96.2

92.0 93.2 NT 89.0 24.1

a Binding assays were performed for 2 hr at 37” using 1.0 x lo6 uninfected CCL64 fetal lung mink cells and saturation levels of **jI-labeled envelope glycoproteins (1 x lo6 cpm). Results are expressed as a percentage of maximum level of binding obtained using uninfected cells and represent mean values of triplicate determinations.

380

MOLDOW ET AL.

wild mouse amphotropic virus has a different affinity for the same site or alternatively, binds a separate class of receptor(s). Interference with Receptor Site Recognition by gp70 Expressed in NonproductivelyInfected Cell Clones In a previous report, we described the isolation and preliminary characterization of a number of cell clones nonproductively infected by representative type-C and type-D retrovirus isolates (Sackset al., 1978). Certain of these mutant clones, when analyzed by specific radioimmunoassay, are characterized by envelope glycoprotein expression in the absence of complete virus, or in some clones, even gag gene translational products (Table 5). We thus extended the above studies to an analysis of representative mutant clones to examine whether glycopro-

tein expression in itself is sufficient to block gp’i’0 binding. lz51-Labeled SMRV gp70 was analyzed for binding to either uninfected FCf2Th cells and to representative nonproductively infected mutant clones. In agreement with data presented above, SMRV gp70 efficiently bound uninfected FCf2Th cells but failed to adhere to more than a limited extent to mutant infected cells positive for either RD114 or M7-baboon glycoprotein expression (Fig. 4). The binding of SMRV gp70 to a RD114 mutant clone lacking detectable gp70 (~8 ng/mg), although still highly significant, was 30% lower than that observed with uninfected cells, possibly reflecting gp70 expression at levels below the limits of detectability by competition immunoassay. The results of an extension of such studies to additional mutant clones and different gp70 isolates are summarized in Table 5.

TABLE 5 BINDING OF 1251-L~~~~~~ENVELOPE GLYCOPROTEINSTO PRODUCTIVELYAND NONPRODUCTIVELYINFECTED CANINE THYMUS CELLS

Cell line Control FCf2Th Nonproductivelyinfected FCf2Th-RD-54-5 FCf2Th-RD-52-1 FCf2Th-M7-24-3 FCf2Th-M7-114-7 Productivelyinfected FCf2Th (RD114) FCf2Th (M7)

Virus production: supernatant reverse Expression of transcriptase gp70 (ng/mg [3H]dTMP incorporated (cpm x 10-3)lmlh cell protein)”

1251-labeledgp70 bound (o/oof uninfected control)’ M’l’lbaboon RD114 SMRV

MPMV

4070-A

100

100

8

0.8

8 64 32 32

1.1 1.3 0.9 0.8

17.7 2.0 1.6 0.7

33.3 5.9 9.8 5.6

66.6 16.5 11.6 9.5

41.0 7.8 9.2 6.1

66.8 22.2 31.3 25.2

1800 1500

503.0 4.96.0

11.2 8.4

13.2 16.8

25.3 22.0

14.2 17.6

96.2 87.3

100

100

100

a Cell extracts were prepared as described under Materials and Methods and tested at serial twofold dilution in heterologous competition immunoassays using goat antiserum to M7/baboon virus for precipitation of ‘ZJI-labeled RD114 gp’70 (Stephenson et al., 1976). Results are expressed as ng viral antigen/mg ceil protein and represent mean values from three separate determinations. b Virus production was measured by analysis of culture fluids for virion-associated reverse transcriptase activity using 0.1 mM MnZ+ as the divalent cation and poly(rA)-oligo(dT),Z-,B as the template primer. The reverse transcriptase values in italics are considered to be significantly above background. r Binding assays were performed using 1.0 x lo6 uninfected cells and 1.0 x 10s cpm ‘Y-labeled gp70. Results are expressed as percentage of maximal binding obtained using uninfected cells and represent mean values of triplicate determinations.

PRIMATE RETROVIRUS

ENVELOPE

Clones expressing either RD114 or M7baboon virus gp70 in the absence of virus exhibited lower extents of lz51-labeledRD114 or M7 gp’70 binding than even productively infected cultures. Binding of SMRV gp70, and to a lesser extent, 4070-A gp70, was also reduced in the RD114 and M7 gp70 positive clones. These studies indicate that glycoproteins expressed in the mutant cell clones may occupy cell surface receptors, thereby preventing the binding of exogenously applied glycoproteins. Cross-interference of RD114 or M7-baboon glycoprotein expression with SMRV gp70 binding supports the contention that these viruses recognize a similar class of receptor sites on the host cell surface. It should be noted that binding interference was more pronounced in gp70 positive mutant clones than in productively infected cells. In fact, crossinterference of RD114 and M7 with 4070-A gp70 binding is only seen with RD114 and M7 gp70 positive mutant clones and not using cells productively infected with these viruses. DISCUSSION

GLYCOPROTEINS

TIME (min)

381

I

FIG. 4. Influence of RD114 and M7 glycoprotein expression on binding of SMRV gp70 to FCf2Th canine thymus cell receptors. Binding assays were carried out for 2 hr at 37” using 1 x lo6 cpm 1251-labeledSMRV gp70 as described under Materials and Methods. Cell lines were used at concentrations of 1 x lo6 cells per reaction and include uninfected FCf2Th canine thymus cells (A), as well as the following nonproductively infected FCf2Th mutant clones: RD-54-5 (O), RD-52-1 (O), M7-24-3 (A), and M7-114-7 (0) clones. Results are corrected for nonspecific background binding and represent mean values from three separate determinations.

Envelope glycoproteins from several mammalian retroviruses have been purified under conditions which result in preservation of their antigenic and cell surface bind- ficulties in accurately measuring specific ing activities. Since the amount of 1251-labeled activities of 1251-labeledproteins and in obgp70 bound plateaus when gp70 concentra- tained saturating levels of binding. Furthertion is progressively increased to saturation more, the values for receptor site numbers levels, an approximation of the number of reported in the present study were detergp70 molecules bound under conditions of mined using cells in suspension subsequent gp70 excess, and by inference, the number to mild trypsin treatment which may cause of binding sites per cell could be deduced. loss of a portion of the receptors. By use of The binding site number on mink cells cal- nontrypsinized culture cells in petri dishes culated by this means for type-C viruses 15-25% higher values are obtained. Howincluding M7, RD114, and 4070-A, and for ever, it is much more difficult to control cell number using attached cells and saturatwo representative type-D retroviruses, SMRV and MPMV, ranged from 1.0 x lo5 tion takes longer to occur. Several lines of evidence argue for speciflcto 5.0 x lo5 per cell. This value is in close agreement with previous reports of around ity of the gp70 binding reactivity observed. lo5 Rauscher-MuLV gp70 binding sites on Productive, as well as nonproductive infecmouse cells (De Larco and Todaro, 1976; tion results in inhibition of binding of the Bishayee et al., 1978). Such determinations homologous gp7Os. Interference by nonproof binding site numbers must, however, ductively infected mutant clones is much be considered as only approximate and more efficient than that observed with probably represents underestimates of the productively infected clones, suggesting actual values. This is due in part to the dif- preferential localization of gp70 on the cell

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surface in the mutant clones. Possibly much of the gp70 immunologic reactivity observed in productive infection is intracellular or virion associated and thus not available for receptor binding. Alternatively, the chronically infected lines might contain subpopulations of gp’70 negative cells responsible for the binding observed. We are presently recloning these cultures to explore this possibility. Further evidence for specificity of binding is indicated by the fact that other type-C and type-D viral proteins such as the major internal antigens fail to bind cell surface receptors and the finding that binding by envelope glycoproteins can be inhibited by use of specific antisera. In general, gp70 receptor site binding correlates closely with viral host range. In each instance where a recipient cell line is susceptible to infection, receptor binding is also observed. However, the converse is not necessarily the case. For instance, despite their strikingly different host ranges, the two type-D retroviruses studied, MPMV and SMRV, exhibit very similar binding characteristics. Thus, the site of restriction to establishment of productive infection by MPMV probably does not reside at the cell surface. Such a block could be at the level of penetration as has been reported in the avian system (Weiss, 1976), or involve intracellular regulatory controls such as the murine type-C viral Fv-1 regulatory gene (Pincus et al., 1971). The availability of highly sensitive and reproducible assays for recognition of cellular receptors by a broad range of nonprimate and primate retroviruses should be of value in attempts to characterize cellular blocks to virus infection. The primate (P. cynocephalus baboon)derived type-C virus isolate, M7, and the highly related endogenous cat virus, RD114, exhibit virtually complete reciprocal crossinterference in receptor site binding. Further, each interferes to a lesser but highly significant extent with the two representative type-D retroviruses studied, SMRV and MPMV. In contrast, minimal, if any, cross-interference is observed between glycoproteins of the murine amphotropic virus, 4070-A, and the two primaterelated type-C isolates and none between envelope glycoproteins of 4070-A and the

type-D virus isolates. Type-C viruses of the M7/RD114 primate group and type-D retroviruses thus apparently recognize the same surface receptors although with slightly differing specificities. This observation is consistent with our earlier demonstration of a class of interspecies gp70 antigenic determinants shared by all type-C retroviruses and a second less pronounced group of determinants specific to envelope glycoproteins of primate-derived type-C and type-D retroviruses (Stephenson et al., 1976; Devare et al., 1978b). It is possible that within the gp70 molecule these latter determinants are identical or located in close proximity to sequences involved in cell receptor site recognition. One explanation for these findings is that the existence of similar sequences within the env genes of type-D and primatederived, type-C viruses is due to genetic recombination. An alternative, and equally plausible possibility, is that because of the recognition of common receptor sites by type-C and type-D retroviruses, genetic sequences within env which encode determinants involved in receptor site recognition may be evolutionarily conserved. Whichever model proves to be the case, our demonstration that highly divergent type-C and type-D retroviruses share gp70 receptor sites should be of value in efforts to intracistronically map regions of the type-C viral env gene defining receptor site recognition. ACKNOWLEDGMENTS The authors thank K. Lundberg and S. VanLaninghamMiller for excellent technical assistance. This work was supported under Public Health Service Grant CH-13722 and Contract NOl-CO-75330,National Cancer Institute, NIH, Bethesda, Maryland 20205. REFERENCES BENVENISTE, R. E., and TODARO,G. J. (1974). Evolution of C-type viral genes: Inheritance of exogenously acquired viral genes. Nature (London) 252, 456459.

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