INTRODUCTION

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Recent evidence has demonstrated that a single cell surface receptor (the X-receptor) from humans mediates infections by both xenotropic and polytropic host range groups of murine leukemia viruses (X-MLVs and P-MLVs, respectively) (1, 37, 40), consistent with previous evidence that these classes of MLVs might use a common receptor. For example, laboratory mice including NIH Swiss are highly susceptible to P-MLVs (also called mink cell focus-forming viruses [MCFs]) (5, 11) but are resistant to X-MLVs (21), whereas most wild mice, including Mus spretus, are variably susceptible to both X-MLVs and P-MLVs (19). Crosses of NIH Swiss mice with M. spretus implied that susceptibility to X-MLVs is caused by a dominant allele of the P-MLV receptor gene Rmc1 (18, 19). Moreover, expression of a P-MLV-related envelope glycoprotein in M. spretus caused weak interference to infections by both groups of virus (19). Cross-interference between X-MLVs and P-MLVs has also been reported for Mus dunni fibroblasts (2, 27). In contrast, Mus castaneus is resistant to both X-MLVs and P-MLVs (24, 25). In crosses of M. castaneus with NIH Swiss or DBA/2 laboratory mice, resistance was inherited as a recessive allele of the Rmc1 gene (24). Surprisingly, however, recent evidence has demonstrated that resistance to X-MLVs and P-MLVs is dominant in crosses of M. castaneus with mice that contain the M. spretus Rmc1 allele (25). Apparently, an X-MLV-related envelope glycoprotein that is endogenously inherited in M. castaneus interferes with the M. spretus X-receptor but not with the NIH Swiss X-receptor (25). Together, these results imply that the X-receptors of mice must be polymorphic and that susceptibilities to infections by X-MLVs and P-MLVs are regulated by these polymorphisms and also by inherited interference factors that differentially interact with X-receptors of distinct mouse strains. In addition, a lipoprotein factor in the sera of most mouse strains specifically inactivates X-MLVs and P-MLVs but not other host range classes of MLVs (15, 21-23). X-MLV gene expression is also partially silenced in certain mouse cells (13, 19a). This evidence has implied that multiple mechanisms of defense have evolved to control diseases caused by the X-MLV/P-MLV family of retroviruses. Indeed, P-MLVs (MCFs) are exceptionally pathogenic and have been implicated as critical causal factors in murine retroviral leukemogenesis and lymphomagenesis (4, 8, 11, 12, 14, 32, 34, 36).

Although it was recently shown that the human X-receptor mediates infections by both X-MLVs and P-MLVs (1, 37, 40) and that an X-receptor from NIH Swiss mice functions as a P-MLV receptor when expressed in hamster cells (40), the latter receptor was not tested for mediation of X-MLV infections. Such testing is necessary because susceptibility of mice to X-MLVs and P-MLVs can be regulated not only by receptors but also, as described above, by many other host factors. Moreover, X-receptor cDNAs from other mouse strains have not been previously described. To address these issues, we cloned and functionally analyzed X-receptor cDNAs from a representative group of mouse strains that are differentially susceptible to X-MLVs and P-MLVs, as well as from mink cells that are morphologically altered by P-MLVs and from Chinese hamster ovary (CHO) cells. Moreover, we constructed and tested a series of mutations in the X-receptors of M. dunni and NIH Swiss mice in order to identify the amino acid sequence differences that are responsible for resistance of laboratory mice to X-MLVs.

	MATERIALS AND METHODS

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Mice, cell lines, and viruses. NIH Swiss inbred NFS/N, M. spretus, and M. castaneus wild-derived mice were obtained from Jackson Laboratory, Bar Harbor, Maine. M. dunni tail fibroblasts and SC-1, BALB/3T3, and mink Mv-1-Lu (CCL64) cells were grown in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum (FBS). Chinese hamster ovary (CHO) cells were grown in -modified minimal essential medium supplemented with 10% FBS.

Receptor cDNA cloning. X-receptor cDNAs were isolated by reverse transcription-PCR amplification with total RNAs. Total RNAs were prepared from M. dunni, SC-1, mink Mv-1-Lu, and hamster (CHO) cells with the RNeasy Midi kit (Qiagen, Valencia, Calif.), whereas total RNAs from mouse kidney tissue, isolated from NIH Swiss, M. spretus, and M. castaneus, were prepared by the cesium chloride method (3). The 2.1-kb X-receptor cDNAs were amplified by using primers complementary to the 5' and 3' ends of the human X-receptor coding region (upstream primer, 5'-GGGGGATCCATGAAGTTCGCCGAGCACCTC-3', containing a BamHI restriction site [underlined sequence]; downstream primer, 5'-GGTCGAGGAAAGGATTGTAG-3'). PCR was run for 30 cycles with a 60°C annealing temperature for 1 min and an extension temperature of 72°C for 3 min. The amplified X-receptor DNAs were cloned into the mammalian expression vector pcDNA3.1.V5His-TOPO (Invitrogen, Carlsbad, Calif.). The DNA sequences were determined at the Microbiology and Molecular Immunology Core Facility on a PE/ABD 377 sequencer with dye terminator cycle sequencing chemistry (Applied Biosystems, Foster City, Calif.).

Transfection and infection assays. Mouse BALB/3T3 and CHO cells expressing NIH Swiss, M. dunni, SC-1, M. castaneus, mink, and hamster X-receptors were generated by transient or stable transfection of the corresponding cDNAs with SuperFect transfection reagent (Qiagen). Transient transfectants were challenged with either LacZ(X-MLV) or LacZ(MCF247) pseudotype virus, with overnight incubations with virus beginning 24 h after transfection. Infected cells were stained by treating cells with 0.25% glutaraldehyde and assayed for -galactosidase activity with X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside) as a substrate (26). Blue CFU were counted, and the titer of infection was expressed as the number of CFU per milliliter of virus supernatant. Stable transfectants were generated by selection with G418 (1 mg/ml). G418-resistant clones were then pooled and tested for sensitivity to LacZ(X-MLV), LacZ(MCF13), and LacZ(MCF247), as outlined above.

Mutagenesis of mouse xenotropic receptor cDNAs. NIH Swiss and M. dunni X-receptor residues were mutated by PCR mutagenesis with two complementary mutagenic primers containing the targeted point mutation and the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, Calif.). Plasmid DNA from three independent clones was sequenced to confirm the mutations. The DNA sequence was determined as described above. The mutants were designated by the parental X-receptor name followed by the mutated amino acid followed by the residue number and the new amino acid.

Cross-interference assay. CHO cells were transfected with either FBXsalf (xenotropic envelope gene NZB expression vector [37]) or phMCF (MCF247 envelope gene cloned into the FBsalf expression vector, kindly provided by Jean-Michel Heard, Pasteur Institute, Paris, France) DNAs by the calcium phosphate-DNA precipitation method (9). Transfectants were selected with phleomycin (50 µg/ml), and resistant clones were screened for envelope expression by immunofluorescence assay using goat anti-Bv2 polyclonal antibody (for xenotropic envelope glycoprotein) and goat anti-gp70 antibody (for MCF247 envelope glycoprotein) (20). The highest envelope-expressing clones were used in the cross-interference assay.

Nucleotide sequence accession numbers. The X-receptor cDNA sequences have been assigned GenBank accession numbers as follows: NIH Swiss, AF131096; M. dunni, AF131097; SC-1, AF131098; CHO, AF131099; CCL64, AF131100; M. spretus, AF13101; M. castaneus, AF131102.

	RESULTS

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Sequences of X-receptors. Seven new X-receptor cDNAs were cloned by reverse transcriptase PCR, as described in Materials and Methods. A comparison of the predicted amino acid sequences of these X-receptors with the human X-receptor is shown in Fig. 1, which also indicates the eight hydrophobic potential membrane-spanning sequences and seven NX(S/T) sites for potential N-linked glycosylation. Based on the absence of a recognizable signal sequence in these proteins and on evidence for a cytosolic localization of the hydrophilic amino-terminal region (35, 37) we have inferred a topology for the X-receptor that includes the presumptive extracellular loop (ECL) regions indicated (Fig. 1). Interestingly, the mouse X-receptor sequences are polymorphic, and each contains multiple nonconservative substitutions, deletions, and/or novel glycosylation sites. All of the seven NX(S/T) sites in the human protein are conserved in the other X-receptors except for the unique NNS sequence at position 432. However, additional potential glycosylation sites occur in the M. castaneus protein at position 402, in the Chinese hamster protein at position 426, and in the NIH Swiss, M. dunni, M. spretus, and M. castaneus X-receptors at position 503. Although many of the distinguishing sequences are scattered throughout these X-receptors, almost all of the nonconservative changes likely to be functionally important for virus infections occur between positions 430 and 590, with particular clustering in the hydrophilic regions labeled ECL 3 and ECL 4. For example, the laboratory mouse (NIH Swiss), M. castaneus, and hamster X-receptors all have nonconservative substitutions and/or deletions in ECL 4. Moreover, the NIH Swiss X-receptor has additional nonconservative substitutions in ECL 3. 

View larger version (49K): [in this window] [in a new window]   FIG. 1.   Amino acid sequence comparison of human, mouse, mink, and hamster X-receptors. The mouse sequences were derived from an NIH Swiss laboratory strain and from the wild mouse strains M. dunni, SC-1, M. spretus, and M. castaneus. The hamster sequence was from CHO cells, and the mink sequence was from CCL64 lung fibroblasts. Positions of identity to the human sequence are indicated by dots, whereas deletions in the NIH Swiss and M. castaneus sequences are indicated by dashes. The eight hydrophobic potential membrane-spanning sequences are indicated by bars underlying the human sequence and are identified by numbers 1 to 8. The putative ECL regions are indicated. Numbers at the right of the sequences correspond to the position of the last amino acid shown. Potential N-linked glycosylation sites are indicated by asterisks.

Properties of X-receptor cDNAs expressed in resistant cells. We assayed the viral receptor functions of these X-receptors by expressing them in naturally resistant cells and assaying for susceptibilities to -galactosidase-encoding X-MLV (NZB) (29), MCF13 (5), and MCF247 (16) pseudotype viruses, as previously described (37). For this purpose, we used CHO cells, which are fully resistant to P-MLVs (but weakly susceptible to X-MLVs) and mouse BALB/3T3 fibroblasts, which are fully resistant to X-MLVs (Table 1). All of the X-receptor cDNAs, including the 2.1-kbp human coding region, were expressed with the pcDNA3.1.V5His-TOPO vector. These results clearly established that the X-receptors from humans, minks, the wild mice M. dunni and M. spretus, and SC-1 cells all conferred significant but variably efficient susceptibilities to the MCFs and X-MLV used in these assays. In contrast, the X-receptor from NIH Swiss mice mediated infections of MCFs but not of X-MLV, whereas the X-receptors from M. castaneus and Chinese hamsters were inactive as viral receptors in these assays. Interestingly, these patterns of infectivity mimic the susceptibilities of the animals from which the cDNAs were derived (see Discussion). Consequently, we infer that the resistances of these animals to infections by X-MLVs and P-MLVs are principally determined by the specific sequences of their X-receptors, with additional effects possibly caused by other mechanisms, such as interfering glycoproteins (19, 25), serum lipoprotein factors (15, 21, 23), or transcriptional silencing (13, 19a). Thus, xenotropism in this system (i.e., the resistance of inbred laboratory mice to X-MLVs) is caused, at least partly, by an inherent property of the X-receptor protein in these mice.

Identification of two amino acids within the presumptive ECL 3 and ECL 4 that specifically control X-MLV infections. To determine which region(s) of the M. dunni and NIH Swiss X-receptors is responsible for their different abilities to mediate X-MLV infections, we used PCR site-directed mutagenesis to substitute sequences between their presumptive extracellular regions. The NIH Swiss X-receptor differs from that of M. dunni at seven positions, including five nonconservative differences. Of these, one (V210D) is in the intracellular amino-terminal region, two (D469N and K500E) are in ECL 3, and two (T582 and D590N) are in ECL 4 (Fig. 1). Furthermore, we considered the ECL 4 region potentially important because the M. castaneus X-receptor is inactive in mediating infections and contains a deletion of five amino acids that overlaps the single T582 deletion in the NIH Swiss protein (Fig. 1).

Cross-interference assay. Previous interference analyses of X-MLV and P-MLV viruses used various cells that expressed only their endogenous receptors (2, 5, 27). For example, the susceptibility of laboratory mouse cells to P-MLVs was unaffected by expression of an X-MLV (NZB) envelope glycoprotein (2). This is not surprising, because the X-receptors of laboratory mice presumably cannot interact with the X-MLV glycoprotein. In contrast, varying degrees of nonreciprocal cross-interference between X-MLVs and P-MLVs have been observed in cells that are susceptible to both groups of virus, with X-MLVs generally interfering strongly with P-MLVs and P-MLVs only interfering weakly with X-MLVs (2, 27). In several assays it was also reported that P-MLV-related envelope glycoproteins only weakly interfered with P-MLVs (2, 19). We have also observed only weak interference with P-MLV in M. dunni, NIH 3T3, and mink CCL64 cells expressing P-MLV envelope glycoproteins (data not shown). Based on this information, it was proposed that X-MLVs and P-MLVs may share a common receptor and that X-MLVs may in addition have a unique receptor (27). However, an alternative interpretation of this evidence, as discussed elsewhere, is that X-MLVs and P-MLVs may compete unequally for a common receptor (37).

View larger version (45K): [in this window] [in a new window]   FIG. 2.   Studies of interference with wild-type and mutant human, M. dunni, and NIH Swiss mouse X-receptors. The assays were done with control CHO cells or CHO derivative clones that stably express either the X-MLV (NZB) envelope glycoprotein or the MCF247 envelope glycoprotein. These cell clones are called CHO.Xenv and CHO.MCFenv, respectively (see Materials and Methods). These cells were transiently transfected with expression vectors for the human, M. dunni, M. dunni T582, M. dunni K500E-T582, NIH Swiss, and NIH Swiss 582T X-receptors. Infections with LacZ pseudotype viruses were initiated 24 h after beginning the transfections. (A) Infections of LacZ(MCF247) virus; (B) infections of LacZ(X-MLV) virus.

	DISCUSSION

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Polymorphisms in X-receptors of mice control susceptibilities to X-MLV and P-MLV infections. In this investigation we have cloned and functionally characterized X-receptor cDNAs from five representative strains of mice that differ in their susceptibilities to X-MLV and P-MLV infections, from mink CCL64 cells that form foci after infection with P-MLVs (MCFs), and from Chinese hamster cells that are completely resistant to P-MLVs and only slightly susceptible to X-MLVs. Importantly, our results substantiate previous genetic evidence, which implied that mouse X-receptors must be polymorphic in their sequences and that these polymorphisms help to control infections by the P-MLV/X-MLV family of retroviruses (18, 19, 24, 25). In particular, we have found that these cloned X-receptors mediate the same patterns of viral susceptibility as the cells from which the cDNAs were derived (Table 1). In addition, the differential activities of these X-receptors in X-MLV infections were closely similar when the assays were done with BALB/3T3 and CHO cells (e.g., see Table 1 and Fig. 2). Thus, the X-receptor from NIH Swiss mice conferred susceptibility to P-MLVs but resistance to X-MLVs; the X-receptors from M. dunni, SC-1 cells, M. spretus, and minks conferred susceptibilities with different efficiencies to both of these host range groups of MLVs; and the corresponding proteins from M. castaneus and CHO cells were inactive as virus receptors in our transient expression and infection assays. The only minor discrepancy in these results was the inactivity of the CHO X-receptor in our assays, despite the weak susceptibility of CHO cells to X-MLVs (compare Table 1 and Fig. 2). This could reflect a relative insensitivity of our transient transfection-infection assays or perhaps an inactivity of this receptor in BALB/3T3 fibroblasts. There is some cell type specificity in assays for X-receptor activities, as illustrated by our observation that resistance of HeLa cells to P-MLVs is dominant (see Results).

Molecular basis for X-MLV xenotropism. By site-directed mutagenesis, we have compared the X-receptors of M. dunni and NIH Swiss mice, which differ in their abilities to mediate X-MLV infections but are both capable of mediating P-MLV infections. Thus, testing for P-MLV infections provided a positive control to demonstrate cell surface expression of the mutant X-receptors used in this investigation. In essence, these studies strongly implied that K500 in presumptive ECL 3 and T582 in ECL 4 make important but somewhat redundant contributions to infections mediated by the M. dunni X-receptor (Tables 2 and 3). Thus, in the context of the M. dunni X-receptor, either K500 or T582 was sufficient to allow X-MLV infections. However, none of the mutations had any effect on P-MLV infections. Consistent with this evidence, a single 582T insertion mutation in the NIH Swiss X-receptor was sufficient to confer activity in X-MLV infections (Table 2). These results imply that NIH Swiss mice contain two mutations, K500E and T582, that are necessary to prevent X-MLV infections and that either K500 or T582 is sufficient for X-MLV susceptibility (Tables 2 and 3 and Fig. 2).

Interference between X-MLVs and P-MLVs. It is striking that X-MLVs and P-MLVs interact so differently with the same X-receptor proteins, as indicated by our infectivity and interference assays. Most significantly, X-MLVs rely heavily on K500 and T582 in the M. dunni X-receptor, and the double mutant K500E-T582 is completely inactive (Tables 2 and 3 and Fig. 2). In contrast, these mutations have no effect on P-MLV infections. Moreover, the same amino acids are critical for interference by the X-MLV (NZB) envelope glycoprotein (Fig. 2), suggesting that they may be necessary for strong glycoprotein-receptor binding. Thus, the amino acids in the X-receptor that are most critical for X-MLVs are ignored by P-MLVs. However, X-receptors may lack alternative sites for strong virus attachments because P-MLVs are very weak in their interference properties (e.g., see Fig. 2).

Potential relevance to P-MLV pathogenesis. It is surprising that P-MLV formation in mice has been highly correlated with the onset of severe pathogenesis whereas X-MLVs have not been associated with disease even in susceptible strains of wild mice (8, 11, 21, 32). Moreover, the only common feature of P-MLVs that distinguishes them from ecotropic and xenotropic MLVs is the amino-terminal receptor-determining region of their envelope glycoproteins (7, 17, 30, 38). Thus, class I P-MLVs typically are recombinants that contain long terminal repeat sequences derived from endogenous X-MLVs, MCF-specific env gene regions derived from endogenously inherited sequences, and a remainder that is derived from an ecotropic MLV (7, 17, 30, 38). These results have implied that P-MLV pathogenesis may involve interaction of the viral glycoprotein with its cell surface receptor. Consequently, the recent demonstration that X-MLVs and P-MLVs use a common cell surface receptor despite their distinct pathogenic effects implies that a difference in their interactions with the X-receptor could have a pathogenic consequence. From this perspective, our observation that X-MLVs and P-MLVs interact in a distinct manner with X-receptors is intriguing. Moreover, several other exceptionally pathogenic retroviruses, in addition to P-MLVs, cause only weak interference with superinfection. For example, the feline leukemia virus FeLV-FAIDS-EECC causes rapid immunodeficiency and T-cell destruction compared to closely related nonpathogenic isolates (28). The most distinguishing characteristic of FeLV-FAIDS-EECC is its inability to cause interference with superinfection (6, 28). Similar results occur with some cytopathic isolates of avian leukosis virus (39), ovine visna lentivirus (10), and human immunodeficiency virus type 1 (33). In agreement with our proposal that there may be a failure of interference in P-MLV-induced diseases, the process of thymic lymphomagenesis in AKR mice has been associated with massive superinfections by MCFs within single cells (12, 36). Hallmarks of this superinfection include numerous MCF proviruses integrated into the DNA of single cells and large quantities of unintegrated MCF-specific proviral DNA (12, 36). Additional investigations will be required to evaluate this hypothesis concerning the mechanism of P-MLV-induced diseases.