Analysis of the murine ecotropic leukemia virus receptor reveals a common biochemical determinant on diverse cell surface receptors that is essential to retrovirus entry.

Two residues, tyrosine 235 and glutamic acid 237, of the ecotropic murine leukemia virus receptor (ATRC1) have been shown to be essential for receptor-mediated virus envelope binding and entry. We performed genetic analyses to examine the biochemical contribution of these residues in a productive virus-receptor interaction. Altered ATRC1 receptors bearing either a phenylalanine, a tryptophan, a histidine, or a methionine at position 235 mediated ecotropic virus entry comparable to that mediated by ATRC1. In contrast, altered ATRC1 receptors bearing alanine, threonine, serine, or proline at position 235 exhibited a 300- to 10,000-fold decrease in receptor capability. Furthermore, substitution of tyrosine or phenylalanine into the corresponding position (242) of the homologous human protein that lacks ecotropic virus receptor capability resulted in acquisition of ecotropic virus receptor function comparable to that of ATRC1. Substitution of a tryptophan or a histidine at that position of the human protein, however, resulted in a much-reduced receptor capability, suggesting a preference for a benzene ring in the hydrophobic side chain. A similar analysis of proteins substituted at position 237 revealed that aspartic acid, but not arginine or lysine, can functionally substitute for glutamic acid 237 in ATRC1 or at the corresponding position in the human protein. These results suggest a requirement for an acidic and a nearby hydrophobic amino acid for efficient ecotropic virus entry. Similar motifs have been identified in the virus binding sites of other retrovirus receptors, suggesting that the initial step of retrovirus entry may be governed by a common mechanism.     

Targeted entry via somatostatin receptors using a novel modified retrovirus glycoprotein that delivers genes at levels comparable to those of wild-type viral glycoproteins

Here we report a novel viral glycoprotein created by replacing a natural receptor-binding sequence of the ecotropic Moloney murine leukemia virus envelope glycoprotein with the peptide ligand somatostatin. This new chimeric glycoprotein, which has been named the Sst receptor binding site (Sst-RBS), gives targeted transduction based on three criteria: (i) a gain of the use of a new entry receptor not used by any known virus; (ii) targeted entry at levels comparable to gene delivery by wild-type ecotropic Moloney murine leukemia virus and vesicular stomatitis virus (VSV) G glycoproteins; and (iii) a loss of the use of the natural ecotropic virus receptor. Retroviral vectors coated with Sst-RBS gained the ability to bind and transduce human 293 cells expressing somatostatin receptors. Their infection was specific to target somatostatin receptors, since a synthetic somatostatin peptide inhibited infection in a dose-dependent manner and the ability to transduce mouse cells bearing the natural ecotropic receptor was effectively lost. Importantly, vectors coated with the Sst-RBS glycoprotein gave targeted entry of up to 1 × 10(6) transducing U/ml, a level comparable to that seen with infection of vectors coated with the parental wild-type ecotropic Moloney murine leukemia virus glycoprotein through the ecotropic receptor and approaching that of infection of VSV G-coated vectors through the VSV receptor. To our knowledge, this is the first example of a glycoprotein that gives targeted entry of retroviral vectors at levels comparable to the natural capacity of viral envelope glycoproteins.     

Identification of a critical basic residue on the ecotropic murine leukemia virus receptor

Susceptibility to ecotropic murine leukemia viruses (MLV) is restricted to mice and rats at the level of virus binding to the host cell receptor. Asparagine 232, valine 233, tyrosine 235, and glutamic acid 237 in the third extracellular domain (EL3) of the receptor are critical determinants of the host range difference between mice and humans. However, placing these residues in the human homolog confers only partial binding, indicating that other divergent sequences are involved. We sought to determine if the other sequences lie within or outside EL3. Here we report the identification of lysine 234 as another critical residue that influences virus binding and infection, as well as evidence that the unidentified sequences lie outside EL3. Each of the four basic residues in the third extracellular domain were changed to an acidic residue and initially examined in combination with a change at position 235 or position 237. Substitution of lysine 211, 215, or 222 combined with substitution of the critical tyrosine 235 or glutamic acid 237 did not affect virus infection. However, combined substitution of lysine 234, a conserved residue between mice and humans, and tyrosine 235 resulted in a marked decrease in virus infection and binding. A lysine 234 change alone reduced virus binding, contrary to previous observations that at least two of the other four residues must be changed before binding is reduced. Interestingly, there was no decrease in infection when lysine 234 was replaced in combination with glutamic acid 237. This result suggests that residue 234 may act by influencing the local structure of residues 233 to 235, whereas the presence of a glycine at position 236 may prevent this influence from extending to residue 237. With this report, the involvement of all the residues divergent between mice and humans in the third extracellular domain has been ruled out, suggesting that as yet unidentified determinants lie in other extracellular domains.     

Complementation of a binding-defective retrovirus by a host cell receptor mutant

The entry of ecotropic murine leukemia virus (MLV) into cells requires the interaction of the envelope protein (Env) with its receptor, mouse cationic amino acid transporter 1 (mATRC1). An aspartic acid-to-lysine change at position 84 (D84K) of ecotropic Moloney MLV Env abolishes virus binding and infection. We recently identified lysine 234 (rK234) in mATRC1 as a residue that influences virus binding and infection. Here we show that D84K virus infection increased 3,000-fold on cells expressing receptor with an rK234A change and 100,000-fold on cells expressing an rK234D change. The stronger complementation of D84K virus infection by rK234D than by the rK234A receptor suggests that although the major reason for loss of infection of D84K and D84R virus is due to steric hindrance and charge repulsion, the loss of an interaction of D84 with receptor appears to contribute as well. Taken together, these results indicate that D84 is very close to rK234 of mATRC1 in the bound complex and there is likely an interaction between them. The definitive localization of the receptor binding site on SU should facilitate the design of chimeric envelope proteins that target infection to new receptors by replacing the receptor binding site with an exogenous ligand sequence.     

An amino-terminal fragment of the Friend murine leukemia virus envelope glycoprotein binds the ecotropic receptor.

Retrovirus entry into cells is mediated by specific binding of the envelope glycoprotein to a cell membrane receptor. Constitutive envelope gene expression prevents infection by interfering with the binding of viruses which recognize the same receptor. We have used this property to investigate the receptor binding capacities of deleted or truncated murine leukemia virus ecotropic envelope glycoproteins. Friend murine leukemia virus envelope glycoproteins bearing internal amino-terminal deletions, or a soluble 245-amino-acid gp70 amino-terminal fragment, were expressed in NIH 3T3 cells. The susceptibility of these cells to ecotropic and amphotropic virus infection was determined. We observed that both membrane-bound and soluble forms of the gp70 245-amino-acid amino-terminal domain induced resistance to ecotropic virus, indicating that this fragment binds the ecotropic receptor. Binding occurs both at the cell surface and in the endoplasmic reticulum, as shown by the use of soluble envelope fragments either secreted in the culture supernatants or retained in the endoplasmic reticulum lumen by a KDEL sequence. These results suggest that the gp70 amino-terminal domain folds into a structure which recognizes the ecotropic receptor regardless of the carboxy-terminal part of the molecule.     

Functional dissection of the Moloney murine leukemia virus envelope protein gp70.

The envelope protein of Moloney murine leukemia virus (Mo-MLV) is a complex glycoprotein that mediates receptor binding and entry via fusion with cell membranes. By using a series of substitution mutations and truncations in the Mo-MLV external envelope surface protein gp70, we have identified regions important for these processes. Firstly, truncations of gp70 revealed that the minimal continuous receptor-binding region is amino acids 9 to 230, in broad agreement with other studies. Secondly, within this region there are two key basic amino acids, Arg-83 and Arg-95, that are essential for receptor binding and may interact with a negatively charged residue(s) or with the pi electrons of the aromatic ring on a hydrophobic residue(s) in the basic amino acid transporter protein that is the Mo-MLV ecotropic receptor. Finally, we showed that outside the minimal receptor-binding region at amino acids 2 to 8, there is a region that is essential for postbinding fusion events.     

Modulation of ecotropic murine retroviruses by N-linked glycosylation of the cell surface receptor/amino acid transporter.

The cell surface receptor for ecotropic host-range (infection limited to mice or rats) murine leukemia viruses (MuLVs) is the widely expressed system y+ transporter for cationic amino acids (CAT-1). Like other retroviruses, ecotropic MuLV infection eliminates virus-binding sites from cell surfaces and results in complete interference to superinfection. Surprisingly, infection causes only partial (ca 40 to 60%) loss of mouse CAT-1 transporter activity. The NIH/Swiss mouse CAT-1 (mCAT-1) contains 622 amino acids with 14 hydrophobic potential membrane-spanning sequences, and it is known that the third extracellular loop from the amino terminus is required for virus binding. Although loop 3 is hypervariable in different species and mouse strains, consistent with its proposed role in virus-host coevolution, loop 3 sequences of both susceptible and resistant species contain consensus sites for N-linked glycosylation. Both of the consensus sites in loop 3 of mCAT-1 are known to be glycosylated and to contain oligosaccharides with diverse sizes (J. W. Kim and J. M. Cunningham, J. Biol. Chem. 268:16316-16320, 1993). We confirmed by several lines of evidence that N-linked glycosylation occludes a potentially functional virus-binding site in the CAT-1 protein of hamsters, thus contributing to resistance of that species. To study the role of receptor glycosylation in animals susceptible to infection, we eliminated loop 3 glycosylation sites by mutagenesis of an mCAT-1 cDNA clone, and we expressed wild-type and mutant receptors in mink fibroblasts and Xenopus oocytes. These receptors had indistinguishable transport properties, as determined by kinetic and voltage-jump electrophysiological studies of arginine uptake in oocytes and by analyses Of L-[3H]arginine uptake in mink cells. Bindings of ecotropic envelope glycoprotein gp7O to the accessible receptor sites on surfaces of mink cells expressing wild-type or mutant mCAT-1 were not significantly different in kinetics or in equilibrium affinities (i.e., K(D) approximately 3.7 X 10(-10) to 7.5 X 10(-10) M). However, when values were normalized to the same levels of mCAT-1 transporter expression, cells with wild-type glycosylated mCAT-1 had only approximately 50% as many sites for gp70 binding as cells with unglycosylated mCAT-1. Although infection with ecotropic MuLV had no effect on activity of the mink CAT-1 transporter that does not bind virus, it caused partial down-modulation of wild-type mCAT-1 and complete down-modulation of unglycosylated mutant mCAT-1. These results suggest that N-linked glycosylation causes wild-type mCAT-1 heterogeneity and that a significant proportion is inaccessible to virus. In part because only the interactive fraction of mCAT-1 can be down-modulated, infected murine cells conserve an amino acid transport capability that supports their viability.     

Fv-4: identification of the defect in Env and the mechanism of resistance to ecotropic murine leukemia virus

Mice expressing the Fv-4 gene are resistant to infection by ecotropic murine leukemia viruses (MuLVs). The Fv-4 gene encodes an envelope (Env) protein whose putative receptor-binding domain resembles that of ecotropic MuLV Env protein. Resistance to ecotropic MuLVs appears to result from viral interference involving binding of the endogenously expressed Fv-4 env-encoded protein to the ecotropic receptor, although the immune system also plays a role in resistance. The Fv-4 env-encoded protein is processed normally and can be incorporated into virus particles but is unable to promote viral entry. Among the many sequence variations between the transmembrane (TM) subunit of the Fv-4 env-encoded protein and the TM subunits of other MuLV Env proteins, there is a substitution of an arginine residue in the Fv-4 env-encoded protein for a glycine residue (gly-491 in Moloney MuLV Env) that is otherwise conserved in all of the other MuLVs. This residue is present in the MuLV TM fusion peptide sequence. In this study, gly-491 of Moloney MuLV Env has been replaced with other residues and a mutant Env bearing a substitution for gly-487 was also created. G491R recapitulates the Fv-4 Env phenotype in cell culture, indicating that this substitution is sufficient for creation of an Env protein that can establish the interference-mediated resistance to ecotropic viruses produced by the Fv-4 gene. Analysis of the mutant MuLV Env proteins also has implications for an understanding of the role of conserved glycine residues in fusion peptides and for the engineering of organismal resistance to retroviruses.     

Mutational Analysis of the Fusion Peptide of Moloney Murine Leukemia Virus Transmembrane Protein p15E

Fusion peptides are hydrophobic sequences located at the N terminus of the transmembrane (TM) envelope proteins of the orthomyxoviruses and paramyxoviruses and several retroviruses. The Moloney murine leukemia virus TM envelope protein, p15E, contains a hydrophobic stretch of amino acids at its N terminus followed by a region rich in glycine and threonine residues. A series of single amino acid substitutions were introduced into this region, and the resulting proteins were examined for their abilities to be properly processed and transported to the cell surface and to induce syncytia in cells expressing the ecotropic receptor. One substitution in the hydrophobic core and several substitutions in the glycine/threonine-rich region that prevented both cell-cell fusion and the transduction of NIH 3T3 cells when incorporated into retroviral vector particles were identified. In addition, one mutation that enhanced the fusogenicity of the resulting envelope protein was identified. The fusion-defective mutants trans dominantly interfered with the ability of the wild-type envelope protein to cause syncytium formation in a cell-cell fusion assay, although no trans-dominant inhibition of transduction was observed. Certain substitutions in the hydrophobic core that prevented envelope protein processing were also found. These data indicate that the N-terminal region of p15E is important both for viral fusion and for the correct processing and cell surface expression of the viral envelope protein.     

Identification of the receptor binding domain of the mouse mammary tumor virus envelope protein

Mouse mammary tumor virus (MMTV) is a betaretrovirus that infects rodent cells and uses mouse transferrin receptor 1 for cell entry. To characterize the interaction of MMTV with its receptor, we aligned the MMTV envelope surface (SU) protein with that of Friend murine leukemia virus (F-MLV) and identified a putative receptor-binding domain (RBD) that included a receptor binding sequence (RBS) of five amino acids and a heparin-binding domain (HBD). Mutation of the HBD reduced virus infectivity, and soluble heparan sulfate blocked infection of cells by wild-type pseudovirus. Interestingly, some but not all MMTV-like elements found in primary and cultured human breast cancer cell lines, termed h-MTVs, had sequence alterations in the putative RBS. Single substitution of one of the amino acids found in an h-MTV RBS variant in the RBD of MMTV, Phe(40) to Ser, did not alter species tropism but abolished both virus binding to cells and infectivity. Neutralizing anti-SU monoclonal antibodies also recognized a glutathione S-transferase fusion protein that contained the five-amino-acid RBS region from MMTV. The critical Phe(40) residue is located on a surface of the MMTV RBD model that is distant from and may be structurally more rigid than the region of F-MLV RBD that contains its critical binding site residues. This suggests that, in contrast to other murine retroviruses, binding to its receptor may result in few or no changes in MMTV envelope protein conformation.     

Identification of key residues in subgroup A avian leukosis virus envelope determining receptor binding affinity and infectivity of cells expressing chicken or quail Tva receptor

To better understand retroviral entry, we have characterized the interactions between subgroup A avian leukosis virus [ALV(A)] envelope glycoproteins and Tva, the receptor for ALV(A), that result in receptor interference. We have recently shown that soluble forms of the chicken and quail Tva receptor (sTva), expressed from genes delivered by retroviral vectors, block ALV(A) infection of cultured chicken cells ( approximately 200-fold antiviral effect) and chickens (>98% of the birds were not infected). We hypothesized that inhibition of viral replication by sTva would select virus variants with mutations in the surface glycoprotein (SU) that altered the binding affinity of the subgroup A SU for the sTva protein and/or altered the normal receptor usage of the virus. Virus propagation in the presence of quail sTva-mIgG, the quail Tva extracellular region fused to the constant region of the mouse immunoglobulin G (IgG) protein, identified viruses with three mutations in the subgroup A hr1 region of SU, E149K, Y142N, and Y142N/E149K. These mutations reduced the binding affinity of the subgroup A envelope glycoproteins for quail sTva-mIgG (32-, 324-, and 4,739-fold, respectively) but did not alter their binding affinity for chicken sTva-mIgG. The ALV(A) mutants efficiently infected cells expressing the chicken Tva receptor but were 2-fold (E149K), 10-fold (Y142N), and 600-fold (Y142N/E149K) less efficient at infecting cells expressing the quail Tva receptor. These mutations identify key determinants of the interaction between the ALV(A) glycoproteins and the Tva receptor. We also conclude from these results that, at least for the wild-type and variant ALV(A)s tested, the receptor binding affinity was directly related to infection efficiency.     

The amphotropic and ecotropic murine leukemia virus envelope TM subunits are equivalent mediators of direct membrane fusion: implications for the role of the ecotropic envelope and receptor in syncytium formation and viral entry.

The murine leukemia virus (MuLV) envelope protein was examined to determine which sequences are responsible for the differences in direct membrane fusion observed with the ecotropic and amphotropic MuLV subtypes. These determinants were studied by utilizing amphotropic-ecotropic chimeric envelope proteins that have switched their host range but retain their original fusion domain (TM subunit). Fusion was tested both in rodent cells and in 293 cells bearing the human homolog of the ecotropic MuLV receptor. The results demonstrate that the amphotropic TM is able to mediate cell-to-cell fusion to an extent equivalent to that mediated by the ecotropic TM, indicating that their fusion domains are equivalent. The "murinized" human homolog of the ecotropic receptor supports syncytium formation as well as the native murine receptor. These findings suggest that interactions between the ecotropic envelope protein and conserved sequences in the ecotropic receptor are the principal determinants of syncytium formation. The relationship of the fusion phenotype to pH-dependent infection and the route of viral entry was examined by studying virions bearing the chimeric envelope proteins. Such virions appear to enter cells via a pathway that is directed by the host range-determining region of their envelope rather than by sequences that confer pH dependence. Therefore, the pH dependence of infection may not reflect the initial steps in viral entry. Thus, it appears that both the syncytium phenotype and the route of viral entry are properties of the viral receptor, the amino-terminal half of the ecotropic envelope protein, or the interaction between the two.     

Detection of receptor-specific murine leukemia virus binding to cells by immunofluorescence analysis.

Four classes of murine leukemia virus (MuLV) which display distinct cellular tropisms and bind to different retrovirus receptors to initiate virus infection have been described. In the present study, we describe a rapid, sensitive immunofluorescence assay useful for characterizing the initial binding of MuLV to cells. By using the rat monoclonal antibody 83A25 (L. H. Evans, R. P. Morrison, F. G. Malik, J. Portis, and W. J. Britt, J. Virol. 64:6176-6183, 1990), which recognizes an epitope of the envelope gp70 molecule common to the different classes of MuLV, it is possible to analyse the binding of ecotropic, amphotropic, or xenotropic MuLV by using only a single combination of primary and secondary antibodies. The MuLV binding detected by this assay is envelope receptor specific and matches the susceptibility to infection determined for cells from a variety of species. The binding of amphotropic MuLV to NIH 3T3 cells was shown to be rapid, saturable, and temperature dependent. Chinese hamster ovary (CHO-K1) cells normally lack the ability to bind ecotropic virus and are not infectible by ecotropic vectors. Expression of the cloned ecotropic retrovirus receptor gene (Rec) in CHO-K1 cells confers high levels of ecotropic virus-specific binding and confers susceptibility to infection. Characterization of MuLV binding to primary cells may provide insight into the infectibility of cells by retroviruses and aid in the selection of appropriate vectors for gene transfer experiments.     

Identification of a receptor-binding pocket on the envelope protein of friend murine leukemia virus

Based on previous structural and functional studies, a potential receptor-binding site composed of residues that form a pocket at one end of the two long antiparallel helices in the receptor-binding domain of Friend 57 murine leukemia virus envelope protein (RBD) has been proposed. To test this hypothesis, directed substitutions for residues in the pocket were introduced and consequences for infection and for receptor binding were measured. Receptor binding was measured initially by a sensitive assay based on coexpression of receptor and RBD in Xenopus oocytes, and the findings were confirmed by using purified proteins. Three residues that are critical for both binding and infection (S84, D86, and W102), with side chains that extend into the pocket, were identified. Moreover, when mCAT-1 was overexpressed, the infectivity of Fr57-MLV carrying pocket substitutions was partially restored. Substitutions for 18 adjacent residues and 11 other previously unexamined surface-exposed residues outside of the RBD pocket had no detectable effect on function. Taken together, these findings support a model in which the RBD pocket interacts directly with mCAT-1 (likely residues, Y235 and E237) and multiple receptor-envelope complexes are required to form the fusion pore.     

A point mutation in the binding subunit of a retroviral envelope protein arrests virus entry at hemifusion

The transmembrane subunits of viral envelope proteins are thought to perform all of the functions required for membrane fusion during entry of enveloped viruses. However, changes in a conserved SPHQ motif near the N terminus of the receptor binding subunit of a murine leukemia virus (MLV) envelope protein block infection and induction of cell-cell fusion but not receptor binding. Here we report evidence that a histidine-to-arginine change at position 8 (H8R) in the SPHQ motif of Moloney MLV blocks infection by arresting virus-cell fusion at the hemifusion state. In cell-cell fusion assays, H8R envelope protein induced mixing of membrane outer leaflet lipids but did not lead to content mixing, a finding indicative of fusion pore formation. Kinetic studies of virus-cell fusion showed that lipid mixing of H8R virus membranes begins much later than for wild-type virus. The length of the delay in lipid mixing decreased upon addition of two second-site changes that increase H8R virus infection to 100-fold less than the wild-type virus. Finally, chlorpromazine, dibucaine, and trifluoperazine, agents that induce pores in an arrested hemifusion state, rescued infection by H8R virus to within 2.5-fold of the level of wild-type virus infection and cell-cell fusion to half that mediated by wild-type envelope protein. We interpret these results to indicate that fusion progressed to the hemifusion intermediate but fusion pore formation was inhibited. These results establish that membrane fusion of Moloney MLV occurs via a hemifusion intermediate. We also interpret these findings as evidence that histidine 8 is a key switch-point residue between the receptor-induced conformation changes that expose fusion peptide and those that lead to six-helix bundle formation.     

Glycosylation-dependent inactivation of the ecotropic murine leukemia virus receptor.

The ecotropic murine leukemia virus (E-MuLV) receptor expressed on Mus dunni tail fibroblast (MDTF) cells is a receptor for all E-MuLVs with the notable of Moloney murine leukemia virus (Mo-MuLV). Substitution of isoleucine for valine at position 214 in the third extracellular region (the putative E-MuLV binding site) of the MDTF receptor molecule allows this molecule to function as a Mo-MuLV receptor (M.V. Eiden, K. Farrell, J. Warsowe, L. A. Mahan, and C. A. Wilson, J. Virol. 67:4056-4061, 1993). We have now determined that treating MDTF cells with tunicamycin, an inhibitor of N-linked glycosylation, also renders them susceptible to Mo-MuLV infection. Two potential N-linked glycosylation sites are present in the third extracellular regions of both the NIH 3T3 and MDTF ecotropic receptors. The glycosylation site at position 229 of the MDTF receptor cDNA was eliminated by substituting a threonine codon for the asparagine codon. Mo-MuLV-resistant human HOS cells, expressing this form of the receptor, are susceptible to Mo-MuLV infection. Thus, our studies suggest that without a glycan moiety at position 229, the valine residue at 214 is no longer restrictive for Mo-MuLV infection. BHK-21 and CHO K1 hamster cells also express glycosylation-inactivated forms of the ecotropic receptor. Sequence analysis of these receptors together with our analysis of MDTF receptor function suggests that a single asparagine-linked glycosylation site is responsible for glycosylation inactivation of these receptors.     

Identification of amino acid residues critical for infection with ecotropic murine leukemia retrovirus.

The murine cationic amino acid transporter is also the receptor for murine ecotropic leukemia retrovirus (MuLV-E). Recently, we have cloned a human gene (H13) homologous to the murine ecotropic retroviral receptor (ERR). Although the human homolog is very similar to murine ERR in sequence (87.6% amino acid identity) and structure (14 transmembrane-spanning domains), the human protein fails to function as a receptor for MuLV-E. To identify amino acid residues critical for MuLV-E infection, we took advantage of this species difference and substituted human H13 and murine ERR amino acid residues. Mouse-human chimeric receptor molecules were generated by taking advantage of using common restriction sites. These studies demonstrated that extracellular domains 3 and/or 4 contain the critical amino acid residues. Oligonucleotide-directed mutagenesis was then used to create 13 individual ERR mutants containing one or two amino acids substitutions or insertions within these two extracellular domains. Substitution of as few as one amino acid residue (Tyr) at position 235 in ERR with the corresponding H13 amino acid residue Pro abrogates the ability to function as a receptor for MuLV-E infection. Conversely, substitution of just two amino acid residues at positions 240 and 242 or 242 and 244 in H13 with the corresponding amino acid residues in ERR endows H13 with the ability to function as the receptor. This observation can be utilized to significantly improve the safety of retrovirus-mediated gene therapy in humans.     

trans-dominant interference with virus infection at two different stages by a mutant envelope protein of Friend murine leukemia virus.

A dominant negative mutant Friend murine leukemia virus (FMLV) env gene was cloned from an immunoselected Friend erythroleukemia cell. The mutant env had a point mutation which resulted in a Cys-to-Arg substitution at the 361st amino acid in the FMLV envelope protein (Env). The mutant Env was retained in the endoplasmic reticulum (ER) and accumulated because of its slow degradation. The NIH 3T3 cells expressing the mutant env were resistant to ecotropic Moloney MLV (MoMLV) penetration, suggesting that the mutant Env traps the ecotropic MLV receptors in the ER. When the mutant env gene was transfected into and expressed in the cells persistently infected with MoMLV, the wild-type Env was trapped in the ER, and the MoMLV production was suppressed. Thus, the mutant Env accumulating in the ER trans-dominantly and efficiently interfered with the ecotropic MLV infection at both the early and the late stages.     

The YXXL Sequences of a Transmembrane Protein of Bovine Leukemia Virus Are Required for Viral Entry and Incorporation of Viral Envelope Protein into Virions

The cytoplasmic domain of an envelope transmembrane glycoprotein (gp30) of bovine leukemia virus (BLV) has two overlapping copies of the (YXXL)₂ motif. The N-terminal motif has been implicated in in vitro signal transduction pathways from the external to the intracellular compartment and is also involved in infection and maintenance of high viral loads in sheep that have been experimentally infected with BLV. To determine the role of YXXL sequences in the replication of BLV in vitro, we changed the tyrosine or leucine residues of the N-terminal motif in an infectious molecular clone of BLV, pBLV-IF, to alanine to produce mutated proviruses designated Y487A, L490A, Y498A, L501A, and Y487/498A. Transient transfection of African green monkey kidney COS-1 cells with proviral DNAs that encoded wild-type and mutant sequences revealed that all of the mutated proviral DNAs synthesized mature envelope proteins and released virus particles into the growth medium. However, serial passages of fetal lamb kidney (FLK) cells, which are sensitive to infection with BLV, after transient transfection revealed that mutation of a second tyrosine residue in the N-terminal motif completely prevented the propagation of the virus. Similarly, Y498A and Y487/498A mutant BLV that was produced by the stably transfected COS-1 cells exhibited significantly reduced levels of cell-free virion-mediated transmission. Analysis of the protein compositions of mutant viruses demonstrated that lower levels of envelope protein were incorporated by two of the mutant virions than by wild-type and other mutant virions. Furthermore, a mutation of a second tyrosine residue decreased the specific binding of BLV particles to FLK cells and the capacity for viral penetration. Our data indicate that the YXXL sequences play critical roles in both viral entry and the incorporation of viral envelope protein into the virion during the life cycle of BLV.     

A putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection

Murine type C ecotropic retrovirus infection is initiated by virus envelope binding to a membrane receptor expressed on mouse cells. We have identified a cDNA clone that may encode for this receptor through a strategy combining gene transfer of mouse NIH 3T3 DNA into nonpermissive human EJ cells, selection of EJ clones that have acquired susceptibility to infection by retrovirus vectors containing drug resistance genes, and identification of the putative receptor cDNA clone through linkage to a mouse repetitive DNA sequence. Human EJ cells that express the cDNA acquire a million-fold increase in MuLV infectivity. The predicted 622 amino acid sequence of the putative receptor protein is extremely hydrophobic; 14 potential membrane-spanning domains have been identified. A computer-based search of sequence data banks did not identify a protein with significant similarity to the putative receptor. We conclude that a novel membrane protein determines susceptibility to ecotropic MuLV infection by binding and/or fusion with the virus envelope.