Analysis of the unique hamster cell tropism of ecotropic murine leukemia virus PVC-211.

PVC-211 murine leukemia virus (MuLV) is a neuropathogenic variant of Friend MuLV (F-MuLV). Previous studies from our laboratory demonstrated that unlike the parental F-MuLV, PVC-211 MuLV can infect rat brain capillary endothelial cells efficiently and that it has acquired genetic changes responsible for its expanded cellular tropism. To determine if PVC-211 MuLV also has expanded its host range, we tested its infectivity on Chinese hamster ovary-derived CHO-K1 cells, which are generally resistant to ecotropic MuLV. The results indicated that PVC-211 MuLV, but not F-MuLV, was highly infectious for CHO-K1 cells. Studies using glycosylation inhibitors and glycosylation mutants of CHO-K1 cells, as well as interference studies, suggested that PVC-211 MuLV has acquired the ability to interact with the ecotropic MuLV receptor on CHO-K1 cells that has undergone glycosylation-dependent modification. Using chimeric viruses between PVC-211 MuLV and F-MuLV, we were able to localize the viral genetic element crucial for CHO-K1 cell tropism within the env gene of PVC-211 MuLV and show that glycine at position 116 and lysine at position 129 of the envelope glycoprotein SU were important. These viral determinants also appear to confer tropism for other hamster cells resistant to ordinary ecotropic MuLVs. Further studies on the interaction between PVC-211 MuLV and the receptor on hamster cells may provide novel insights into the molecular mechanisms for receptor recognition and binding by viral envelope glycoproteins.     

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.     

Definition of an amino-terminal domain of the human T-cell leukemia virus type 1 envelope surface unit that extends the fusogenic range of an ecotropic murine leukemia virus

Murine leukemia viruses (MuLV) and human T-cell leukemia viruses (HTLV) are phylogenetically highly divergent retroviruses with distinct envelope fusion properties. The MuLV envelope glycoprotein surface unit (SU) comprises a receptor-binding domain followed by a proline-rich region which modulates envelope conformational changes and fusogenicity. In contrast, the receptor-binding domain and SU organization of HTLV are undefined. Here, we describe an HTLV/MuLV envelope chimera in which the receptor-binding domain and proline-rich region of the ecotropic MuLV were replaced with the potentially corresponding domains of the HTLV-1 SU. This chimeric HTLV/MuLV envelope was processed, specifically interfered with HTLV-1 envelope-mediated fusion, and similar to MuLV envelopes, required cleavage of its cytoplasmic tail to exert significant fusogenic properties. Furthermore, the HTLV domain defined here broadened ecotropic MuLV envelope-induced fusion to human and simian cell lines.     

Mechanism of restriction of ecotropic and xenotropic murine leukemia viruses and formation of pseudotypes between the two viruses.

Ecotropic and xenotropic murine leukemia viruses (MuLV's) constitute separate interference groups; within each group there is cross-interference, but between the groups there is no detectable interference. Interference is manifest against pseudotypes in which the vesicular stomatitis virus genome is contained within the coat of one of the murine leukemia viruses. The pseudotypes display the cell specificity of the leukemia viruses: pseudotypes with an ecotropic MuLV coat infect mouse cells but not rabbit or mink cells; pseudotypes with a xenotropic MuLV coat infect rabbit or mink cells well but mouse cells very poorly. Efficient pseudotype formation also occurs between the two MuLV classes, and both the interference patterns and the cell specificity of these pseudotypes are entirely determined by their envelope. Using these pseudotypes, ecotropic MuLV infection could be established in xenogeneic cells, and the resulting progeny could be scored by using a conventional XC cell assay. Also, xenotropic MuLV infection could be established in a mouse cell, showing that no absolute intracellular barrier against xenotropic virus growth exists in murine cells. The major barriers against both xenotropic and ecotropic MuLV therefore are cell surface barriers. Xenogeneic cells probably lack receptors for ecotropic MuLV, but murine cells may either lack receptors for xenotropic MuLV or have receptors that are blocked by endogenous expression of the glycoprotein of endogenous xenotropic MuLV.     

Cell fusion induced by the murine leukemia virus envelope glycoprotein.

To determine whether ecotropic murine leukemia virus (MuLV) envelope glycoproteins are sufficient to cause cell-to-cell fusion when expressed in the absence of virus production, we used an ecotropic MuLV, AKV, to construct env expression vectors that lack the gag and pol genes. The rat cell line XC, which undergoes cell-to-cell fusion upon infection with ecotropic MuLV, was transfected with wild-type env expression vectors, and high levels of syncytium formation resulted. Transfection of the murine cell line NIH 3T3 with expression vectors containing the wild-type or mutated env region did not result in syncytium formation. Immunoprecipitation analysis of the envelope glycoproteins expressed in NIH 3T3 and XC cells showed that the mature surface glycoprotein expressed in XC cells was of a much lower apparent molecular weight than that expressed in NIH 3T3 cells. Further characterization showed that most if not all of this difference was the result of differences in glycosylation. Finally, site-directed mutagenesis was used to introduce several conservative and nonconservative changes into the amino-terminal region of the transmembrane protein. Analysis of the effect of these mutations confirmed that this region is a fusion domain.     

Glycosylation and intracellular transport of membrane glycoproteins encoded by murine leukemia viruses. Inhibition by amino acid analogues and by tunicamycin

Addition of asparagine-linked oligosaccharides to nascent murine leukemia virus (MuLV)-encoded membrane glycoproteins was inhibited either completely by tunicamycin or specifically at Asn-X-Thr glycosylation sites by incorporation of the threonine analogue beta-hydroxynorvaline. In conditions of partial analogue substitution, a series of subglycosylated components is formed which are related by a constant apparent Mr difference when assayed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The total number of asparagine-linked oligosaccharides is then estimated by dividing the measured apparent Mr of one oligosaccharide into the total apparent Mr difference between the complete glycoprotein and the polypeptide chain that is synthesized in cells incubated with tunicamycin. Correct results were obtained using glycoproteins with known numbers of oligosaccharides. Our analyses indicate that the gp70 membrane envelope glycoproteins of certain ecotropic MuLVs contain seven oligosaccharides, whereas the GIX+ antigen-containing variant gp70 contains one fewer Asn-X-Thr-linked oligosaccharide. The membrane glycoprotein encoded by the gag gene of Friend MuLV contains only one asparagine-linked oligosaccharide. Similarly, the gp55 membrane glycoprotein encoded by Friend erythroleukemia virus contains four asparagine-linked oligosaccharides. Pulse-chase and cell surface iodination analyses indicate that MuLV membrane envelope glycoprotein processing by partial proteolysis and transport to the cell surface can be efficiently blocked by structural perturbations caused by incorporation of different amino acid analogues or by loss of oligosaccharides. Our data also suggest that loss of oligosaccharides may expose new antigenic sites in viral membrane glycoproteins and increase their susceptibility to intracellular proteolysis.     

Receptor-binding domain of murine leukemia virus envelope glycoproteins.

The surface glycoprotein (SU) of murine leukemia viruses (MuLVs) comprises two domains connected by a proline-rich hinge. The interaction of MuLV particles with subgroup-specific cell surface receptors depends primarily on two variable regions (VRA and VRB) located in the amino-terminal domain. To delineate the minimal receptor-binding domains, we examined the capacity of soluble envelope fragments to compete with the entry of virus particles. Amphotropic, ecotropic, polytropic, and xenotropic truncated SUs were produced by inserting stop codons in the env gene of the 4070A, Friend, MCF247 and NZB MuLVs, respectively. These fragments, as well as full-length envelope glycoproteins, were stably expressed in cells bearing the corresponding receptor. Synthesis, posttranslational modifications, transport, and secretion of the env gene products were monitored by immunoprecipitation. Cells expressing the modified SUs or naive cells preincubated with SU-containing conditioned media were infected with different pseudotypes of a retroviral vector carrying a beta-galactosidase marker gene. Reduction of cell susceptibility to infection in the presence of SU was used as a measure of receptor occupancy. The results indicated that the amphotropic and ecotropic envelope amino-terminal domains contain all of the determinants required for receptor binding. In contrast, additional sequences in the proline-rich region were needed for efficient interaction of the polytropic and xenotropic amino-terminal domains with the receptors.     

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.     

Basis for receptor specificity of nonecotropic murine leukemia virus surface glycoprotein gp70SU.

Murine leukemia viruses (MuLVs) initiate infection of NIH 3T3 cells by binding of the viral envelope (Env) protein to a cell surface receptor. Interference assays have shown that MuLVs can be divided into four groups, each using a distinct receptor: ecotropic, polytropic, amphotropic, and 10A1. In this study, we have attempted to map the determinants within viral Env proteins by constructing chimeric env genes. Chimeras were made in all six pairwise combinations between Moloney MCF (a polytropic MuLV), amphotropic MuLV, and 10A1, using a conserved EcoRI site in the middle of the Env coding region. The receptor specificity of each chimera was determined by using an interference assay. We found that amphotropic receptor specificity of each chimera was determined by using an interference assay. We found that amphotropic receptor specificity seems to map to the N-terminal portion of surface glycoprotein gp70SU. The difference between amphotropic and 10A1 receptor specificity can be attributed to one or more of only six amino acid differences in this region. Nearly all other cases showed evidence of interaction between Env domains in the generation of receptor specificity. Thus, a chimera composed exclusively of MCF and amphotropic sequences was found to exhibit 10A1 receptor specificity. None of the chimeras were able to infect cells by using the MCF receptor; however, two chimeras containing the C-terminal portion of MCF gp70SU could bind to this receptor, while they were able to infect cells via the amphotropic receptor. This result raises the possibility that receptor binding maps to the C-terminal portion of MCF gp70SU but requires MCF N-terminal sequences for a functional interaction with the MCF receptor.     

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.     

Sequences determining the pH dependence of viral entry are distinct from the host range-determining region of the murine ecotropic and amphotropic retrovirus envelope proteins.

The entry of ecotropic and amphotropic murine leukemia retroviruses (MuLV) into cells was investigated by using viral vector particles carrying chimeric amphotropic-ecotropic envelope glycoproteins on their surface. Chimeras were made by joining, at or near the polyproline hinge, the N-terminal portion of the amphotropic (4070A) gp70 onto the C-terminal portion of the ecotropic (Moloney) gp70 and p15E (constructs AE2 and AE4) or vice versa (AE12). Transduction efficiency of the constructs was tested on target cells that either have only ecotropic receptors (CHO-2 and CHO-11 cells), only amphotropic receptors (mink lung fibroblasts and Cos 1 cells), or both types of receptors (NIH 3T3 cells). The assay made use of the fact that the mechanism for viral entry of ecotropic viruses is pH dependent while that of amphotropic viruses is pH independent. Treatment of target cells with NH4Cl, which prevents the reduction of pH within endosomes, reduced the titers of viral particles bearing the C-terminal moiety from the ecotropic envelope but did not reduce the titers of particles which had a C-terminal moiety from the amphotropic envelope. In addition, in contrast to other low-pH-dependent enveloped viruses, brief acid treatment did not allow surface-bound viruses to bypass the NH4Cl block. The results indicate that the pH dependence of viral entry is a property of the sequences C terminal to the polyproline hinge.     

Molecular characterization of the Akvr-1 restriction gene: a defective endogenous retrovirus-borne gene identical to Fv-4r.

A dominant restriction allele, Akvr-1r, from California wild mice (Mus musculus domesticus) confers resistance to exogenous ecotropic murine leukemia virus (MuLV) infection. The presence of an ecotropic MuLV envelope-related glycoprotein in uninfected virus-resistant cells suggests that viral interference is a possible mechanism for this resistance. We molecularly cloned the ecotropic MuLV envelope-related sequence from the genomic DNA of a wild mouse homozygous for the Akvr-1r locus. The cloned provirus was defective and contained a C-terminal end of the pol gene, a complete envelope gene, and a 3' long terminal repeat. The presence of this provirus was directly correlated with Akvr-1r-mediated virus resistance in cell cultures and hybrid mice. The Akvr-1r provirus restriction map and partial DNA sequence were identical to those of the Fv-4r allele, an ecotropic MuLV resistance locus from Japanese feral mice (M. musculus molossinus), which was previously shown to be allelic with the Akvr-1r gene. The 3' host flanking sequences of Fv-4r and Akvr-1r also had identical restriction maps. These findings indicate that Akvr-1r and Fv-4r are the same gene. It was probably acquired by interbreeding of these feral species in recent times. Conservation of this locus might be favored by the useful function that it performs in protection against ecotropic MuLV infection endemic in both populations of wild mice.     

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.     

Genetic control of retroviral disease in aging wild mice

Different populations of wild mice (Mus musculus domesticus) in Los Angeles and Ventura Counties were observed over their lifespan in captivity for expression of infectious murine leukemia virus (MuLV) and murine mammary tumor virus (MMTV) and for the occurrence of cancer and other diseases. In most populations of feral mice these indigenous retroviruses were infrequently expressed and cancer seldom occurred until later in life (>2 years old). MMTV was found in the milk of about 50% of wild mice, but was associated with only a low incidence (>1%) of breast cancer after one year of age. By contrast, in several populations, most notably at a squab farm near Lake Casitas (LC), infectious MuLV acquired at birth via milk was highly prevalent, and the infected mice were prone to leukemia and a lower motor neuron paralytic disease after one year of age. These two diseases were both caused by the same infectious (ecotropic)strain of MuLV and were the principal cause of premature death in these aging LC mice. A dominant gene called FV-4^R restricting the infection with ecotropic MuLV was found segregating in LC mice. Mice inheriting this FV-4^R allele were resistant to the ecotropic MuLV associated lymphoma and paralysis. The FV-4^R allele represents a defective endogenous MuLV provirus DNA segment that expresses an ecotropic MuLV envelope-related glycoprotein (gp70) on the cell surface. This FV-4^R encoded gp70 presumably occupies the receptor for ecotropic MuLV and blocks entry of the virus. The FV-4^R gene was probably acquired by the naturally occurring crossbreeding of LC feral mice with another species of feral mice (Mus castaneus) from Southeast Asia. The FV-4^R gp70 does not block entry of the amphotropic MuLV that uses a separate cell surface receptor. Therefore LC mice continued to be susceptible to the highly prevalent but weakly lymphogenic and nonparalytogenic amphotropic strain of MuLV. The study points out the potential of feral populations to reveal genes associated with specific disease resistance.     

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.     

A Unique Heparin-Binding Domain in the Envelope Protein of the Neuropathogenic PVC-211 Murine Leukemia Virus May Contribute to Its Brain Capillary Endothelial Cell Tropism

Previous studies from our laboratory demonstrated that PVC-211 murine leukemia virus (MuLV), a neuropathogenic variant of Friend MuLV (F-MuLV), had undergone genetic changes which allowed it to efficiently infect rat brain capillary endothelial cells (BCEC) in vivo and in vitro. Two amino acid changes from F-MuLV in the putative receptor binding domain (RBD) of the envelope surface protein of PVC-211 MuLV (Glu-116 to Gly and Glu-129 to Lys) were shown to be sufficient for conferring BCEC tropism on PVC-211 MuLV. Recent examination of the unique RBD of PVC-211 MuLV revealed that the substitution of Lys for Glu at position 129 created a new heparin-binding domain that overlapped a heparin-binding domain common to ecotropic MuLVs. In this study we used heparin-Sepharose columns to demonstrate that PVC-211 MuLV, but not F-MuLV, can bind efficiently to heparin and that one or both of the amino acids in the RBD of PVC-211 MuLV that are associated with BCEC tropism are responsible. We further showed that heparin can enhance or inhibit MuLV infection and that the mode of action is dependent on heparin concentration, sulfation of heparin, and the affinity of the virus for heparin. Our results suggest that the amino acid changes that occurred in the envelope surface protein of PVC-211 MuLV may allow the virus to bind strongly to the surface of BCEC via heparin-like molecules, increasing the probability that the virus will bind to its cell surface receptor and efficiently infect these cells.     

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.     

Role of the mutation Q252R in activating membrane fusion in the murine leukemia virus surface envelope protein

Entry of retroviruses into host cells requires the fusion between the viral and cellular membranes. It is unclear how receptor binding induces conformational changes within the surface envelope protein (SU) that activate the fusion machinery residing in the transmembrane envelope protein (TM). In this report, we have isolated a point mutation, Q252R, within the proline-rich region of the 4070A murine leukemia virus SU that altered the virus-cell binding characteristics and induced cell-cell fusion. Q252R displays a SU shedding-sensitive phenotype. Cell-cell fusion is receptor dependent and is observed only in the presence of MuLV Gag-Pol. Both cellular binding and fusion by Q252R are greatly enhanced in conjunction of G100R, a mutation within the SU variable region A which increases viral binding through an independent mechanism. Deletion of a conserved histidine (His36) at the SU N terminus abolished cell-cell fusion by G100R/Q252R Env without compromising virus-cell binding. Although G100R/Q252R virus has no detectable titer, replacement of the N-terminal nine 4070A SU amino acids with the equivalent ecotropic MuLV sequence restored viral infectivity. These studies provide insights into the functional cooperation between multiple elements of SU required to signal receptor binding and activate the fusion machinery.