Mutation of amino acids within the gibbon ape leukemia virus (GALV) receptor differentially affects feline leukemia virus subgroup B, simian sarcoma-associated virus, and GALV infections.

The three type C retroviruses, gibbon ape leukemia virus (GALV), simian sarcoma-associated virus (SSAV), and feline leukemia virus subgroup B (FeLV-B), infect human cells by interacting with the same cell surface receptor, GLVR1. Using LacZ retroviral pseudotypes and murine cells transfected with mutant GLVR1 expression vectors, we show that the same 9-amino-acid region of human GLVR1 is critical for infection by the three viruses. Rat cells were not susceptible to infection by LacZ (FeLV-B) pseudotypes because of a block at the receptor level. We found multiple amino acid differences from human GLVR1 in the 9-amino-acid critical region of rat GLVR1. Expression of a human-rat chimeric GLVR1 in murine cells demonstrated that rat GLVR1 could function as a receptor for GALV and SSAV but not for FeLV-B. Substitution of human GLVR1 amino acids in the critical region of rat GLVR1 identified three amino acids as responsible for resistance to FeLV-B infection; two of these affect SSAV infection, but none affects GALV infection.     

Feline leukemia virus subgroup B uses the same cell surface receptor as gibbon ape leukemia virus.

Pseudotypes of gibbon ape leukemia virus/simian sarcoma-associated virus (GALV/SSAV) and feline leukemia virus subgroup B (FeLV-B) have been constructed by rescuing a Moloney murine leukemia virus vector genome with wild-type GALV/SSAV or FeLV-B. The resulting recombinant viruses utilized core and envelope proteins from the wild-type virus and conferred resistance to growth in L-histidinol upon infected cells by virtue of the HisD gene encoded by the vector genome. They displayed the host range specificity of the rescuing viruses and could be neutralized by virus-specific antisera. Receptor cross-interference was observed when the GALV/SSAV or FeLV-B pseudotypes were used to superinfect cells productively infected with either GALV/SSAV or FeLV-B. Although murine cells are resistant to FeLV-B infection, murine cells expressing the human gene for the GALV/SSAV receptor became susceptible to FeLV-B infection. Therefore GALV/SSAV and FeLV-B utilize the same cell surface receptor.     

Identification of Envelope Determinants of Feline Leukemia Virus Subgroup B That Permit Infection and Gene Transfer to Cells Expressing Human Pit1 or Pit2

The retroviral vector systems that are in common use for gene therapy are designed to infect cells expressing either of two widely expressed phosphate transporter proteins, Pit1 or Pit2. Subgroup B feline leukemia viruses (FeLV-Bs) use the gibbon ape leukemia virus receptor, Pit1, as a receptor for entry. Our previous studies showed that some chimeric envelope proteins encoding portions of FeLV-B could also enter cells by using a related receptor protein, Pit2, which serves as the amphotropic murine leukemia virus receptor (S. Boomer, M. Eiden, C. C. Burns, and J. Overbaugh, J. Virol. 71:8116--8123, 1997). Here we show that an arginine at position 73 within variable region A (VRA) of the FeLV-B envelope surface unit (SU) is necessary for viral entry into cells via the human Pit2 receptor. However, C-terminal SU sequences have a dominant effect in determining human Pit2 entry, even though this portion of the protein is outside known receptor binding domains. This suggests that a combination of specific VRA sequences and C-terminal sequences may influence interactions between FeLV-B SU and the human Pit2 receptor. Binding studies suggest that the C-terminal sequences may affect a postbinding step in viral entry via the Pit2 receptor, although in all cases, binding of FeLV-B SU to human Pit2 was weak. In contrast, neither the arginine 73 nor specific C-terminal sequences are required for efficient binding or infection with Pit1. Taken together, these data suggest that different residues in SU may interact with these two receptors. The specific FeLV-Bs described here, which can enter cells using either human Pit receptor, may be useful as envelope pseudotypes for viruses used in gene therapy.     

Fusion-defective gibbon ape leukemia virus vectors can be rescued by homologous but not heterologous soluble envelope proteins

Murine leukemia virus (MLV)-derived envelope proteins containing alterations in or adjacent to the highly conserved PHQ motif present at the N terminus of the envelope surface subunit (SU) are incorporated into vector particles but are not infectious due to a postbinding block to viral entry. These mutants can be rendered infectious by the addition of soluble receptor-binding domain (RBD) proteins in the culture medium. The RBD proteins that rescue the infectivity of these defective MLV vectors can be derived from the same MLV or from other MLVs that use distinct receptors to mediate entry. We have now constructed functional immunologically reactive gibbon ape leukemia virus (GALV) envelope proteins, tagged with a feline leukemia virus (FeLV)-derived epitope tag, which are efficiently incorporated into infectious particles. Tagged GALV envelope proteins bind specifically to cells expressing the phosphate transporter protein Pit1, demonstrating for the first time that Pit1 is the binding receptor for GALV and not a coreceptor or another type of GALV entry factor. We have also determined that GALV particles bearing SU proteins with an insertion C-terminal to the PHQ motif (GALV I(10)) bind Pit1 but fail to infect cells. Incubation with soluble GALV RBD renders GALV I(10) particles infectious, whereas incubation with soluble RBDs from MLV or FeLV-B does not. This finding is consistent with the results obtained by Lauring et al. using FeLV-T, a virus that employs Pit1 as a receptor but requires soluble FeLV RBD for entry. MLV and GALV RBDs are not able to render FeLV-T infectious (A. S. Lauring, M. M. Anderson, and J. Overbaugh, J. Virol. 75:8888-8898, 2001). Together, these results suggest that fusion-defective FeLV-T and GALV are restricted to homologous RBD rescue of infectivity.     

Characterization of the Proline-Rich Region of Murine Leukemia Virus Envelope Protein

Mammalian type C retroviral envelope proteins contain a variable proline-rich region (PRR), located between the N-terminal receptor-binding domain and the more highly conserved C-terminal portion of the surface (SU) subunit. We have investigated the role of the PRR in the function of murine leukemia virus (MuLV) envelope protein. In the MuLVs, the PRR contains a highly conserved N-terminal sequence and a hypervariable C-terminal sequence. Despite this variability, the amphotropic PRR could functionally substitute for the ecotropic PRR. The hypervariable region of the PRR was not absolutely required for envelope protein function. However, truncations in this region resulted in decreased levels of both the SU and TM proteins in viral particles and increased amounts of the uncleaved precursor protein, Pr85. In contrast, the N-terminal conserved region was essential for viral infectivity. Deletion of this region prevented the stable incorporation of envelope proteins into viral particles in spite of normal envelope protein processing, wild-type levels of cell surface expression, and a wild-type ability to induce syncytia in an XC cell cocultivation assay. However, higher levels of the SU protein were shed into the supernatant, suggesting a defect in SU-TM interactions. Our data are most consistent with a role for the PRR in stabilizing the overall structure of the protein, thereby affecting the proper processing of Pr85, SU-TM interactions, and the stable incorporation of envelope proteins into viral particles. In addition, we have demonstrated that the PRR can tolerate the insertion of a peptide-binding domain, making this a potentially useful site for constructing targetable retroviral vectors.     

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.     

Gibbon ape leukemia virus receptor functions of type III phosphate transporters from CHOK1 cells are disrupted by two distinct mechanisms

The Chinese hamster cell lines E36 and CHOK1 dramatically differ in susceptibility to amphotropic murine leukemia virus (A-MuLV) and gibbon ape leukemia virus (GALV); E36 cells are highly susceptible to both viruses, CHOK1 cells are not. We have previously shown that GALV can infect E36 cells by using both its own receptor, HaPit1, and the A-MuLV receptor, HaPit2. Given that the two cell lines are from the same species, the loss of function of both of these receptors in CHOK1 cells is surprising. Other studies have shown that CHOK1 cells secrete proteins that block A-MuLV entry into CHOK1 as well as E36, suggesting the two A-MuLV receptors are functionally identical. However, CHOK1 conditioned medium does not block GALV entry into E36, indicating the secreted inhibitors do not block HaPit1. HaPit1 and ChoPit1 therefore differ as receptors for GALV; ChoPit1 is either inactivated by secreted factors or intrinsically nonfunctional. To determine why GALV cannot infect CHOK1, we cloned and sequenced ChoPit1 and ChoPit2. ChoPit2 is almost identical to HaPit2, which explains why CHOK1 conditioned medium blocks A-MuLV entry via both receptors. Although ChoPit1 and HaPit1 are 91% identical, a notable difference is at position 550 in the fourth extracellular region, shown by several studies to be crucial for GALV infection. Pit1 and HaPit1 have aspartate at 550, whereas ChoPit1 has threonine at this position. We assessed the significance of this difference for GALV infection by replacing the aspartate 550 in Pit1 with threonine. This single substitution rendered Pit1 nonfunctional for GALV and suggests that threonine at 550 inactivates ChoPit1 as a GALV receptor. Whether native ChoPit1 functions for GALV was determined by interference assays using Lec8, a glycosylation-deficient derivative of CHOK1 that is susceptible to both viruses and that has the same receptors as CHOK1. Unlike with E36, GALV and A-MuLV exhibited reciprocal interference when infecting Lec8, suggesting that they use the same receptor. We conclude both viruses can use ChoPit2 in the absence of the inhibitors secreted by CHOK1 and ChoPit1 is nonfunctional.     

Porcine endogenous retroviruses infect cells lacking cognate receptors by an alternative pathway: implications for retrovirus evolution and xenotransplantation

A PHQ motif near the amino termini of gammaretroviral envelope glycoprotein surface (SU) subunits is important for infectivity but not for incorporation into virions or binding to cognate receptors. The H residue of this motif is most critical, with all substitutions we tested being inactive. Interestingly, porcine endogenous retroviruses (PERVs) of all three host-range groups, A, B, and C, lack full PHQ motifs, but most members have an H residue at position 10. H10A PERV mutants are noninfectious but were efficiently transactivated by adding to the assays a PHQ-containing SU or receptor-binding subdomain (RBD) derived from a gibbon ape leukemia virus (GALV). A requirement of this transactivation was a functional GALV receptor on the cells. In contrast to this heterologous transactivation, PERV RBDs and SUs were inactive in all tested cells, including porcine ST-IOWA cells. Surprisingly, transactivation by GALV RBD enabled wild-type or H10A mutant PERVs of all three host-range groups to efficiently infect cells from humans and rodents that lack functional PERV receptors and it substantially enhanced infectivities of wild-type PERVs, even for cells with PERV receptors. Thus, PERVs can suboptimally infect cells that contain cognate receptors or they can employ a transactivation pathway to more efficiently infect all cells. This ability to infect cells lacking cognate receptors was previously demonstrated only for nontransmissible variant gammaretroviruses with recombinant and mutant envelope glycoproteins. We conclude that some endogenously inherited mammalian retroviruses also have a receptor-independent means for overcoming host-range and interference barriers, implying a need for caution in xenotransplantation, especially of porcine tissues.     

Detailed Mapping of Determinants within the Porcine Endogenous Retrovirus Envelope Surface Unit Identifies Critical Residues for Human Cell Infection within the Proline-Rich Region

Replication-competent porcine endogenous retroviruses (PERVs) are either human cell tropic (PERV-A and PERV-B) or non-human cell tropic (PERV-C). We previously demonstrated that PERV in vitro cell tropism is modulated by 2 residues within the C terminus of SU and that the PERV receptor binding domain (RBD) extends beyond the variable regions A and B (VRA and VRB, respectively), to include the proline rich-region (PRR) of SU (M. Gemeniano et al., Virology 346:108-117, 2000; T. Argaw et al., J. Virol. 82:7483-7489, 2008). The present study aimed to identify the specific elements within the PERV RBD that interact with the C-terminal elements of SU to facilitate human cell infection. We constructed a series of chimeric and mutated envelopes between PERV-A and PERV-C and using pseudotyped retroviral vectors to map the human cell tropism-determining sequences within the PERV RBD. We show that the PRR from PERV-A is both necessary and sufficient to allow human cell infection when substituted into the homologous region of the PERV-C envelope carrying two C-terminal amino acid substitutions shown to influence human cell tropism, Q374R and I412V (PERV-Crv). Furthermore, substitution of a single amino acid residue in the PRR of the non-human-tropic PERV-Crv envelope allows vectors carrying this envelope to infect human cells. Receptor interference assays showed that these modified PERV-C envelopes do not bind either of the human PERV-A receptors, suggesting the presence of a distinct human PERV-C receptor. Finally, vectors carrying these modified PERV-C envelopes infect primary human endothelial cells, a cell type likely to be exposed to PERV in clinical use of certain porcine xenotransplantation products.     

Primate gammaretroviruses require an ancillary factor not required for murine gammaretroviruses to infect BHK cells

BHK cells remain resistant to xenotropic murine retrovirus-related virus (XMRV) or gibbon ape leukemia virus (GALV) infection, even when their respective receptors, Xpr1 or PiT1, are expressed. We set out to determine the stage at which viral infection is blocked and whether this block is mediated by a dominant-negative factor or the absence of a requisite ancillary factor. BHK cells bind neither XMRV nor GALV envelope proteins. BHK cells expressing the appropriate receptors bind XMRV or GALV envelope proteins. BHK cells can be infected by NZB-XMV(New Zealand Black mouse xenotropic murine virus)-enveloped vectors, expressing an envelope derived from a xenotropic retrovirus that, like XMRV, employs Xpr1 as a receptor, and also by vectors bearing the envelope of 10A1 murine leukemia virus (MLV), a murine retrovirus that can use PiT1 as a receptor. The retroviral vectors used in these analyses differ solely in their viral envelope proteins, suggesting that the block to XMRV and GALV infection is mediated at the level of envelope-receptor interactions. N-linked glycosylation of the receptors was not found to mediate resistance of receptor-expressing BHK cells to GALV or XMRV, as shown by tunicamycin treatment and mutation of the specific glycosylation site of the PiT1 receptor. Hybrid cells produced by fusing BHKXpr1 or BHKPiT1 to XMRV- or GALV-resistant cells, respectively, can mediate efficient XMRV or GALV infection. These findings indicate that BHK cells lack a factor that is required for infection by primate xenotropic viruses. This factor is not required for viruses that use the same receptors but were directly isolated from mice.     

Reassessing the role of region A in Pit1-mediated viral entry

The mammalian gammaretroviruses gibbon ape leukemia virus (GALV) and feline leukemia virus subgroup B (FeLV-B) can use the same receptor, Pit1, to infect human cells. A highly polymorphic nine-residue sequence within Pit1, designated region A, has been proposed as the virus binding site, because mutations in this region abolish Pit1-mediated cellular infection by GALV and FeLV-B. However, a direct correlation between region A mutations deleterious for infection and loss of virus binding has not been established. We report that cells expressing a Pit1 protein harboring mutations in region A that abolish receptor function retain the ability to bind virus, indicating that Pit1 region A is not the virus binding site. Furthermore, we have now identified a second region in Pit1, comprising residues 232 to 260 (region B), that is required for both viral entry and virus binding. Epitope-tagged Pit1 proteins were used to demonstrate that mutations in region B result in improper orientation of Pit1 in the cell membrane. Compensatory mutations in region A can restore proper orientation and full receptor function to these region B mutants. Based on these results, we propose that region A of Pit1 confers competence for viral entry by influencing the topology of the authentic binding site in the membrane and hence its accessibility to a viral envelope protein. Based on glycosylation studies and results obtained by using N- and C-terminal epitope-tagged Pit1, region A and region B mutants, and the transmembrane helices predicted with the PHD PredictProtein algorithm, we propose a new Pit1 topology model.     

Identification of residues outside of the receptor binding domain that influence the infectivity and tropism of porcine endogenous retrovirus

Identification of determinants of human tropism of porcine endogenous retrovirus (PERV) is critical to understanding the risk of transmission of PERV to recipients of porcine xenotransplantation products. Previously, we showed that a chimeric envelope cDNA encoding the 360 N-terminal residues of the human-tropic PERV envelope class A (PERV-A) SU and the 130 C-terminal residues of the pig-tropic PERV-C SU and all of TM (PERV-A/C) showed a 100-fold decrease in infectivity titer on human cells (M. Gemeniano, O. Mpanju, D. R. Salomon, M. V. Eiden, and C. A. Wilson, Virology 346:108-117, 2006). To identify residues important for human cell infection, we performed site-directed mutagenesis on each of the nine residues, singly or in combination, that distinguish the C-terminal region of PERV-C from PERV-A. Of the nine amino acids, two single-amino-acid substitutions, Q374R and I412V, restored the infectivity of human cells to the chimeric PERV-A/C to a titer equivalent to that of PERV-A. In contrast, PERV-A/C mutant envelope Q439P resulted in undetectable infection of human cells and an approximately 1,000-fold decrease in control pig cells. Mutation of K441R rescued mutants that carried Q439P, suggesting an incompatibility between the proline residue at this position and the presence of KK in the proteolytic cleavage signal. We confirmed this incompatibility with vectors carrying PERV-A envelope mutant R462K that were also rendered noninfectious. Finally, tropism of vectors carrying PERV-C envelope mutants with only four amino acid changes in the C terminus of PERV-C envelope, NHRQ436YNRP plus K441R, was shifted to one similar to that of PERV-A. Our results show an important and previously unrecognized role for infectivity and tropism for residues at the C terminus of SU.     

Substitution of a single amino acid residue is sufficient to allow the human amphotropic murine leukemia virus receptor to also function as a gibbon ape leukemia virus receptor.

We have previously reported the unique properties of a receptor for amphotropic murine leukemia viruses (A-MuLVs) expressed on Chinese hamster E36 cells (C.A. Wilson, K.B. Farrell, and M.V. Eiden, J. Virol. 68:7697-7703, 1994). This receptor, HaPiT2 (formerly designated EAR), in contrast to the human form of the A-MuLV receptor (PiT2), functions as a receptor not only for A-MuLVs but also for gibbon ape leukemia virus (GALV). Comparison of the deduced amino acid sequences of the HaPiT2 and PiT2 proteins suggested that differences in the amino acid composition of the extracellular region(s) of the hamster and human proteins account for their functional differences. We substituted extracellular regions of HaPiT2 for those of PiT2 to map the region of the HaPiT2 protein required for GALV receptor function. Only those PiT2-HaPiT2 chimeric receptors containing the fourth and fifth extracellular regions of HaPiT2 functioned as GALV receptors. We have now determined that the substitution of a single amino acid residue, glutamic acid, for the lysine residue at position 522 in the fourth extracellular region of the PiT2 protein is sufficient to render PiT2 functional as a GALV receptor.     

Gene therapy for malignant glioma using Sindbis vectors expressing a fusogenic membrane glycoprotein

BACKGROUND: Malignant glioma has a dismal prognosis. It was previously shown that glioma cells are efficiently killed when they express a gene coding for a hyperfusogenic mutant of the gibbon ape leukemia virus envelope glycoprotein (GALV.fus). However, production of viral vectors expressing GALV.fus has proven problematic because the transgene is toxic to vector-producing cells of human origin. We reasoned that Sindbis-virus-based vectors might be ideal for GALV.fus gene transfer because high-titer stocks can easily be generated in hamster cells and Sindbis virus efficiently infects human tumor cells through the high-affinity 67 kDa laminin receptor. In addition, Sindbis virus nonstructural proteins are potent inducers of apoptosis, and Sindbis vector RNAs expressing fusogenic viral proteins have been shown to spread from cell-to-cell in membrane-formed infectious particles. METHODS: Sindbis virus replicon-containing particles were generated by co-transfecting vector and helper RNAs into baby hamster kidney (BHK-21) cells. Packaged beta-galactosidase and GALV.fus expressing Sindbis vectors were used to infect glioma cell lines, which were then compared for syncytial cytopathic effect, cell killing, and release of infectious virus-like particles containing the vector genome. Finally, the efficacy of GALV.fus and beta-galactosidase Sindbis vectors was compared in an orthotopic intracerebral U87 glioma xenograft model in nude mice. RESULTS: High-titer stocks (>10(9) infectious units (iu)/ml) of the GALV.fus and beta-galactosidase vectors were obtained. Glioma cells infected with the GALV.fus vector formed large syncytia which died rapidly by apoptosis and released infectious membrane-formed particles that could transfer vector genomes to uninfected cells. The GALV.fus vector had significantly greater antitumor therapeutic potency than the beta-galactosidase vector in the U87 glioma xenograft model. CONCLUSIONS: Sindbis vectors expressing GALV.fus can be packaged into infectious viral particles to high titer, they exhibit potent bystander cytopathic potential and are active against U87 glioma xenografts. Sindbis-virus-based replicons appear to be efficient vector systems for delivery and expression of fusogenic membrane glycoproteins.     

Gene Therapy of Lung Adenocarcinoma using Herpes Virus Expressing a Fusogenic Membrane Glycoprotein

The aim of this study is to observe the in vitro-targeted destruction of lung adenocarcinoma using recombinant Type I herpes simplex virus (HSV-I)-mediated gibbon ape leukemia virus envelope glycoprotein (GALV.fus), controlled by UL38 promoter and cytomegalovirus promoter (CMVP). A recombinant HSV-I plasmid encoding the GALV.fus was transfected into green monkey kidney cells, the lung adenocarcinoma line A549, and the human fetal fibroblast cell line HFL-I GNHu5 in various doses. The effects and expression of in vitro GALV.fus were observed using an inverted microscope. Enhanced green fluorescence protein expression served as the contro1 for GALV.fus. Recombinant HSV-I virus was produced. Fusogenic recombinant virus infection led to cell fusions in A549 in a dose-dependent manner. Nonfusogenic viruses only produced conventional cytotoxic effects. Recombinant HSV-I with the CMVP initiated cell fusions in HFL-1 GNHu5 cells with arrested cell cycles or as quiescence. HSV-I regulated by UL38p caused cell fusion only in growing cells. Protein expression of GALV.fus was confirmed by Western Blot in infected A549 and HFL-1 GNHu5. Delivery and tumor-specific expression of GALV.fus gene can selectively and safely target lung cancer in vitro, and may prove to be a novel gene therapy for lung cancer.     

Characterization of chimeras between the ecotropic Moloney murine leukemia virus and the amphotropic 4070A envelope proteins.

A series of 22 chimeric envelope (env) genes were generated between the ecotropic Moloney murine leukemia virus and the amphotropic 4070A isolate. The chimeric envelopes were expressed within the complete, replication-competent provirus and tested for virus viability by transient expression assays. Eleven of the 22 viruses were viable. Five of these chimeric viruses showed an ecotropic host range, and six exhibited an amphotropic host range and viral interference. The host range determinants map to the first half of the surface (SU) protein. The N-terminal 72 amino acids of 4070A (42 of processed SU) are not required for amphotropic receptor usage. Ecotropic and amphotropic viruses differ in their ability to form large, multinucleated syncytia when cocultured with the rat XC cell line. Ecotropic murine leukemia virus forms large syncytia with XC cells, whereas no syncytia are reported for amphotropic virus. All chimeras which contained the N-terminal half of the ecotropic SU protein, encoding the receptor binding domain, formed the large multinucleated syncytia with XC cells.     

Chimeras of receptors for gibbon ape leukemia virus/feline leukemia virus B and amphotropic murine leukemia virus reveal different modes of receptor recognition by retrovirus.

Glvr1 encodes the human receptor for gibbon ape leukemia virus (GALV) and feline leukemia virus subgroup B (FeLV-B), while the related gene Glvr2 encodes the human receptor for amphotropic murine leukemia viruses (A-MLVs). The two proteins are 62% identical in their amino acid sequences and are predicted to have 10 transmembrane domains and five extracellular loops. A stretch of nine amino acids (region A) in the predicted fourth extracellular loop was previously shown to be critical for the function of Glvr1 as receptor for GALV and FeLV-B. Glvr1 and -2 show clusters of amino acid differences in several of their predicted extracellular loops, with the highest degree of divergence in region A. Chimeras were made between the two genes to further investigate the role of Glvr1 region A in defining receptor specificity for GALV and FeLV-B and to map which regions of Glvr2 control receptor specificity for A-MLVs. Region A from Glvr1 was sufficient to confer receptor specificity for GALV upon Glvr2, with the same chimera failing to act as a receptor for FeLV-B. However, introduction of additional N- or C-terminal Glvr1-encoding sequences in addition to Glvr1 region A-encoding sequences resulted in functional FeLV-B receptors. Therefore, FeLV-B is dependent on Glvr1 sequences outside region A for infectivity. The receptor specificity of Glvr2 for A-MLV could not be mapped to a single critical region; rather, N-terminal as well as C-terminal Glvr2-encoding sequences could confer specificity for A-MLV infection upon Glvr1. Surprisingly, though GALV/FeLV-B and A-MLV belong to different interference groups, some chimeras functioned as receptors for all three viruses.     

In vitro characterization of a koala retrovirus

Recently, a new endogenous koala gammaretrovirus, designated KoRV, was isolated from koalas. The KoRV genome shares 78% nucleotide identity with another gammaretrovirus, gibbon ape leukemia virus (GALV). KoRV is endogenous in koalas, while GALV is exogenous, suggesting that KoRV predates GALV and that gibbons and koalas acquired the virus at different times from a common source. We have determined that subtle adaptive differences between the KoRV and GALV envelope genes account for differences in their receptor utilization properties. KoRV represents a unique example of a gammaretrovirus whose envelope has evolved to allow for its expanded host range and zoonotic potential.     

Dissection of gammaretroviral receptor function by using type III phosphate transporters as models

Gammaretroviruses that enter cells via binding to a surface receptor use one of two fundamental mechanisms. In the first, binding of the virus particle to its cognate receptor is followed by fusion and internalization. The second, less common mechanism requires the addition of an accessory protein in order to achieve fusion and entry into the target cells; this protein is usually the soluble form of the envelope protein containing the receptor-binding domain (RBD). For some viruses, such as amphotropic murine leukemia virus (A-MLV), particles with fusion-defective envelope proteins can enter cells in the presence of their own RBD or that of another viral envelope, regardless of its cognate receptor, suggesting that these viruses share a common entry mechanism. A notable exception is gibbon ape leukemia virus (GALV). Fusion-impaired GALV envelope mutants can be trans-activated for infectivity only by GALV RBDs. Using dually functional GALV/A-MLV receptors, we examined the role of receptor with respect to which RBD could overcome fusion impaired virus entry.     

Retroviral display of urokinase-binding domain fused to amphotropic envelope protein

Tumors frequently express urokinase (uPA) receptor (uPAR). To investigate whether uPAR can efficiently target cancerous cells using amphotropic retroviral vectors, we generated a retrovirus displaying the amino-terminal fragment (ATF) of uPA as an N-terminal extension of viral envelope protein. We also made use of a "two-step strategy" by inserting a uPA cleavage site between the ATF moiety and the envelope. We measured the ability of ATF-bearing chimeric envelopes to infect huPAR-overexpressing Madin-Darby canine kidney (MDCK) and control MDCK II cells. The ATF-viruses infected both MDCK cell lines with an equivalent efficiency, suggesting that the chimeric viruses were not sequestered by uPAR and infect cells preferentially via the Pit-2 receptor. The addition of a uPA cleavage site increased the infection level of huPAR-MDCK cells by 2-fold when uPA was present in the infection medium. Surprisingly, ATF-env viruses infected huPAR-MDCK cells 5.5-fold more efficiently in the presence of exogenous uPA. This stimulatory effect of uPA on infection of huPAR-MDCK cells by the ATF-env virus was completely abolished by methyl-beta-cyclodextrin, suggesting that this effect involves the caveolar endocytosis pathway.