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.     

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.     

Single Amino Acid Insertion in Loop 4 Confers Amphotropic Murine Leukemia Virus Receptor Function upon Murine Pit1

Pit1 is the human receptor for gibbon ape leukemia virus (GALV) and feline leukemia virus subgroup B (FeLV-B), while the related human protein Pit2 is a receptor for amphotropic murine leukemia virus (A-MuLV). The A-MuLV-related isolate 10A1 can utilize both Pit1 and Pit2 as receptors. A stretch of amino acids named region A was identified in Pit1 (residues 550 to 558 in loop 4) as critical for GALV and FeLV-B receptor function. We have here investigated the role of region A in A-MuLV and 10A1 entry. Insertion of a single amino acid in region A of mouse Pit1 resulted in a functional A-MuLV receptor, showing that region A plays a role in A-MuLV infection. Moreover, the downregulation of 10A1 receptor function by changes in region A of human Pit1 indicates that this region is also involved in 10A1 entry. Therefore, region A seems to play a role in infection by all viruses utilizing Pit1 and/or Pit2 as receptors.     

A 13-amino-acid Pit1-specific loop 4 sequence confers feline leukemia virus subgroup B receptor function upon Pit2

Feline leukemia virus subgroup B (FeLV-B) and gibbon ape leukemia virus (GALV) utilize the human protein Pit1 but not the related protein, Pit2, as receptor. A stretch of 9 amino acids, named region A, was identified in the putative fourth extracellular loop of Pit1 (residues 550 through 558) as critical for FeLV-B and GALV receptor function. However, the presence of Pit1 region A did not confer receptor function for FeLV-B upon Pit2, while it did so for GALV. We have here shown that the presence of two Pit1-specific loop 4 residues (tyrosine 546 and valine 548) in addition to Pit1 region A is sufficient to make Pit2 an efficient FeLV-B receptor; that is, a stretch of 13 amino acids encompassing all loop 4 amino acid differences between Pit1 and Pit2 comprises a C-terminal determinant for FeLV-B receptor function. Thus, the same limited receptor region is sufficient to confer receptor function for both viruses upon Pit2.     

The Japanese feral mouse Pit1 and Pit2 homologs lack an acidic residue at position 550 but still function as gibbon ape leukemia virus receptors: implications for virus binding motif.

Murine cells are typically resistant to gibbon ape leukemia virus (GALV). MMMol, a Japanese feral mouse cell line, is an exception in that these cells are susceptible to infection by GALV. We show here that MMMol cells are further distinguished by their unusual receptor properties. MMMol cells infected by GALV are resistant to subsequent infection not only by GALV but also by amphotropic murine leukemia virus. This suggests that GALV can enter MMMol via not only the GALV receptor (MolPit1) but also the amphotropic murine leukemia virus receptor (MolPit2). Therefore, MolPit2 was cloned, sequenced, and compared with the previously reported sequence of MolPit1. Earlier studies have shown that a stretch of nine residues (position 550 to 558) in the fourth extracellular domain of Pit1 is crucial for GALV entry and that an acidic residue at position 550 is indispensable. However, MolPit1 has isoleucine at this position and MolPit2 has glutamine at the corresponding position (position 522), thus breaking this consensus. To determine what effect these specific changes in the fourth extracellular domain of MolPit1 and MolPit2 have on GALV receptor function, chimeric receptors were made by substituting the fourth extracellular domain of either MolPit1 or MolPit2 for the same region of Pit2, a nonfunctional receptor for GALV. These chimeras were then tested in MDTF, a cell line that lacks functional GALV receptors and is resistant to GALV. Results show that MDTF expressing these chimeras became susceptible to GALV, whereas cells expressing wild-type Pit2 remained resistant. Further, the MolPit1 chimera was identical to Pit1 in efficiency, but the MolPit2 chimera proved substantially less efficient.     

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.     

Simian Sarcoma-Associated Virus Fails To Infect Chinese Hamster Cells despite the Presence of Functional Gibbon Ape Leukemia Virus Receptors

We have sequenced the envelope genes from each of the five members of the gibbon ape leukemia virus (GALV) family of type C retroviruses. Four of the GALVs, including GALV strain SEATO (GALV-S), were originally isolated from gibbon apes, whereas the fifth member of this family, simian sarcoma-associated virus (SSAV), was isolated from a woolly monkey and shares 78% amino acid identity with GALV-S. To determine whether these viruses have identical host ranges, we evaluated the susceptibility of several cell lines to either GALV-S or SSAV infection. GALV-S and SSAV have the same host range with the exception of Chinese hamster lung E36 cells, which are susceptible to GALV-S but not SSAV. We used retroviral vectors that differ only in their envelope composition (e.g., they contain either SSAV or GALV-S envelope protein) to show that the envelope of SSAV restricts entry into E36 cells. Although unable to infect E36 cells, SSAV infects GALV-resistant murine cells expressing the E36-derived viral receptor, HaPit2. These results suggest that the receptors present on E36 cells function for SSAV. We have constructed several vectors containing GALV-S/SSAV chimeric envelope proteins to map the region of the SSAV envelope that blocks infection of E36 cells. Vectors bearing chimeric envelopes comprised of the N-terminal region of the GALV-S SU protein and the C-terminal region of SSAV infect E36 cells, whereas vectors containing the N-terminal portion of the SSAV SU protein and C-terminal portion of GALV-S fail to infect E36 cells. This finding indicates that the region of the SSAV envelope protein responsible for restricting SSAV infection of E36 cells lies within its amino-terminal region.     

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.     

Reevaluation of host ranges of feline leukemia virus subgroups

We reevaluated the host ranges of feline leukemia virus (FeLV) subgroups A, B and C using pseudotype assays based on recombinant NB-tropic murine leukemia virus, which is not usually blocked after viral entry in mammalian cells. Pseudotype viruses of FeLV-B and -C infected a variety of cell lines from many mammalian species. Unexpectedly, FeLV-A pseudotype viruses of two independent isolates from the UK and US also infected a variety of non-feline cell lines including cells from humans, rabbits, pigs and minks. Moreover, both isolates of FeLV-A productively infected human embryonic kidney 293 and mink Mv-1-Lu cells. We conclude that FeLV-A is not strictly ecotropic.     

Variable regions A and B in the envelope glycoproteins of feline leukemia virus subgroup B and amphotropic murine leukemia virus interact with discrete receptor domains.

The surface (SU) envelope glycoproteins of feline leukemia virus subgroup B (FeLV-B) and amphotropic murine leukemia virus (A-MLV) are highly related, even in the variable regions VRA and VRB that have been shown to be required for receptor recognition. However, FeLV-B and A-MLV use different sodium-dependent phosphate symporters, Pit1 and Pit2, respectively, as receptors for infection. Pit1 and Pit2 are predicted to have 10 membrane-spanning domains and five extracellular loops. The close relationship of the retroviral envelopes enabled us to generate pseudotype virions carrying chimeric FeLV-B/A-MLV envelope glycoproteins. We found that some of the pseudotype viruses could not use Pit1 or Pit2 proteins but could efficiently utilize specific chimeric Pit1/Pit2 proteins as receptors. By studying Mus dunni tail fibroblasts expressing chimeric Pit1/Pit2 proteins and pseudotype virions carrying chimeric FeLV-B/A-MLV envelopes, we show that FeLV-B and A-MLV VRA and VRB interact in a modular manner with specific receptor domains. Our results suggest that FeLV-B VRA interacts with Pit1 extracellular loops 4 and 5 and that residues Phe-60 and Pro-61 of FeLV-B VRA are essential for receptor choice. However, this interaction is insufficient for infection, and an additional interaction between FeLV-B VRB and Pit1 loop 2 is essential. Similarly, A-MLV infection requires interaction of A-MLV VRA with Pit2 loops 4 and 5 and VRB with Pit2 loop 2, with residues Tyr-60 and Val-61 of A-MLV VRA being critical for receptor recognition. Together, our results suggest that FeLV-B and A-MLV infections require two major discrete interactions between the viral SU envelope glycoproteins and their respective receptors. We propose a common two-step mechanism for interaction between retroviral envelope glycoproteins and cell surface receptors.     

Gibbon ape leukemia virus and the amphotropic murine leukemia virus 4070A exhibit an unusual interference pattern on E36 Chinese hamster cells.

The gibbon ape leukemia virus (GaLV), the amphotropic mouse leukemia virus (A-MLV) 4070A, and the xenotropic mouse leukemia virus (X-MLV) exhibit wide but not identical species host ranges. However, most Chinese hamster cells resist infection by all three viruses. We have now determined that the Chinese hamster cell line E36 differs from other Chinese hamster cell lines in that it is susceptible to infection by wild-type GaLV, A-MLV, and X-MLV. Surprisingly, analysis of the interference pattern of GaLV and A-MLV in E36 cells indicated that GaLV and A-MLV interfere in a nonreciprocal fashion. E36 cells productively infected with GaLV were resistant to superinfection by both GaLV and amphotropically packaged recombinant retroviral vectors. In contrast, E36 cells infected with A-MLV were resistant to superinfection with an amphotropic vector but could still be infected by a GaLV vector. These results imply the existence of a receptor on E36 cells that interacts with both GaLV and A-MLV.     

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.     

Specificity in receptor usage by T-cell-tropic feline leukemia viruses: implications for the in vivo tropism of immunodeficiency-inducing variants

Cytopathic, T-cell-tropic feline leukemia viruses (FeLV-T) evolve from FeLV-A in infected animals and demonstrate host cell specificities that are distinct from those of their parent viruses. We recently identified two cellular proteins, FeLIX and Pit1, required for productive infection by these immunodeficiency-inducing FeLV-T variants (M. M. Anderson, A. S. Lauring, C. C. Burns, and J. Overbaugh, Science 287:1828-1830, 2000). FeLV-T is the first example of a naturally occurring type C retrovirus that requires two proteins to gain entry into target cells. FeLIX is an endogenous protein that is highly related to the N-terminal portion of the FeLV envelope protein, which includes the receptor-binding domain. Pit1 is a multiple-transmembrane phosphate transport protein that also functions as a receptor for FeLV-B. The FeLV-B envelope gene is derived by recombination with endogenous FeLV-like sequences, and its product can functionally substitute for FeLIX in facilitating entry through the Pit1 receptor. In the present study, we tested other retrovirus envelope surface units (SUs) with their cognate receptors to determine whether they also could mediate infection by FeLV-T. Cells were engineered to coexpress the transmembrane form of the envelope proteins and their cognate receptors, or SU protein was added as a soluble protein to cells expressing the receptor. Of the FeLV, murine leukemia virus, and gibbon ape leukemia virus envelopes tested, we found that only those with receptor-binding domains derived from endogenous FeLV could render cells permissive for FeLV-T. We also found that there is a strong preference for Pit1 as the transmembrane receptor. Specifically, FeLV-B SUs could efficiently mediate infection of cells expressing the Pit1 receptor but could only inefficiently mediate infection of cells expressing the Pit2 receptor, even though these SUs are able to bind to Pit2. Expression analysis of feline Pit1 and FeLIX suggests that FeLIX is likely the primary determinant of FeLV-T tropism. These results are discussed in terms of current models for retrovirus entry and the interrelationship among FeLV variants that evolve in vivo.     

Cellular and species resistance to murine amphotropic, gibbon ape, and feline subgroup C leukemia viruses is strongly influenced by receptor expression levels and by receptor masking mechanisms

Chinese hamster ovary (CHO) cells are resistant to infections by gibbon ape leukemia virus (GALV) and amphotropic murine leukemia virus (A-MLV) unless they are pretreated with tunicamycin, an inhibitor of N-linked glycosylation. These viruses use the related sodium-phosphate symporters Pit1 and Pit2, respectively, as receptors in nonhamster cells, and evidence has suggested that the corresponding transporters of CHO cells may be masked by tunicamycin-sensitive secreted inhibitors. Although the E36 line of Chinese hamster cells was reported to secrete the putative Pit2 inhibitor and to be sensitive to the inhibitory CHO factors, E36 cells are highly susceptible to both GALV and A-MLV in the absence of tunicamycin. Moreover, expression of E36 Pit2 in CHO cells conferred tunicamycin-independent susceptibilities to both viruses. Based on the latter results, it was suggested that E36 Pit2 must functionally differ from the endogenous Pit2 of CHO cells. To test these ideas, we analyzed the receptor properties of CHO Pit1 and Pit2 in CHO cells. Surprisingly, and counterintuitively, transfection of a CHO Pit2 expression vector into CHO cells conferred strong susceptibility to both GALV and A-MLV, and similar overexpression of CHO Pit1 conferred susceptibility to GALV. Thus, CHO Pit2 is a promiscuous functional receptor for both viruses, and CHO Pit1 is a functional receptor for GALV. Similarly, we found that the natural resistance of Mus dunni tail fibroblasts to subgroup C feline leukemia viruses (FeLV-C) was eliminated simply by overexpression of the endogenous FeLV-C receptor homologue. These results demonstrate a novel and simple method to unmask latent retroviral receptor activities that occur in some cells. Specifically, resistances to retroviruses that are caused by subthreshold levels of receptor expression or by stoichiometrically limited masking or interference mechanisms can be efficiently overcome simply by overexpressing the endogenous receptors in the same cells.     

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.     

Host cell range of T-lymphotropic feline leukemia virus in vitro

We compared the host cell range of T-lymphotropic feline leukemia virus (FeLV-T) with that of FeLV subgroup B (FeLV-B) by pseudotype assay in the presence of FeLIX, a truncated envelope glycoprotein of endogenous FeLV. Although both viruses use Pit1 as a receptor and FeLIX does not hamper FeLV-B infection by receptor interference, the host ranges of FeLV-T and -B were not exactly the same, suggesting a different Pit1 usage at the post-binding level. A comparison of Pit1 sequences of various mammalian species indicated that extracellular loop 1 in a topology model deduced with the PHD PredictProtein algorism may be one of the regions responsible for efficient infection by FeLV-T.     

Leukemogenicity and cell transformation mechanisms in vitro by Gross murine leukemia virus: analysis of virus subpopulations.

The leukemogenic activity of Gross murine leukemia virus adapted to rats was tested in W/Fu rats and NIH/Swiss mice. All animals infected with this virus developed thymic and nonthymic T-cell leukemia with a short latency period. It was observed that cell-free extracts from thymic lymphoma tissue of mice and rats, induced by either Gross murine leukemia virus or Gross murine leukemia virus adapted to rats, consisted of both small-plaque-forming and large-plaque-forming viruses, as determined by the XC plaque test. MCF-type virus was found in these virus complexes. Transformed cell foci were induced in SC-1 cell layers by double infection of the cloned MCF-type virus and an ecotropic virus. SC-1 cells containing transformed cell foci were shown to be tumorigenic upon inoculation into nude mice. The formation of transformed cell foci in mink lung cells was also observed after double infection with the cloned MCF-type virus and a xenotropic virus. The possible mechanism of leukemogenesis by endogenous viruses is discussed.     

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.     

Change of tropism of SL3-2 murine leukemia virus, using random mutational libraries

SL3-2 is a polytropic murine leukemia virus with a limited species tropism. We cloned the envelope gene of this virus, inserted it into a bicistronic vector, and found that the envelope protein differs from other, similar envelope proteins that also utilize the polytropic receptor (Xpr1) in that it is severely impaired in mediating infection of human and mink cells. We found that two adjacent amino acid mutations (G212R and I213T), located in a previously functionally uncharacterized segment of the surface subunit, are responsible for the restricted tropism of the SL3-2 wild-type envelope. By selection from a two-codon library, several hydrophobic amino acids at these positions were found to enable the SL3-2 envelope to infect human TE 671 cells. In particular, an M212/V213 mutant had a titer at least 6 orders of magnitude higher than that of the wild-type envelope for human TE 671 cells and infected human, mink, and murine cells with equal efficiencies. Notably, these two amino acids are not found at homologous positions in known murine leukemia virus isolates. Functional analysis and library selection were done on the basis of sequence and tropism analyses of the SL3-2 envelope gene. Similar approaches may be valuable in the design and optimization of retroviral envelopes with altered tropisms for biotechnological purposes.