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.     

Genetic and biochemical analyses of receptor and cofactor determinants for T-cell-tropic feline leukemia virus infection

Entry by retroviruses is mediated through interactions between the viral envelope glycoprotein and the host cell receptor(s). We recently identified two host cell proteins, FeLIX and Pit1, that are necessary for infection by cytopathic, T-cell-tropic feline leukemia viruses (FeLV-T). Pit1 is a classic multiple transmembrane protein used as a receptor by several other simple retroviruses, including subgroup B FeLV (FeLV-B), and FeLIX is a secreted cellular protein expressed from endogenous FeLV-related sequences (enFeLV). FeLIX is nearly identical to FeLV-B envelope sequences that encode the N-terminal half of the viral surface unit (SU), because these FeLV-B sequences are acquired by recombination with enFeLV. FeLV-B SUs can functionally substitute for FeLIX in mediating FeLV-T infection. Both of these enFeLV-derived cofactors can efficiently facilitate FeLV-T infection only of cells expressing Pit1, not of cells expressing the related transport protein Pit2. We therefore have used chimeric Pit1/Pit2 receptors to map the determinants for cofactor binding and FeLV-T infection. Three distinct determinants appear to be required for cofactor-dependent infection by FeLV-T. We also found that Pit1 sequences within these same domains were required for binding by FeLIX to the Pit receptor. In contrast, these determinants were not all required for receptor binding by the FeLV-B SU cofactors used in this study. These data indicate that cofactor binding is not sufficient for FeLV-T infection and suggest that there may be a direct interaction between FeLV-T and the Pit1 receptor.     

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.     

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.     

Envelope determinants for dual-receptor specificity in feline leukemia virus subgroup A and T variants

Gammaretroviruses, including the subgroups A, B, and C of feline leukemia virus (FeLV), use a multiple-membrane-spanning transport protein as a receptor. In some cases, such as FeLV-T, a nonclassical receptor that includes both a transport protein (Pit1) and a soluble cofactor (FeLIX) is required for entry. To define which regions confer specificity to classical versus nonclassical receptor pathways, we engineered mutations found in either FeLV-A/T or FeLV-T, individually and in combination, into the backbone of the transmissible form of the virus, FeLV-A. The receptor specificities of these viruses were tested by measuring infection and binding to cells expressing the FeLV-A receptor or the FeLV-T receptors. FeLV-A receptor specificity was maintained when changes at amino acid position 6, 7, or 8 of the mature envelope glycoprotein were introduced, although differences in infection efficiency were observed. When these N-terminal mutations were introduced together with a C-terminal 4-amino-acid insertion and an adjacent amino acid change, the resulting viruses acquired FeLV-T receptor specificity. Additionally, a W-->L change at amino acid position 378, although not required, enhanced infectivity for some viruses. Thus, we have found that determinants in the N and C termini of the envelope surface unit can direct entry via the nonclassical FeLV-T receptor pathway. The region that has been defined as the receptor binding domain of gammaretroviral envelope proteins determined entry via the FeLV-A receptor independently of the presence of the N- and C-terminal FeLV-T receptor determinants.     

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.     

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.     

Pathogenesis of feline leukemia virus T17: contrasting fates of helper, v-myc, and v-tcr proviruses in secondary tumors.

A naturally occurring feline thymic lymphosarcoma (T17) provided the unique observation of a T-cell antigen receptor beta-chain gene (v-tcr) transduced by a retrovirus. The primary tumor contained three classes of feline leukemia virus (FeLV) provirus, which have now been characterized in more detail as (i) v-tcr-containing recombinant proviruses, (ii) v-myc-containing recombinant proviruses, and (iii) apparently full-length helper FeLV proviruses. The two transductions appear to have been independent events, with distinct recombinational junctions and no sequence overlap in the host-derived inserts. The T17 tumor cell line releases large numbers of FeLV particles of low infectivity; all three genomes are encapsidated, but passage of FeLV-T17 on feline fibroblast and lymphoma cells led to selective loss of the recombinant viruses. The oncogenic potential of the T17 virus complex was, therefore, tested by infection of neonatal cats with virus harvested directly from the primary T17 tumor cell line. A single inoculation of FeLV-T17 caused persistent low-grade infection culminating in thymic lymphosarcoma and acute thymic atrophy, which was accelerated by coinfection with the weakly pathogenic FeLV subgroup A (FeLV-A)/Glasgow-1 helper. Molecularly cloned FeLV-tcr virus (T-31) rescued for replication by a weakly pathogenic FeLV-A/Glasgow-1 helper virus was similarly tested in vivo and induced thymic atrophy and thymic lymphosarcomas. Most FeLV-T17-induced tumors manifested either v-myc or an activated c-myc allele and had undergone rearrangement of endogenous T-cell antigen receptor beta-chain genes, supporting the proposition that the oncogenic effects of c-myc linked to the FeLV long terminal repeat are targeted to a specific window in T-cell differentiation. However, neither the FeLV-T17-induced tumors nor the T-31 + FeLV-A-induced tumors contained clonally represented v-tcr sequences. Only one of the FeLV-T17-induced tumors contained detectable v-tcr proviruses, at a low copy number. While v-tcr does not have a readily transmissible oncogenic function, a more restricted role is not excluded, perhaps involving antigenic peptide-major histocompatibility complex recognition by the T-cell receptor complex. Such a function could be obscured by the genetic diversity of the outbred domestic cat host.     

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.     

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.     

Nucleotide sequences of a feline leukemia virus subgroup A envelope gene and long terminal repeat and evidence for the recombinational origin of subgroup B viruses.

Molecular clones of the subgroup A feline leukemia virus FeLV-A/Glasgow-1 have been obtained. Nucleotide sequence analysis of the 3' end of the proviral genome and comparison with the published sequence of FeLV-B/Gardner-Arnstein showed that the most extensive differences are located within the 5' domain of the env gene. Within this domain, several divergent regions of env are separated by more conserved segments. The 3' end of env is highly conserved, with only a single amino acid coding difference in p15env. The proviral long terminal repeats are also highly conserved, differing by only eight base substitutions and one base insertion. Specific probes constructed from the FeLV-A or FeLV-B env genes were used to compare the env genes of various exogenous FeLV isolates and the endogenous FeLV-related proviruses of normal cat DNA. An FeLV-A-derived env probe showed no hybridization to normal cat DNA but detected all FeLV-A and FeLV-C isolates tested. In contrast, an FeLV-B env probe detected independent FeLV-B isolates and a family of endogenous FeLV-related proviruses. Our observations provide strong evidence to support the hypothesis that FeLV-B viruses have arisen by recombination between FeLV-A and endogenous proviral elements in cat DNA.     

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.     

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.     

Defective endogenous proviruses are expressed in feline lymphoid cells: evidence for a role in natural resistance to subgroup B feline leukemia viruses.

Endogenous feline leukemia virus (FeLV)-related sequences (enFeLV) are a family of proviral elements found in domestic cats and their close relatives. These elements can recombine with exogenous, infectious FeLVs of subgroup A (FeLV-A), giving rise to host range variants of FeLV-B. We found that a subset of defective enFeLV proviruses is highly expressed in lymphoma cell lines and in a variety of primary tissues, including lymphoid tissues from healthy specific-pathogen-free cats. At least two RNA species were detected, a 4.5-kb RNA containing gag, env, and long terminal repeat sequences and a 2-kb RNA containing env and long terminal repeat sequences. Cloning of enFeLV cDNA from two FeLV-free lymphoma cell lines (3201 and MCC) revealed a long open reading frame (ORF) encoding a truncated env gene product corresponding to the N-terminal portion of gp70env. Interestingly, all of three natural FeLV-B isolates include 3' env sequences which are missing from the highly transcribed subset and hence must be derived from other enFeLV elements. The enFeLV env ORF cDNA clones were closely similar to a previously characterized enFeLV provirus, CFE-16, but were polymorphic at a site corresponding to an exogenous FeLV neutralization epitope. Site-specific antiserum raised to a C-terminal 30-amino-acid peptide of the enFeLV env ORF detected an intracellular product of 35 kDa which was also shed from cells in stable form. Expression of the 35-kDa protein correlated with enFeLV RNA levels and was negatively correlated with susceptibility to infection with FeLV-B. Cell culture supernatant containing the 35-kDa protein specifically blocked infection of permissive fibroblast cells with FeLV-B isolates. We suggest that the truncated env protein mediates resistance by receptor blockade and that this form of enFeLV expression mediates the natural resistance of cats to infection with FeLV-B in the absence of FeLV-A.     

Host range and receptor binding properties of vectors bearing feline leukemia virus subgroup B envelopes can be modulated by envelope sequences outside of the receptor binding domain

To evaluate host range differences between two different strains of feline leukemia virus subgroup B (FeLV-B), we compared the binding and infectivity patterns of retrovirus vectors bearing either FeLV-B-90Z or FeLV-B-GA envelopes. We report here that the ability of these envelopes to utilize different Pit1 orthologs is mediated primarily by the receptor binding domain; however, in the case of FeLV-B-90Z, the C terminus also contributes to the recognition of certain Pit1 orthologs.     

Subtle mutational changes in the SU protein of a natural feline leukemia virus subgroup A isolate alter disease spectrum

FeLV-945 is a representative isolate of the natural feline leukemia virus (FeLV) variant predominant in non-T-cell malignant, proliferative, and degenerative diseases in a geographic cohort. The FeLV-945 surface glycoprotein (SU) is closely related to natural horizontally transmissible FeLV subgroup A (FeLV-A) but was found to differ from a prototype to a larger extent than the members of FeLV-A differ among themselves. The sequence differences included point mutations restricted largely to the functional domains of SU, i.e., VRA, VRB, and PRR. Despite the sequence differences in these critical domains, measurements of receptor utilization, including host range and superinfection interference, confirmed the assignment of FeLV-945 to subgroup A. Other proviruses isolated from the cohort contained similar sequence hallmarks and were assigned to FeLV subgroup A. A provirus from cat 1046 contained a histidine-to-proline change at SU residue 6 within an SPHQ motif that was previously identified as a critical mediator of fusion events during virus entry. The 1046 pseudotype virus entered cells only in the presence of the soluble cofactor FeLIX provided in trans, but it retained an ecotropic host range even in the presence of FeLIX. The mutational changes in FeLV-945 were shown to confer significant functional differences compared to prototype FeLV-A viruses. The substitution of FeLV-945 envelope gene sequences for FeLV-A/61E sequences conferred a small but statistically significant replicative advantage in some feline cells. Moreover, substitution of the unique FeLV-945 long terminal repeat and envelope gene for those of FeLV-A/61E altered the disease spectrum entirely, from a thymic lymphoma of a T-cell origin to an as yet uncharacterized multicentric lymphoma that did not contain T cells.     

A Putative Cell Surface Receptor for Anemia-Inducing Feline Leukemia Virus Subgroup C Is a Member of a Transporter Superfamily

Domestic cats infected with the horizontally transmitted feline leukemia virus subgroup A (FeLV-A) often produce mutants (termed FeLV-C) that bind to a distinct cell surface receptor and cause severe aplastic anemia in vivo and erythroblast destruction in bone marrow cultures. The major determinant for FeLV-C-induced anemia has been mapped to a small region of the surface envelope glycoprotein that is responsible for its receptor binding specificity. Thus, erythroblast destruction may directly or indirectly result from FeLV-C binding to its receptor. To address these issues, we functionally cloned a putative cell surface receptor for FeLV-C (FLVCR) by using a human T-lymphocyte cDNA library in a retroviral vector. Expression of the 2.0-kbp FLVCR cDNA in naturally resistant Swiss mouse fibroblasts and Chinese hamster ovary cells caused substantial susceptibility to FeLV-C but no change in susceptibilities to FeLV-B and other retroviruses. The predicted FLVCR protein contains 555 amino acids and 12 hydrophobic potential membrane-spanning sequences. Database searches indicated that FLVCR is a member of the major-facilitator superfamily of transporters and implied that it may transport an organic anion. RNA blot analyses showed that FLVCR mRNA is expressed in multiple hematopoietic lineages rather than specifically in erythroblasts. These results suggest that the targeted destruction of erythroblasts by FeLV-C may derive from their greater sensitivity to this virus rather than from a preferential susceptibility to infection.     

Feline leukemia virus infection requires a post-receptor binding envelope-dependent cellular component

Gammaretrovirus receptors have been suggested to contain the necessary determinants to mediate virus binding and entry. Here, we show that murine NIH 3T3 and baby hamster kidney (BHK) cells overexpressing receptors for subgroup A, B, and C feline leukemia viruses (FeLVs) are weakly susceptible (10(1) to 10(2) CFU/ml) to FeLV pseudotype viruses containing murine leukemia virus (MLV) core (Gag-Pol) proteins, whereas FeLV receptor-expressing murine Mus dunni tail fibroblast (MDTF) cells are highly susceptible (10(4) to 10(6) CFU/ml). However, NIH 3T3 cells expressing the FeLV subgroup B receptor PiT1 are highly susceptible to gibbon ape leukemia virus pseudotype virus, which differs from the FeLV pseudotype viruses only in the envelope protein. FeLV resistance is not caused by a defect in envelope binding, low receptor expression levels, or N-linked glycosylation. Resistance is not alleviated by substitution of the MLV core in the FeLV pseudotype virus with FeLV core proteins. Interestingly, FeLV resistance is alleviated by fusion of receptor-expressing NIH 3T3 and BHK cells with MDTF or human TE671 cells, suggesting the absence of an additional cellular component in NIH 3T3 and BHK cells that is required for FeLV infection. The putative FeLV-specific cellular component is not a secreted factor, as MDTF conditioned medium does not alleviate the block to FeLV infection. Together, our findings suggest that FeLV infection requires an additional envelope-dependent cellular component that is absent in NIH 3T3 and BHK cells but that is present in MDTF and TE671 cells.     

Feline Leukemia Virus-B Envelope Together With its GlycoGag and Human Immunodeficiency Virus-1 Nef Mediate Resistance to Feline SERINC5

Human SERINC5 (SER5) protein is a recently described restriction factor against human immunodeficiency virus-1 (HIV-1), which is antagonized by HIV-1 Nef protein. Other retroviral accessory proteins such as the glycosylated Gag (glycoGag) from the murine leukemia virus (MLV) can also antagonize SER5. In addition, some viruses escape SER5 restriction by expressing a SER5-insensitive envelope (Env) glycoprotein. Here, we studied the activity of human and feline SER5 on HIV-1 and on the two pathogenic retroviruses in cats, feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV). HIV-1 in absence of Nef is restricted by SER5 from domestic cats and protected by its Nef protein. The sensitivity of feline retroviruses FIV and FeLV to human and feline SER5 is considerably different: FIV is sensitive to feline and human SER5 and lacks an obvious mechanism to counteract SER5 activity, while FeLV is relatively resistant to SER5 inhibition. We speculated that similar to MLV, FeLV-A or FeLV-B express glycoGag proteins and investigated their function against human and feline SER5 in wild type and envelope deficient virus variants. We found that the endogenous FeLV recombinant virus, FeLV-B but not wild type exogenous FeLV-A envelope mediates a strong resistance against human and feline SER5. GlycoGag has an additional but moderate role to enhance viral infectivity in the presence of SER5 that seems to be dependent on the FeLV envelope. These findings may explain, why in vivo FeLV-B has a selective advantage and causes higher FeLV levels in infected cats compared to infections of FeLV-A only.