Recombinant feline leukemia virus (FeLV) variants establish a limited infection with altered cell tropism in specific-pathogen-free cats in the absence of FeLV subgroup A helper virus.
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Feline leukemia virus subgroup B (FeLV-B) is commonly associated with feline lymphosarcoma and arises through recombination between endogenous retroviral elements inherited in the cat genome and corresponding regions of the envelope (env) gene from FeLV subgroup A (FeLV-A). In vivo infectivity for FeLV-B is thought to be inefficient in the absence of FeLV-A. Proposed FeLV-A helper functions include enhanced replication efficiency, immune evasion, and replication rescue for defective FeLV-B virions. In vitro analysis of the recombinant FeLV-B-like viruses (rFeLVs) employed in this study confirmed these viruses were replication competent prior to their use in an in vivo study without FeLV-A helper virus. Eight specific-pathogen-free kittens were inoculated with the rFeLVs alone. Subsequent hematology and histology results were within normal limits, however, in the absence of detectable viremia, virus expression, or significant seroconversion, rFeLV proviral DNA was detected in bone marrow tissue of 4/4 (100%) cats at 45 weeks postinoculation (pi), indicating these rFeLVs established a limited but persistent infection in the absence of FeLV-A. Altered cell tropism was also noted. Focal infection was seen in T-cell areas of the splenic follicles in 3/4 (75%) rFeLV-infected cats analyzed, while an FeLV-A-infected cat showed focal infection in B-cell areas of the splenic follicles. Nucleotide sequence analysis of the surface glycoprotein portion of the rFeLV env gene amplified from bone marrow tissue collected at 45 weeks pi showed no sequence alterations from the original rFeLV inocula. (+info)
Feline leukemia virus long terminal repeat activates collagenase IV gene expression through AP-1.
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Leukemia and lymphoma induced by feline leukemia viruses (FeLVs) are the commonest forms of illness in domestic cats. These viruses do not contain oncogenes, and the source of their pathogenic activity is not clearly understood. Mechanisms involving proto-oncogene activation subsequent to proviral integration and/or development of recombinant viruses with enhanced replication properties are thought to play an important role in their disease pathogenesis. In addition, the long terminal repeat (LTR) regions of these viruses have been shown to be important determinants for pathogenicity and tissue specificity, by virtue of their ability to interact with various transcription factors. Previously, we have shown that, in the case of Moloney murine leukemia virus, the U3 region of the LTR independently induces transcriptional activation of specific cellular genes through an LTR-generated RNA transcript (S. Y. Choi and D. V. Faller, J. Biol. Chem. 269:19691-19694, 1994; S.-Y. Choi and D. V. Faller, J. Virol. 69:7054-7060, 1995). In this report, we show that the U3 region of exogenous FeLV LTRs can induce transcription from collagenase IV (matrix metalloproteinase 9) and monocyte chemotactic protein 1 (MCP-1) promoters up to 12-fold. We also show that AP-1 DNA-binding activity and transcriptional activity are strongly induced in cells expressing FeLV LTRs and that LTR-specific RNA transcripts are generated in those cells. Activation of mitogen-activated protein kinase kinases 1 and 2 (MEK1 and -2) by the LTR is an intermediate step in the FeLV LTR-mediated induction of AP-1 activity. These findings thus suggest that the LTRs of FeLVs can independently activate transcription of specific cellular genes. This LTR-mediated cellular gene transactivation may play an important role in tumorigenesis or preleukemic states and may be a generalizable activity of leukemia-inducing retroviruses. (+info)
A putative cell surface receptor for anemia-inducing feline leukemia virus subgroup C is a member of a transporter superfamily.
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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. (+info)
The FeLV-945 LTR confers a replicative advantage dependent on the presence of a tandem triplication.
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Feline leukemia virus (FeLV), like other naturally occurring retroviruses, is characterized by a high degree of genetic diversity. FeLV-945 is a natural isolate derived from non-B-cell non-T-cell lymphomas classified anatomically as multicentric. FeLV-945 exhibits a unique structural motif in the LTR composed of a 21-bp tandem triplication downstream of a single copy of enhancer. The unique FeLV-945 LTR is precisely conserved among eight independent multicentric lymphomas collected in a geographic cluster. Previous studies using reporter gene constructs predict that the FeLV-945 LTR would confer a replicative advantage on the virus that contains it, particularly in primitive hematopoietic cells. Such an advantage may account for the precise conservation of the unique LTR sequence. To test that prediction, a set of recombinant, infectious FeLVs was developed that are isogenic other than the presence of the FeLV-945 LTR or mutations of it. Replication assays show that the FeLV-945 LTR confers a distinct growth advantage in K-562, FEA, and 3201 cells and implicate the 21-bp triplication in that function. Replacement of two copies of the triplicated element with random sequence greatly diminished the replicative capacity, thus implicating the triplicated sequence itself in LTR function. The 21-bp triplication was shown to contain specific nuclear protein binding sites, which may account for the selective pressure to conserve the sequence. (+info)
X-ray diffraction study of feline leukemia virus fusion peptide and lipid polymorphism.
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The structural effects of the fusion peptide of feline leukemia virus (FeLV) on the lipid polymorphism of N-methylated dioleoylphosphatidylethanolamine were studied using a temperature ramp with sequential X-ray diffraction. This peptide, the hydrophobic amino-terminus of p15E, has been proven to be fusogenic and to promote the formation of highly curved, intermediate structures on the lamellar liquid-crystal to inverse hexagonal phase transition pathway. The FeLV peptide produces marked effects on the thermotropic mesomorphic behaviour of MeDOPE, a phospholipid with an intermediate spontaneous radius of curvature. The peptide is shown to reduce the lamellar repeat distance of the membrane prior to the onset of an inverted cubic phase. This suggests that membrane thinning may play a role in peptide-induced membrane fusion and strengthens the link between the fusion pathway and inverted cubic phase formation. The results of this study are interpreted in relation to models of the membrane fusion mechanism. (+info)
A comprehensive approach to mapping the interacting surfaces of murine amphotropic and feline subgroup B leukemia viruses with their cell surface receptors.
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Because mutations in envelope glycoproteins of retroviruses or in their cell surface receptors can eliminate function by multiple mechanisms, it has been difficult to unambiguously identify sites for their interactions by site-directed mutagenesis. Recently, we developed a gain-of-function approach to overcome this problem. Our strategy relies on the fact that feline leukemia virus subgroup B (FeLV-B) and amphotropic murine leukemia virus (A-MLV) have closely related gp70 surface envelope glycoproteins and use related Na(+)-dependent phosphate symporters, Pit1 and Pit2, respectively, as their receptors. We previously observed that FeLV-B/A-MLV envelope glycoprotein chimeras spliced between the variable regions VRA and VRB were unable to use Pit1 or Pit2 as a receptor but could efficiently use specific Pit1/Pit2 chimeras. The latter study suggested that the VRA of A-MLV and FeLV-B functionally interact with the presumptive extracellular loops 4 and 5 (ECL4 and -5) of their respective receptors, whereas VRB interacts with ECL2. We also found that FeLV-B gp70 residues F60 and P61 and A-MLV residues Y60 and V61 in the first disulfide-bonded loop of VRA were important for functional interaction with the receptor's ECL4 or -5. We have now extended this approach to identify additional VRA and VRB residues that are involved in receptor recognition. Our studies imply that FeLV-B VRA residues F60 and P61 interact with the Pit1 ECL5 region, whereas VRA residues 66 to 78 interact with Pit1 ECL4. Correspondingly, A-MLV VRA residues Y60 and V61 interact with the Pit2 ECL5 region, whereas residues 66 to 78 interact with Pit2 ECL4. Similar studies that focused on the gp70 VRB implicated residues 129 to 139 as contributing to specific interactions with the receptor ECL2. These results identify three regions of gp70 that interact in a specific manner with distinct portions of their receptors, thereby providing a map of the functionally interacting surfaces. (+info)
Protein stabilization: a common consequence of mutations in independently derived v-Myc alleles.
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Myc is overexpressed in many cancers as a result of gene rearrangement or amplification, but coding sequence changes which cluster in the N-terminal transactivation domain also appear to play a role in tumour progression. The prototypic v-Myc gene of MC29 virus differs from avian c-Myc by a series of mutations, including a change at a regulatory phosphorylation site within the mutational hotspot (thr-61) which is known to potentiate transformation in vitro. We now show that the mutation at thr-61 stabilizes the v-Myc protein (turnover difference) and that this single mutation is both necessary and sufficient for the phenotype. A major involvement of the proteasome in Myc degradation was confirmed, but surprisingly, a dilysine motif adjacent to thr-61 proved not to be the ubiquitin target. Two other v-Myc genes which carry a mutation at thr-61 (avian MH2) or a large deletion encompassing this domain (feline T17) were found to be stabilized to a similar extent as MC29, showing that stabilization is a common feature of independently derived Myc oncogenes. These results suggest a common selective process in the genesis of these three viral oncoproteins and a mechanistic link with Jun, Fos and Myb oncoproteins which are also stabilized relative to their cellular counterparts. (+info)
A novel truncated env gene isolated from a feline leukemia virus-induced thymic lymphosarcoma.
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We PCR amplified the exogenous feline leukemia virus (FeLV)-related env gene species from lymphosarcomas induced by intradermally administered plasmid DNA of either the prototype FeLV, subgroup A molecular clone, F6A, or a new molecular clone, FeLV-A, Rickard strain (FRA). Of the nine tumors examined, six showed the presence of deleted env species of variable sizes in the tumor DNA. One env mutant, which was detected in a FRA-induced thymic lymphosarcoma, had a large internal deletion beginning from almost the N-terminal surface glycoprotein (SU) up to the middle region of the transmembrane (TM) protein of the env gene. The deduced polypeptide of this truncated env (tenv) retained the complete signal peptide and seven amino acids of the N-terminal mature SU of FRA env gene, followed by eight amino acids from the frameshift in the TM region. To study the biological function of tenv, we used a murine retrovirus vector to produce amphotropic virions. Infection of feline fibroblasts (H927), human fibrosarcoma cells (HT1080), or human B-lymphoma cells (Raji) led to pronounced cytotoxicity, while the tenv virus did not induce significant cytotoxicity to feline T-lymphoma cells (3201B) or human T-lymphoma cells (CEM). Together, these results convincingly demonstrated that the genetic events that led to truncation in the env gene occurred de novo in FeLV lymphomagenesis and that such a product, tenv could induce cytotoxicity to fibroblastic and B-lymphoid cells but not to T-lymphoid tumor cells. This type of selective toxicity might be potentially important in the development of the neoplastic disease. (+info)