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. 2005 Feb;79(3):1351-60.
doi: 10.1128/JVI.79.3.1351-1360.2005.

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

Chandtip Chandhasin  1 , Patricia N Coan, Laura S Levy
Affiliations

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

Chandtip Chandhasin et al. J Virol. 2005 Feb.

Abstract

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.

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Figures

FIG. 1.
FIG. 1.
Diagrammatic representation of FeLV-945 envelope gene and 3′ LTR, indicating surface (SU) and transmembrane (TM) subunits, variable regions A and B (VRA and VRB), and the proline-rich region (PRR). The FeLV-945 LTR is indicated to contain a single enhancer element (hatched box) followed by a 21-bp element triplicated in tandem (open boxes) (23). The percentage of amino acid sequence identity between FeLV-945 and FeLV-A/61E across each designated region is indicated below the diagram.
FIG. 2.
FIG. 2.
Diagrammatic representation of recombinant FeLVs constructed by substituting the envelope gene and/or LTR of FeLV-945 for homologous sequences of FeLV-A/61E.
FIG. 3.
FIG. 3.
Replication kinetics of recombinant viruses containing the LTR of FeLV-945 (61E/945L) or the LTR and the env gene of FeLV-945 (61E/945SL). These recombinants were compared in order to evaluate the impact of FeLV-945 env on virus replication. The replication of recombinant viruses was compared to that of the parental virus, FeLV-A/61E (61E), as a control. Plasmid DNAs containing the indicated FeLV proviruses were introduced into feline FEA cells (A), FC6.BM cells (B), or 3201 cells (C), and culture supernatants were collected at regular intervals thereafter for quantitation of reverse transcriptase activities. The data shown represent the means of three (C) or four (A and B) experiments, expressed as counts per minute of incorporated [3H]TTP per milliliter. Data from replicate assays were analyzed statistically by a one-way analysis of variance and the Bonferroni posttest. In FEA cells, the replication rate of 61E/945SL was significantly higher than that of 61E/945L at days 4, 8, and 12 posttransfection (P < 0.001). The replication kinetics of 61E/945L and 61E/945SL were statistically indistinguishable in other cell lines.
FIG. 4.
FIG. 4.
(A) Comparison of predicted amino acid sequences of VRA, VRB, and PRR domains of SU proteins from previously reported isolates of FeLV-A (11, 37) and from proviruses amplified by PCR from diseased tissues of naturally infected animals. (B) Comparison of predicted amino acid sequence of FeLV-1046 SU to those of previously reported isolates of FeLV-A (11, 37) and FeLV-945. (C) Comparison of predicted amino acid sequence of FeLV-1306 SU to those of previously reported isolates of FeLV-A, -B, and -C (11, 25, 32). Numbers above the sequences correspond to the amino acid positions of the mature FeLV-A/61E Env protein (11). Asterisks indicate amino acid identity.
FIG. 5.
FIG. 5.
Western blot analysis of FeLV SU proteins on pseudotype virus particles harvested from supernatants of producer 293T cells. Virus particles were pelleted from culture supernatants by ultracentrifugation, fractionated by SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. Envelope SU proteins were detected with goat anti-FeLV gp70 followed by incubation with a bovine anti-goat antibody conjugated to horseradish peroxidase. Molecular mass markers (in kilodaltons) are indicated.
FIG. 6.
FIG. 6.
Superinfection interference assays using pseudotype viruses bearing the envelope proteins of FeLV-945, -922, -1049, -1306, -1046, FeLV-C/Sarma, and FeLV-A/61E, as indicated. Uninfected FEA cells or FEA cells that were chronically infected with 61E/945SL or FeLV-B/Gardner-Arnstein were challenged at an MOI of 0.1 with pseudotype particles containing the pRT43.2TnIsβgal genome. Pseudotype entry was quantified (in focus-forming units [FFU]/ml) by enumerating β-galactosidase-expressing cells. For some experiments, conditioned medium from 3201 cells was added at a 1:1 dilution at the time of challenge as a source of FeLIX. The data shown are averages from duplicate (FEA and FEA plus 945SL) or triplicate (FEA plus FeLV-B) experiments.
FIG. 7.
FIG. 7.
Southern blot analyses of DNAs from tumors induced in three animals (O23, O24, and O26) by infection with 61E/945L (A) and from tumors induced in three animals (N90, N91, and N92) by infection with 61E/945SL (B). DNA samples were digested with KpnI (left) and hybridized to a probe for exogenous FeLV LTR (exLTR) or were digested with HincII (right) and hybridized to a probe representing the β chain gene of the murine T-cell receptor (TCRβ). DNAs from the liver of an uninfected cat (lanes C) were included in each set as a control. An internal proviral fragment homologous to the LTR probe (open circles), the 8.4- and 2.9-kb fragments that are definitive of the germ line organization of the feline TCR-β locus (closed circles), and a polymorphism in the TCR-β locus that is characteristic of the litter of animals infected with 91E/945SL (arrowhead) are indicated.
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