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. 2012 May 31:9:48.
doi: 10.1186/1742-4690-9-48.

Identification of novel subgroup A variants with enhanced receptor binding and replicative capacity in primary isolates of anaemogenic strains of feline leukaemia virus

Hazel Stewart  1 , Karen W Adema, Elizabeth L McMonagle, Margaret J Hosie, Brian J Willett
Affiliations

Identification of novel subgroup A variants with enhanced receptor binding and replicative capacity in primary isolates of anaemogenic strains of feline leukaemia virus

Hazel Stewart et al. Retrovirology. .

Abstract

Background: The development of anaemia in feline leukaemia virus (FeLV)-infected cats is associated with the emergence of a novel viral subgroup, FeLV-C. FeLV-C arises from the subgroup that is transmitted, FeLV-A, through alterations in the amino acid sequence of the receptor binding domain (RBD) of the envelope glycoprotein that result in a shift in the receptor usage and the cell tropism of the virus. The factors that influence the transition from subgroup A to subgroup C remain unclear, one possibility is that a selective pressure in the host drives the acquisition of mutations in the RBD, creating A/C intermediates with enhanced abilities to interact with the FeLV-C receptor, FLVCR. In order to understand further the emergence of FeLV-C in the infected cat, we examined primary isolates of FeLV-C for evidence of FeLV-A variants that bore mutations consistent with a gradual evolution from FeLV-A to FeLV-C.

Results: Within each isolate of FeLV-C, we identified variants that were ostensibly subgroup A by nucleic acid sequence comparisons, but which bore mutations in the RBD. One such mutation, N91D, was present in multiple isolates and when engineered into a molecular clone of the prototypic FeLV-A (Glasgow-1), enhanced replication was noted in feline cells. Expression of the N91D Env on murine leukaemia virus (MLV) pseudotypes enhanced viral entry mediated by the FeLV-A receptor THTR1 while soluble FeLV-A Env bearing the N91D mutation bound more efficiently to mouse or guinea pig cells bearing the FeLV-A and -C receptors. Long-term in vitro culture of variants bearing the N91D substitution in the presence of anti-FeLV gp70 antibodies did not result in the emergence of FeLV-C variants, suggesting that additional selective pressures in the infected cat may drive the subsequent evolution from subgroup A to subgroup C.

Conclusions: Our data support a model in which variants of FeLV-A, bearing subtle differences in the RBD of Env, may be predisposed towards enhanced replication in vivo and subsequent conversion to FeLV-C. The selection pressures in vivo that drive the emergence of FeLV-C in a proportion of infected cats remain to be established.

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Figures

Figure 1
Figure 1
Sequence alignment of receptor binding domains from theenvs of anaemogenic strains of FeLV. FEA cells were infected with primary isolates of FeLV from cats with anaemia. Env genes were amplified from total cellular DNA, and the nucleic acid sequences of multiple clones were determined. Sequences in blue were putatively assigned to subgroup A while sequences in red were assigned to subgroup C. Dots indicate conservation with the A (Glasgow-1) sequence. Dashes are included to maintain alignment conservation in the presence of deletion or insertions. Residues 83 and 91 in the N terminal region are highlighted.
Figure 2
Figure 2
The DD mutant of FeLV-A (Glasgow-1) displays a higher infectious titre. (A) Three mutants of the FeLV-A (Glasgow-1) molecular clone, possessing mutations at amino acids 83 and(or) 91, were transfected into HEK293T cells. While FeLV-A (Glasgow-1) (A) has the phenotype D83:N91, the mutants DD, ND and NN were D83:D91, N83:D91 and N83:N91 respectively. Viral supernatant was harvested 72 hours post-transfection, filtered and ultracentrifuged, separated by SDS-PAGE, immunoblotted and probed with either anti-gp70 (SU) or anti-p27 (CA). (B) FeLV-A (Glasgow-1) (A) or the DD, ND and NN mutants were transfected into 293 T cells and the supernatant was recovered. Serial dilutions of each supernatant were prepared and plated onto QN10 S + L- cells. 72 hours post-infection, foci were scored manually, values represent the mean +/− SEM of two independent experiments.
Figure 3
Figure 3
The DD Env supports more efficient infection of HEK293T cells. (A) MLV(FeLV) lacZ pseudotypes were prepared from HEK293T cells. Samples of each pseudotype preparation were pelleted, separated by SDS-PAGE, immunoblotted and probed with either anti-FeLV gp70 or anti-SFFV Gag. (B) Matched inputs (RT activity) of MLV(FeLV) lacZ pseudotypes bearing the Glasgow-1 (A), DD, ND or NN Envs, were plated onto HEK293T cells. 72 hours post-infection, the cells were stained for expression of lacZ and counted manually. Values are presented as infectious units per μl (I.U./ μl ), mean +/− SE of three independent experiments. The increase in titre noted for the DD mutant and the decrease in titre of the ND mutant (asterisks) are statistically significant in comparison with A (Glasgow-1) (unpaired T test, p < 0.05).
Figure 4
Figure 4
The DD Env confers enhanced utilisation of THTR1 homologues. MLV (FeLV) lacZ pseudotypes bearing the A (Glasgow-1), C (Sarma), DD, ND or NN Envs were plated onto MDTF cells expressing the feline (fe), human (hu) or porcine (po) THTR1 homologues, human FLVCR1 or human FLVCR2. 72 hours post-infection, cells were stained for expression of lacZ and counted manually. Values represent the mean +/− SEM of three independent experiments. The increase in titre associated with the DD mutation upon feline, human or porcine THTR1 is statistically significant in comparison with A (Glasgow-1) (unpaired T test, p < 0.05).
Figure 5
Figure 5
Expression of soluble Fc-tagged FeLV-SUs. HEK293T cells were transfected with the pTORSTEN vector into which the SU domains of the Glasgow-1 (A), Gardner-Arnstein (B) and Sarma (C), or the Glasgow-1 mutants DD, ND and NN had been cloned. Matched volumes of supernatant were separated by SDS-PAGE, transferred to nitrocellulose and immunoblotted for both gp70 (4-15% gradient gel, upper) and the human IgG Fc tag (8% linear gel, lower).
Figure 6
Figure 6
The DD mutation confers enhanced binding of Fc-SU to multiple receptors. Matched volumes of supernatant containing the Fc-SU fusion proteins from Glasgow-1 (A), Gardner-Arnstein (B), Sarma (C), or the mutants DD, ND and NN, were added to either MDTF or 104 C1 cells expressing feline, human or porcine THTR1, human FLVCR1 & 2, human Pit1 (MDTF only), or vector only (CON). Fc-SU binding was detected by flow cytometry with PE-conjugated anti-human IgG-Fc. Each histogram represents 10,000 events collected in LIST mode and are representative of two independent analyses. Ordinate displays number of events while abscissa displays fluorescence intensity.
Figure 7
Figure 7
Neutralisation of FeV by either pooled serum from FeLV-recovered cats or anti-gp70 monoclonal antibody. (A) Purified FeLV-A (Glasgow-1) was separated by SDS-PAGE and transferred to nitrocellulose membrane. Strips of membrane were probed with pooled FeLV-positive sera (lane 1), pooled FeLV-negative sera (lane 2) or murine anti-gp70 MAb (lane 3). 100 I.U. of FeLV-A (Glasgow-1) (A) or the DD, ND and NN mutants were incubated for two hours with serially-diluted pooled (B) FeLV-positive serum or (C) anti-gp70 MAb, before being titrated on QN10 cells. 72 hours post-infection, foci were enumerated manually. Values represent the mean +/− SEM of two independent experiments.
Figure 8
Figure 8
Replication of FeLV in the presence of sub-neutralising concentrations of anti-FeLV antibodies. FEA cells were infected with FeLV A(Glasgow-1) (A) or the DD, ND and NN mutants in the presence of sub-optimal neutralising concentrations of either pooled serum from FeLV-positive cats or anti-gp70 MAb. Supernatant was harvested from infected cultures at 10 and 28 days post-infection, concentrated by ultracentrifugation, viral pellets were then separated by SDS-PAGE, transferred to nitrocellulose and stained for capsid protein (p27).
Figure 9
Figure 9
Acquisition of non-synonymous mutations in theenvsof FeLV-A mutants following long-term culture. FEA cultures were infected with FeLV-A (Glasgow-1), or the DD, ND and NN mutants in the presence of pooled feline sera from FeLV-positive cats, anti-gp70 MAbs, or with no antibody (control). 50 days post-infection, env sequences were amplified from purified viral RNA, and their nucleic acid sequence were determined.
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