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. 2009 Jul;83(13):6706-16.
doi: 10.1128/JVI.02317-08. Epub 2009 Apr 15.

Identification of a feline leukemia virus variant that can use THTR1, FLVCR1, and FLVCR2 for infection

Zvi Shalev  1 , Simon P Duffy, Karen W Adema, Rati Prasad, Naveen Hussain, Brian J Willett, Chetankumar S Tailor
Affiliations

Identification of a feline leukemia virus variant that can use THTR1, FLVCR1, and FLVCR2 for infection

Zvi Shalev et al. J Virol. 2009 Jul.

Abstract

The pathogenic subgroup C feline leukemia virus (FeLV-C) arises in infected cats as a result of mutations in the envelope (Env) of the subgroup A FeLV (FeLV-A). To better understand emergence of FeLV-C and potential FeLV intermediates that may arise, we characterized FeLV Env sequences from the primary FY981 FeLV isolate previously derived from an anemic cat. Here, we report the characterization of the novel FY981 FeLV Env that is highly related to FeLV-A Env but whose variable region A (VRA) receptor recognition sequence partially resembles the VRA sequence from the prototypical FeLV-C/Sarma Env. Pseudotype viruses bearing FY981 Env were capable of infecting feline, human, and guinea pig cells, suggestive of a subgroup C phenotype, but also infected porcine ST-IOWA cells that are normally resistant to FeLV-C and to FeLV-A. Analysis of the host receptor used by FY981 suggests that FY981 can use both the FeLV-C receptor FLVCR1 and the feline FeLV-A receptor THTR1 for infection. However, our results suggest that FY981 infection of ST-IOWA cells is not mediated by the porcine homologue of FLVCR1 and THTR1 but by an alternative receptor, which we have now identified as the FLVCR1-related protein FLVCR2. Together, our results suggest that FY981 FeLV uses FLVCR1, FLVCR2, and THTR1 as receptors. Our findings suggest the possibility that pathogenic FeLV-C arises in FeLV-infected cats through intermediates that are multitropic in their receptor use.

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Figures

FIG. 1.
FIG. 1.
The respective receptor-binding domain (RBD), PRR, C domain, and the TM region of the FeLV envelopes are shown for FeLV-A/Glasgow, FY981 variant I, and FeLV-C/Sarma Env proteins. Also shown are the variable regions VRA and VRB and the C2 loop located in the C domain. Both VRA and the C2 loop have been shown to contain residues critical for receptor binding. Asterisks above serine 99 and serine 101 indicate the residues that were mutated to prolines in variant II clones of FY981 Envs. Dots indicate identical residues, and dashes indicate spaces that were introduced for alignment. The Env sequences were aligned using the BioEdit sequence alignment program (http://www.mbio.ncsu.edu/BioEdit/BioEdit.html).
FIG. 2.
FIG. 2.
LacZ-encoding virus bearing either FY981, FeLV-C, or FeLV-A Env was generated by cotransfection of HEK293 cells with MLV Gag-Pol, LacZ, and Env expression constructs. Supernatants were harvested, and infection titers were tested on a panel of cell lines. Titers are represented as the number of CFU per milliliter of virus supernatant, and values are averages of three independent infection studies. The cell lines tested are feline kidney H06T1, guinea pig 104C1, mink lung Mv1Lu, human cervical carcinoma HeLa, porcine testes ST-IOWA, and murine NIH 3T3. Arrows indicate zero infection titers. Standard deviation is shown for each infection.
FIG. 3.
FIG. 3.
CHO cells were stably transduced with human (hu), feline (fe), or porcine (po) FeLV receptor expression constructs using VSV pseudotype virus. ST-IOWA cells were also transduced with VSV pseudotype virus with porcine FeLV receptor expression constructs. Transduced cells were selected using G418, and resistant cells were pooled and tested for susceptibility to lacZ(FY981), lacZ(FeLV-C), or lacZ(FeLV-A). Parental CHO and ST-IOWA cells were included. Titers are averages of three independent infections and are represented as the number of CFU per milliliter of virus supernatant. Standard deviation is shown for each infection. Arrows indicate zero infection titers.
FIG. 4.
FIG. 4.
(A) Receptor-expressing cells were incubated with (white histogram) or without (black histogram) FeLV-C SU (C-SU), FY981 SU (FY-SU), or FeLV-A SU (A-SU) proteins tagged with a double HA epitope. Bound SU protein was detected using mouse anti-HA antibody (HA.11) and fluorescein-conjugated donkey anti-mouse and analyzed by flow cytometry. An increase in fluorescence (white histogram) represents SU binding. (B) Soluble FeLV-A (A), FeLV-C (C), or FY981 (FY) SU proteins in 1 ml of SU supernatant. Relative amount of protein (determined using ImageJ software) is shown below the blot.
FIG. 5.
FIG. 5.
The predicted TM-spanning segments of huFLVCR1 and poFLVCR1, determined using the TMpred program (http://www.ch.embnet.org/software/TMPRED_form.html) are indicated by a line above the amino acid sequences. Presumptive ECLs are indicated above the sequences. The asterisk indicates residue 487 in ECL6 that had previously been shown to be critical for receptor function (7). Dots represent identical residues. The FLVCR1 sequences were aligned using the BioEdit sequence alignment program (http://www.mbio.ncsu.edu/BioEdit/BioEdit.html).
FIG. 6.
FIG. 6.
(A) FeLV receptor protein expression in cell lysate (CL) and crude membrane (M) prepared from CHO cells transduced with VSV pseudotype virus carrying feline (fe), human (hu), or porcine (po) FeLV receptor (FLVCR1, FLVCR2, or THTR1) constructs. Receptor proteins were tagged with an HA epitope, and proteins were detected using an anti-HA HRP-conjugated monoclonal antibody. The loading control for actin and Na+K+ ATPase are shown. The relative membrane receptor expression is shown below the ATPase blot and was determined using ImageJ Software (see Materials and Methods). Membrane expression is relative to huFLVCR1. The asterisk denotes low membrane expression. (B) FeLV receptor protein expression in crude membrane (M) prepared from porcine ST-IOWA cells expressing HA-tagged poFLVCR1, poFLVCR2, and poTHTR1. The Na+K+ ATPase loading control is shown with relative membrane expression.
FIG. 7.
FIG. 7.
(A) Validation of FLVCR2 knockdown by FLVCR2 siRNA. CHO/huFLVCR2 or CHO/huFLVCR1 cells were transiently transfected with huFLVCR2-specific siRNAs S1 or S2 or with Scr siRNA, and expression of the HA-tagged huFLVCR1 or huFLVCR2 proteins was analyzed by Western blotting. (B) FY981 infection titer in porcine ST-IOWA cells transiently expressing Scr, S1, or S2 siRNAs. The percent FY981 infection titer is shown relative to infection observed on ST-IOWA cells expressing Scr siRNA. Infection titers are averages of three infection experiments. Standard deviation bars are shown.
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