Identification of a Feline Leukemia Virus Variant That Can Use THTR1, FLVCR1, and FLVCR2 for Infection (cid:1)

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. of pathogenic FeLV-C in infected cats and potential FeLV variants/intermediates that may arise, we isolated and characterized FeLV Env sequences from a primary FeLV isolate derived from a cat with pure red cell aplasia. In this study, we report the isolation and charac-* are available from Invitrogen) were used to knock down FLVCR2 expression. To validate the specificity of the FLVCR2 siRNAs, CHO cells expressing HA-tagged huFLVCR1 or huFLVCR2 were seeded (1 (cid:2) 10 5 ) in a 12-well plate. The next day, cells were transfected with 100 pmol of S1 or S2 or with control scrambled siRNA (Invitrogen) using a Lipofectamine RNAi Max kit (Invitrogen). Transfected cells were incubated for a further 2 days before preparation of cell lysates and analysis of HA-tagged FLVCR1 and FLVCR2 protein expression by Western blotting (see above). Porcine ST-IOWA cells expressing scrambled, S1, or S2 siRNA were generated by transfection of ST-IOWA cells with 100 pmol of siRNA oligonucleotides. At 2 days posttransfection, cells were infected with serial dilutions of (cid:5) -galactosidase encoding FY981 pseudotype virus, and infected cells were stained 2 days later as described above. Nucleotide sequence accession numbers. The FY981 FeLV Env and poFLVCR1, poFLVCR2, and poTHTR1 cDNA and protein sequences have been deposited in the GenBank database under accession numbers FJ436991, FJ436992, FJ436993, and FJ436994, respectively.