Presence of Endogenous Viral Elements Negatively Correlates with Feline Leukemia Virus Susceptibility in Puma and Domestic Cat Cells

Abstract

While feline leukemia virus (FeLV) has been shown to infect felid species other than the endemic domestic cat host, differences in FeLV susceptibility among species has not been evaluated. Previous reports have noted a negative correlation between endogenous FeLV (enFeLV) copy number and exogenous FeLV (exFeLV) infection outcomes in domestic cats. Since felids outside the genus Felis do not harbor enFeLV genomes, we hypothesized absence of enFeLV results in more severe disease consequences in felid species lacking these genomic elements. We infected primary fibroblasts isolated from domestic cats (Felis catus) and pumas (Puma concolor) with FeLV and quantitated proviral and viral antigen loads. Domestic cat enFeLV env and long terminal repeat (LTR) copy numbers were determined for each individual and compared to FeLV viral outcomes. FeLV proviral and antigen levels were also measured in 6 naturally infected domestic cats and 11 naturally infected Florida panthers (P. concolor coryi). We demonstrated that puma fibroblasts are more permissive to FeLV than domestic cat cells, and domestic cat FeLV restriction was highly related to enFeLV-LTR copy number. Terminal tissues from FeLV-infected Florida panthers and domestic cats had similar exFeLV proviral copy numbers, but Florida panther tissues have higher FeLV antigen loads. Our work indicates that enFeLV-LTR elements negatively correlate with exogenous FeLV replication. Further, Puma concolor samples lacking enFeLV are more permissive to FeLV infection than domestic cat samples, suggesting that endogenization can play a beneficial role in mitigating exogenous retroviral infections. Conversely, presence of endogenous retroelements may relate to new host susceptibility during viral spillover events.IMPORTANCE Feline leukemia virus (FeLV) can infect a variety of felid species. Only the primary domestic cat host and related small cat species harbor a related endogenous virus in their genomes. Previous studies noted a negative association between the endogenous virus copy number and exogenous virus infection in domestic cats. This report shows that puma cells, which lack endogenous FeLV, produce more virus more rapidly than domestic cat fibroblasts following cell culture challenge. We document a strong association between domestic cat cell susceptibility and FeLV long terminal repeat (LTR) copy number, similar to observations in natural FeLV infections. Viral replication does not, however, correlate with FeLV env copy number, suggesting that this effect is specific to FeLV-LTR elements. This discovery indicates a protective capacity of the endogenous virus against the exogenous form, either via direct interference or indirectly via gene regulation, and may suggest evolutionary outcomes of retroviral endogenization.

Keywords: FeLV; Felis catus; Puma concolor; endogenous retroviruses; retroviral interactions.

FIG 1

(A) Puma fibroblasts exposed to FeLV have greater FeLV proviral load compared to that of domestic cat fibroblasts. On both day 5 and day 10, puma cells (Pco) demonstrated significantly higher proviral copy numbers than domestic cat primary fibroblasts (Kruskal-Wallis; **, P < 0.01). Variance between domestic cat (Fca) cells was greater than that of puma cells, while CrFK cell infections displayed the least amount of variation. (B) FeLV viral capsid antigen (p27) production is higher in puma than in domestic cat cells. Beginning day 3 postinfection, puma cells replicated more virus than domestic cat cells (repeated measure ANOVA individual test statistics, *, P < 0.05; **, P < 0.01). One puma, one domestic cat, and one control cat experiment were ended at day 7. Cutoff values were established by 3× standard error above average value for negative control wells (red line). Viable cell numbers and cell mortality increase over time and are similar in puma and domestic cat cultures. (C) Puma (blue) and domestic cat (black) fibroblast cell counts increased over time and reached 100% confluence by day 5. Initial seeding density was 5 × 10⁴ cells per 2 cm². Differences in domestic cat and puma fibroblast cell counts were not significant between day 5 and day 10. Minus signs denote uninfected cell cultures. (D) Puma (blue) and domestic cat (black) fibroblast mortality increased over time following 100% confluence at day 5. Differences in domestic cat and puma fibroblast cell counts were not significant between day 5 and day 10. Minus signs denote uninfected cell cultures.

FIG 2

(A) enFeLV proviral loads vary between individual cats. Domestic cat cells (n = 7) display variation between enFeLV-LTRs and env. In individual cats, LTR copy number was greater than env copy number. enFeLV-LTR ranged between 9 and 13 copies per cell. enFeLV-env was more variable ranging between 32 and 74 copies per cell. (B) Domestic cat enFeLV-LTR copy number was negatively correlated with FeLV antigen production (Pearson’s coefficient = −0.8943; P = 0.0066). (C) Domestic cat enFeLV-env copy number does not correlate with FeLV antigen production (Pearson’s coefficient = −0.1071; P = 0.8397), suggesting a direct role of enLTR in suppression of FeLV replication.

FIG 3

Proviral load does not highly correlate with viral antigen load. Linear regression analysis shows only 44% of variation in antigen production can be explained by proviral load following experimental FeLV infection of domestic cat (black) and puma (blue) cells. This indicates that the amount of viral antigen (a proxy for viral replication) is not governed completely by the degree of infection as measured by proviral load. Horizontal and vertical error bars indicate standard error for proviral load and viral antigen load, respectively.

FIG 4

Lymphoid tissues from naturally infected FeLV-positive domestic cats and pumas have varying viral loads at end-stage disease. (A) Domestic cat lymphoid tissues displayed greater FeLV proviral load compared to puma tissues (Mann-Whitney test; ***, P < 0.0001). Puma tissues had a much wider range of viral loads, which is potentially attributable to sample quality. (B) Puma tissues trended to greater amounts of viral antigen (Mann-Whitney test; P = 0.1495).

FIG 5

Oligonucleotides synthesized for qPCR quantification. Two gBlocks were synthesized commercially (Integrated DNA Technologies, Coralville, IA). Both concatenated gBlocks contained sequences for FeLV-61E (blue) and CCR5 (yellow). One contained enFeLV-LTR sequence, and the second contained enFeLV-env sequence. Bold sequences represent forward and reverse primer binding sites.