BET Inhibitor JQ1 Attenuates Feline Leukemia Virus DNA, Provirus, and Antigen Production in Domestic Cat Cell Lines

Abstract

Feline leukemia virus (FeLV) is a cosmopolitan gammaretrovirus that causes lifelong infections and fatal diseases, including leukemias, lymphomas, immunodeficiencies, and anemias, in domestic and wild felids. There is currently no definitive treatment for FeLV, and while existing vaccines reduce the prevalence of progressive infections, they neither provide sterilizing immunity nor prevent regressive infections that result in viral reservoirs with the potential for reactivation, transmission, and the development of associated clinical diseases. Previous studies of murine leukemia virus (MuLV) established that host cell epigenetic reader bromodomain and extra-terminal domain (BET) proteins facilitate MuLV replication by promoting proviral integration. Here, we provide evidence that this facilitatory effect of BET proteins extends to FeLV. Treatment with the archetypal BET protein bromodomain inhibitor (+)-JQ1 and FeLV challenge of two phenotypically disparate feline cell lines, 81C fibroblasts and 3201 lymphoma cells, significantly reduced FeLV proviral load, total FeLV DNA load, and p27 capsid protein expression at nonlethal concentrations. Moreover, significant decreases in FeLV proviral integration were documented in 81C and 3201 cells. These findings elucidate the importance of BET proteins for efficient FeLV replication, including proviral integration, and provide a potential target for treating FeLV infections.

Keywords: BET inhibitors; bromodomains; epigenetics; feline leukemia virus; gammaretroviruses; integration; provirus; retrovirus; viral load.

Figure 1

Effects of (+)-JQ1 over time on 81C cell cultures challenged with FeLV. (a) No dose-dependent differences in viable adherent cell concentrations were present at 24 and 192 hpi. Dose-dependent decreases in both (b) FeLV p27 concentration per million viable adherent cells and (c) total FeLV DNA load per adherent cell were observed. Neither FeLV DNA nor p27 were detected in FeLV-unexposed controls. D(−)V(+)T(−) indicates an FeLV-exposed, untreated positive control. D(−)V(−)T(−) indicates FeLV-unexposed, untreated cells. One-way ANOVA with Holm-Šídák multiple comparisons test. Error bars represent ± SEM; n = 3 biological replicates. * p < 0.05, ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 2

Effects of (+)-JQ1 over time on proviral integration in 81C cell cultures challenged with FeLV. Dose-dependent decreases in fold-differences of (a) proviral FeLV DNA load and (b) ratio of total-to-integrated FeLV DNA genomes at 24 hpi, suggestive of inhibited proviral integration, were observed. Results were calibrated to D(−)V(+)T(−) at 24 hpi. D(−)V(+)T(−) indicates FeLV-exposed, untreated positive control. One-way ANOVA with Holm-Šídák multiple comparisons test. Error bars represent ± SEM; n = 3 biological replicates. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 3

Effects of (+)-JQ1 over time on 3201 cell cultures challenged with FeLV. Dose-dependent decreases in (a) total cell concentration, (b) FeLV p27 concentration per million viable cells, and (c) total FeLV DNA load per cell were observed. No significant changes in precent cell viability were observed (Figure S1). D(−)V(+)T(−) indicates FeLV-exposed, untreated cells. Neither FeLV p27 antigen nor DNA were detected in FeLV-unexposed controls. D(−)V(−)T(−) indicates FeLV-unexposed, untreated cells. D(+)V(+)T(−) indicates FeLV-exposed, DMSO vehicle-treated cells. D(+)V(−)T(−) indicates FeLV-unexposed, DMSO vehicle-treated cells. One-way ANOVA with Holm-Šídák multiple comparisons test. Error bars represent ± SEM; n = 3 biological replicates. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 4

Effects of (+)-JQ1 concentrations over time on proviral integration in 3201 cell cultures challenged with FeLV. A trend toward a fold-difference decrease in (a) proviral FeLV DNA load with (b) a significant increase in the ratio of total-to-integrated FeLV DNA genomes at 24 hpi was seen. Results were calibrated to D(+)V(+)T(−) at 24 hpi. D(−)V(+)T(−) indicates FeLV-exposed, untreated cells. D(−)V(−)T(−) indicates FeLV-unexposed, untreated cells. D(+)V(+)T(−) indicates FeLV-exposed, DMSO vehicle-treated cells. D(+)V(−)T(−) indicates FeLV-unexposed, DMSO vehicle-treated cells. One-way ANOVA with Holm-Šídák multiple comparisons test. Error bars represent ± SEM; n = 3 biological replicates.; *** p < 0.001.