Inhibitory effect of gamma interferon on BK virus gene expression and replication

Abstract

BK virus (BKV) is widely accepted to be the causative agent of polyomavirus nephropathy. In immunocompromised individuals, especially kidney transplant recipients, BKV can replicate in kidney epithelial cells, causing loss of renal function and eventual destruction of the graft. Advances in immunosuppressive therapies may be partially responsible for the increasing incidence of polyomavirus nephropathy among transplant recipients by more effectively eliminating components of the immune system, such as gamma interferon (IFN-gamma)-producing lymphocytes, that keep BKV infections at a subclinical level. In this study, we investigated the role of IFN-gamma in regulating lytic infection by BKV. Treatment with IFN-gamma inhibited the expression of the viral early protein large tumor antigen (TAg) and the late protein VP1 in a dose-dependent manner. We detected 1.6- and 12-fold reductions in TAg transcripts at 48 and 96 h postinfection, respectively, with 250 U/ml IFN-gamma, suggesting that IFN-gamma-mediated inhibition occurs at the level of transcription. Furthermore, IFN-gamma inhibited the level of viral progeny production as much as 50-fold at a multiplicity of infection (MOI) of 0.5 and 80-fold at an MOI of 0.1. The inhibitory effects of IFN-gamma were similar for three different strains of BKV examined. These results indicate an important role for IFN-gamma in regulating BKV lytic infection.

FIG. 1.

Dose-dependent IFN-γ inhibition of BKV gene expression. RPTE cells were infected with the TU strain of BKV at an MOI of 0.5 and treated with cytokines at 3 to 6 hours postinfection, and total cell protein was harvested at 4 days postinfection. Samples were analyzed by Western blotting, probing for TAg, VP1, and GAPDH. (A) Infected cells were treated with 50 or 250 U/ml IFN-γ or IFN-α, 100 ng/ml IL-6, IL-8, MCP-1, or TNF-α, or 300 ng/ml RANTES. (B) Infected cells were treated with the indicated concentrations of IFN-γ. Mock, mock-infected samples with no cytokine treatment; untreated, BKV-infected samples with no cytokine treatment; T = 0, samples harvested directly after 1 hour of adsorption with BKV.

FIG. 2.

BKV replication kinetics in the presence of IFN-γ. RPTE cells were infected with the TU strain of BKV at an MOI of 0.5 and treated with 50 U/ml (A and B) or 250 U/ml (B) IFN-γ at 3 to 6 hours postinfection. (A) Total cell protein was harvested every 12 h for 4 days. Samples were analyzed by Western blotting, probing for TAg, VP1, and GAPDH. Untreated, BKV-infected samples with no IFN-γ treatment; M, mock-infected samples; hpi, hours postinfection; 0 hpi, samples harvested directly after 1 hour of adsorption with BKV. (B) Viral lysates were harvested at 0, 1, 2, 3, 4, and 6 days postinfection, and progeny production was determined by a fluorescent focus assay. Data are represented as the log of the viral titer in IU/ml, and samples were assayed in triplicate. Error bars are too small to be seen for some samples.

FIG. 3.

Effect of IFN-γ on viral early region transcript levels. RPTE cells were infected with the TU strain of BKV at an MOI of 0.5 and treated with 50 or 250 U/ml IFN-γ at 3 to 6 hours postinfection, and total cell RNA was prepared at (A) 48 or (B) 96 h postinfection. The level of TAg transcripts in each sample was determined using real-time RT-PCR, with normalization to the level of GAPDH transcripts; the levels of samples treated with 250 U/ml IFN-γ were arbitrarily set to 1. Each bar represents the average from two (A) or three (B) independent experiments analyzed in triplicate in the same assay. Mock, mock-infected samples with no IFN-γ treatment; untreated, BKV-infected samples with no IFN-γ treatment.

FIG. 4.

Effect of IFN-γ during infections with different MOIs. RPTE cells were infected with the TU strain of BKV at an MOI of 12.5, 2.5, 0.5, or 0.1 and treated with 50 U/ml (A to C) or 250 U/ml (C) IFN-γ at 3 to 6 hours postinfection. (A and B) Total cell protein was harvested at 2, 3, and 4 days postinfection and analyzed by Western blotting, probing for TAg, VP1, and GAPDH. The analysis of total cell protein harvested from infection at an MOI of 0.5 was repeated for panel B for direct comparison to samples from infection at an MOI of 0.1. M, mock-infected samples with no IFN-γ treatment; dpi, days postinfection. (C) Viral lysates were harvested at time zero and at 4 days postinfection, and viral progeny production was determined by a fluorescent focus assay. Data are represented as the log of the viral titer in IU/ml, and samples were assayed in triplicate. Untreated, BKV-infected samples with no IFN-γ treatment.

FIG. 5.

Response of various BKV strains to IFN-γ treatment. RPTE cells were infected with the TU, Dunlop, or Proto-2 strain of BKV at an MOI of 0.5 and treated with 50 U/ml (A and B) or 250 U/ml (B) IFN-γ at 3 to 6 hours postinfection. (A) Total cell protein was harvested at 2, 3, and 4 days postinfection and analyzed by Western blotting, probing for TAg, VP1, and GAPDH. M, mock-infected samples with no IFN-γ treatment; dpi, days postinfection. (B) Viral lysates were harvested at time zero and at 4 days postinfection, and viral progeny production was determined by a fluorescent focus assay. Data are represented as the log of the viral titer in IU/ml, and samples were assayed in triplicate. Untreated, BKV-infected samples with no IFN-γ treatment.