Murine gammaherpesvirus 68 has evolved gamma interferon and stat1-repressible promoters for the lytic switch gene 50

Abstract

Cytokines regulate viral gene expression with important consequences for viral replication and pathogenesis. Gamma interferon (IFN-gamma) is a key regulator of chronic murine gammaherpesvirus 68 (gammaHV68) infection and a potent inhibitor of gammaHV68 reactivation from latency. Macrophages are the cell type that is responsive to the IFN-gamma-mediated control of gammaHV68 reactivation; however, the molecular mechanism of this IFN-gamma action is undefined. Here we report that IFN-gamma inhibits lytic replication of gammaHV68 in primary bone marrow-derived macrophages and decreases transcript levels for the essential lytic switch gene 50. Interestingly, IFN-gamma suppresses the activity of the two known gene 50 promoters, demonstrating that an inflammatory cytokine can directly regulate the promoters for the gammaHV68 lytic switch gene. Stat1, but not IFN-alpha/beta signaling, is required for IFN-gamma action. Moreover, Stat1 deficiency increases basal gammaHV68 replication, gene 50 expression, and promoter activity. Together, these data identify IFN-gamma and Stat1 as being negative regulators of the gammaHV68 lytic cycle and raise the possibility that gammaHV68 maintains IFN-gamma/Stat1-responsive gene 50 promoters to facilitate cell-extrinsic control over the interchange between the lytic and latent cycles.

FIG. 1.

IFN-γ-induced inhibition of γHV68 lytic replication in bone marrow-derived macrophages is mediated by Stat1. (A to C) Compared to medium alone, IFN-γ treatment inhibited γHV68 replication in wild-type (WT) bone marrow-derived macrophages (A) but not in IFN-γR^−/− (B) or Stat1^−/− (C) macrophages. (D to H) Similar to wild-type cells, IFN-γ suppressed γHV68 replication in IRF1^−/− (D), CIITA^−/− (E), IFN-αβR^−/− (F), IRF3^−/− (G), and IRF7^−/− (H) bone marrow macrophages. Primary bone marrow-derived macrophages were prepared as previously described (57). Day 7 bone marrow derived-macrophages were plated at 1 × 10⁵ cells/ml. On day 10, these cells were pretreated with medium alone or 10 units/ml of IFN-γ for 12 h before infection with γHV68 (multiplicity of infection [MOI] of 10). After 1 h of absorption, cells were washed, and IFN-γ was added back into treated cultures. At the indicated times postinfection the viral titer was measured by plaque assay on 3T12 fibroblasts (63). The limit of detection was 50 PFU/ml. Data were collected from 2 to 5 independent experiments and are presented as means ± standard errors of the means (SEM). Statistical analyses were performed by a Student's t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG. 2.

IFN-γ negatively regulates γHV68 lytic switch gene 50 in a Stat1-dependent manner. (A) IFN-γ treatment decreased gene 50 transcript levels compared to medium alone in wild-type but not Stat1^−/− bone marrow-derived macrophages. (B) In contrast, IFN-γ did not alter gene 73 transcript levels. Day 10 bone marrow-derived macrophages were pretreated for 12 h with 10 units/ml of IFN-γ and then infected with γHV68 at an MOI of 10. Total RNA was isolated 12 h postinfection by using TRIzol reagent. RNA was treated with DNase I (Ambion, Austin, TX) before reverse transcriptase cDNA synthesis was performed using oligo(dT)12-18 and Superscript II (Invitrogen, Carlsbad, CA). qRT-PCR was performed with SYBR green (Invitrogen, Carlsbad, CA) and the following primer sequences: 5′-TGCCCCCATGTTTGTGATG and 5′-TGTGGTCATGAGCCCTTCC for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-AGAAACCCACAGCTCGCACTT and 5′-CAATATGCTGGACAGGCGTATC for gene 50, and 5′-CCAGAAGCTTGTGTACTTGTGGAT and 5′-AAATACCACAGCAGCGTAGAAGGT for gene 73. Transcript levels were normalized to GAPDH within each sample. Data were collected from 4 to 7 independent experiments and calculated using the ΔΔC_T method (29). Data are presented as means ± SEM, and statistical analyses were performed by a Student's t test.

FIG. 3.

Promoters for lytic switch gene 50 are IFN-γ responsive. (A) Schematic representation of the known gene 50 transcripts and promoters. The 410-bp promoter is located between nucleotides (nt) 66242 and 66652, and the 250-bp promoter is located between nucleotides 65667 and 65920 according to the γHV68 sequence (60). (B) Predicted GAS sites in the gene 50 promoters were identified by using MatInspector software (Genomatix, Munich, Germany). Point mutations were introduced to disrupt the predicted GAS sites by using QuikChange site-directed mutagenesis (Stratagene, La Jolla, CA). Mutated nucleotides are underlined, and MatInspector analysis was performed on the mutant promoter sequences to ensure that the indicated mutations did not introduce additional predicted transcription factor binding sites. (C and D) Day 10 wild-type bone marrow-derived macrophages were pretreated for 12 h with IFN-γ doses ranging from 0.001 to 10 units/ml. A total of 1 × 10⁶ cells were cotransfected with 2 μg of a β-galactosidase reporter driven by the ubiquitin promoter (Invitrogen, Carlsbad, CA) and 2 μg of a pGL2-luciferase vector (Promega, Madison, WI) containing the 410-bp promoter (C) or the 250-bp promoter (D). Cells were transfected by using Amaxa Nucleofector (Lonza, Germany), and luciferase activity was measured 12 h posttransfection by using the Luciferase Assay System kit (Promega, Madison, WI). Luciferase activity was normalized to β-galactosidase expression, and the value from cells transfected with empty pGL2 vector was subtracted for each condition. (E and F) Stat1^−/− macrophages were pretreated with medium or 10 units/ml of IFN-γ, and promoter activity for the 410-bp (E) or 250-bp (F) promoter was assessed as described above for C and D. (G and H) Predicted GAS sites in the gene 50 promoters were mutated as described above for B, and promoter activity in wild-type bone marrow-derived macrophages was assessed as described above for C and D. Data were pooled from 4 to 8 independent experiments and are presented as means ± SEM. Statistical analyses were performed by a Student's t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. RLU, relative light units; OD, optical density.