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. 2011 Oct;85(19):10286-99.
doi: 10.1128/JVI.00373-11. Epub 2011 Jul 20.

Reversible inhibition of murine cytomegalovirus replication by gamma interferon (IFN-γ) in primary macrophages involves a primed type I IFN-signaling subnetwork for full establishment of an immediate-early antiviral state

Kai A Kropp  1 , Kevin A Robertson, Garwin Sing, Sara Rodriguez-Martin, Mathieu Blanc, Paul Lacaze, Muhamad F B Noor Hassim, Mizanur R Khondoker, Andreas Busche, Paul Dickinson, Thorsten Forster, Birgit Strobl, Mathias Mueller, Stipan Jonjic, Ana Angulo, Peter Ghazal
Affiliations

Reversible inhibition of murine cytomegalovirus replication by gamma interferon (IFN-γ) in primary macrophages involves a primed type I IFN-signaling subnetwork for full establishment of an immediate-early antiviral state

Kai A Kropp et al. J Virol. 2011 Oct.

Abstract

Activated macrophages play a central role in controlling inflammatory responses to infection and are tightly regulated to rapidly mount responses to infectious challenge. Type I interferon (alpha/beta interferon [IFN-α/β]) and type II interferon (IFN-γ) play a crucial role in activating macrophages and subsequently restricting viral infections. Both types of IFNs signal through related but distinct signaling pathways, inducing a vast number of interferon-stimulated genes that are overlapping but distinguishable. The exact mechanism by which IFNs, particularly IFN-γ, inhibit DNA viruses such as cytomegalovirus (CMV) is still not fully understood. Here, we investigate the antiviral state developed in macrophages upon reversible inhibition of murine CMV by IFN-γ. On the basis of molecular profiling of the reversible inhibition, we identify a significant contribution of a restricted type I IFN subnetwork linked with IFN-γ activation. Genetic knockout of the type I-signaling pathway, in the context of IFN-γ stimulation, revealed an essential requirement for a primed type I-signaling process in developing a full refractory state in macrophages. A minimal transient induction of IFN-β upon macrophage activation with IFN-γ is also detectable. In dose and kinetic viral replication inhibition experiments with IFN-γ, the establishment of an antiviral effect is demonstrated to occur within the first hours of infection. We show that the inhibitory mechanisms at these very early times involve a blockade of the viral major immediate-early promoter activity. Altogether our results show that a primed type I IFN subnetwork contributes to an immediate-early antiviral state induced by type II IFN activation of macrophages, with a potential further amplification loop contributed by transient induction of IFN-β.

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Figures

Fig. 1.
Fig. 1.
Dose and time dependency of the IFN-γ-induced antiviral state. (A) Viral replication was measured by plaque assay in BMDMs that were pretreated for 2 h, 4 h, 6 h, or 8 h with IFN-γ or left untreated (0 h) before infection with MCMV (MOI, 1). Cells were pretreated with 1, 10, or 100 U/ml IFN-γ, as indicated. Dashed lines represent detection limits. Data points located on the x axis represent cultures without detected plaques; data points on the dashed line indicate cultures with single infection events. If not stated differently, in this and all subsequent figures, data points depict averages (n = 3) and error bars represent SDs. d, days. (B) Correlation of pretreatment time and antiviral effect. Plot of pretreatment times against viral titers on day 4 showed a log-linear correlation (R2 = 0.99) for all three IFN-γ concentrations (1 U/ml, 10 U/ml, and 100 U/ml). ET50s were calculated by a best-fit model for comparison of plaque numbers of the respective pretreated samples with pretreatment time.
Fig. 2.
Fig. 2.
The antiviral effects of IFN-γ are dependent on persistent stimulation. BMDMs were continuously treated with 10 U/ml IFN-γ (+IFN-γ; black triangles) or transiently treated (w-IFN-γ; white circles) or were left untreated (−IFN-γ; black squares); cells were subsequently infected (MOI, 1). (A) The IFN-γ-induced antiviral state is reversible. BMDMs were pretreated as described and infected at time point 0 with MCMV. Culture supernatant was analyzed on primary MEFs for secreted infectious virus until the plateau phase for the untreated control was reached. (B) Schematic representation of the m157 region in wild-type MCMV and mutant reporter virus strain GLuc-MCMV. Reporter gene expression in the mutant strain is controlled by an additional MCMV MIEP element. Both reporter genes are expressed as one bicistronic mRNA and autocatalytically cleaved during the translation process by peptide 2A of the foot-and-mouth-disease virus. (C) Replication of both virus strains in BMDMs is comparable. Viral replication of MCMV (black circles) and GLuc-MCMV (white squares) in BMDMs was measured by standard plaque assay on primary MEFs and is indistinguishable for both virus strains. (D) Effects of 10 U/ml IFN-γ pretreatment on viral replication of GLuc-MCMV are already evident in the first 48 h after infection. Viral replication was monitored by repeated measurement of extracellular levels of Gaussia luciferase in 10 μl culture supernatant. Data points represent means (n = 10; error bars are SDs), and the dashed line indicates the level of the background signal. Symbols are as described for panel A.
Fig. 3.
Fig. 3.
Establishing an antiviral state in BMDMs involves a blockage of MIEP activity. (A) The inhibitory effect of IFN-γ is already evident in the immediate-early phase of viral replication. MIEP activity was measured by GLuc synthesis assay. BMDMs that were continuously treated with 10 U/ml IFN-γ (+IFN-γ; black triangles), transiently treated (w-IFN-γ; white circles), or untreated (−IFN-γ; black squares) were infected with GLuc-MCMV (MOI, 1). GLuc activity released into cell culture supernatant was measured in 2-h time windows, and supernatant was completely replaced at each indicated sampling point. Data points represent means (n = 6; error bars are SDs), with the dashed line indicating the level of the background signal. (B) IFN-γ treatment reversibly inhibits expression of the viral major IE genes. Relative comparison of mRNA levels of MIE genes by quantitative real-time PCR. BMDMs were pretreated and infected as described for panel A. Total cell cDNA was analyzed with intron-spanning MIE gene-specific TaqMan probes and compared to that from untreated samples. Cellular GAPDH mRNA was used for normalization. Bars represent mean relative quantification values of 3 independent biological replicates (error bars depict SEMs). (C) Measurement of reporter expression in individual cells. Flow cytometry side scatter and GFP fluorescence dot plots of GFP-positive gated BMDM populations (dot plots of complete populations are available in Fig. S4 in the supplemental material). Cells pretreated with the indicated IFN-γ concentrations for 24 h and subsequently infected (MOI, 1) with GFP-MCMV were harvested at 6 h p.i., and 10,000 cells for respective treatments were analyzed for GFP expression. Autofluorescence of BMDMs infected with MCMV was used to define the threshold for the GFP gate.
Fig. 4.
Fig. 4.
Analysis of gene transcription profiles in BMDMs treated with IFN-γ. BMDMs (BALB/c) were treated with IFN-γ (3 replicates) as described (+IFN-γ or w-IFN-γ) or left untreated (−IFN-γ). Statistical analysis of the array data (GPX accession number GPX-000029.1) then identified 521 probes whose expression was significantly altered. (A) Cluster analysis of the 521 identified targets. Expression profiles of the 521 targets over the three conditions were compared, and a Pearson correlation of 0.95 was used as the cutoff for cluster formation. Profiles grouped into five clusters (clusters C1 to C5), indicated in the corresponding line plots, which show the average expression profile of the corresponding clusters over the three conditions (right). Gene numbers in the groups are shown in parentheses. In the network representation of the clustering (A), gray nodes represent 33 probe sets that were below the cutoff for clustering, and 18 probes were excluded due to incomplete annotation. (B) Heat map of the 521 identified targets with three replicates for each condition (continuously numbered). Expression levels were normalized to the average of the data set. Statistical testing (empirical Bayes test) was used to filter for genes that reversed expression levels completely after withdrawal of IFN-γ. One hundred sixty-three targets which fulfilled the criteria were identified (P ≤ 0.05). The heat map was produced using supervised clustering, which organized 163 targets into group A and the remaining 358 targets into group B. (C) Type I IFN-induced genes form a substantial part of the induced transcriptional network. Probe sets were mapped to a nonredundant list of ENSEMBL identifiers, resulting in 492 discrete candidates for further analysis. This list was used to query the Interferome database to identify genes specifically induced by type I IFNs. For the 154 ENSEMBL identifiers from group A, 27 genes were specifically induced by type I IFNs (I), 38 genes were induced by type I or type II IFNs (I/II), and 89 genes could not be mapped to the database. (D) IFN-β influences basal and induced expression levels of candidate host genes in the IFN-γ BMDM system. Genes of group A were compared to array data from BMDMs transfected with control siRNAs (RISC) or an siRNA targeting IFN-β and subsequently treated with IFN-γ (44). (Left) Average microarray expression data (n = 3) for five exemplary target genes; (right) qRT-PCR data for the same set of genes in WT and IFN-β1−/− BMDMs after treatment with 10 U/ml IFN-γ. A complete list of target genes and expression levels for the microarray and qRT-PCR data set are available (see Table S3 and Fig. S2 in the supplemental material).
Fig. 5.
Fig. 5.
Type I IFNs play a functional role in establishing the IFN-γ-induced antiviral state. (A) Low levels of IFN-β mRNA transcription are induced by IFN-γ stimulation. Normalized IFN-β expression levels were derived from an Agilent V2 array with cDNA from IFN-γ-treated BMDMs (BALB/c). Total cellular RNA was sampled at the indicated time points, and the derived cDNA was analyzed by whole-genome microarray, as described in reference . (B) Minimal IFN-β secretion is induced in IFN-γ-treated cells. The concentration of IFN-β in supernatants of IFN-γ (10 U/ml)-treated BMDM cultures from BALB/c mice was measured by ELISA (white rhombs). Supernatant from mock-treated cultures was used as a negative control (black rhombs). (C) Type I IFNs contribute to the IFN-γ-induced antiviral state in BMDMs. Plaque assay measuring viral replication in BMDMs pretreated with IFN-γ. BMDMs from wild type, IFN-αR1−/−, or Tyk2−/− mice were pretreated with 10 U/ml IFN-γ and infected with MCMV (MOI, 1). Relative plaque formation was calculated at each indicated time point from means of treated and untreated samples. Error bars depict SDs of the ratios. We used the delta method (17) as an approximation to obtain an error estimate for the calculated ratio of treated over untreated. The estimate is calculated under the assumption that the two variables are normally distributed, independent, and not correlated. (D) Type I IFNs contribute to the IFN-γ-induced block of viral IE gene expression. BMDMs from wild type, IFN-β1−/−, IFN-αR1−/−, or STAT1−/− mice were pretreated with IFN-γ and subsequently infected with GFP-MCMV (MOI, 1). Cells were fixed at 6 h p.i. and analyzed by flow cytometry for GFP expression.
Fig. 6.
Fig. 6.
Schematic model of the cross talk between type I and type II signaling. The constitutive subthreshold expression of type I IFNs is mediated by c-Jun binding to the IFN-β enhancer (25) but is not triggering a full-scale type I response. Binding of subthreshold levels of type I IFNs by their cognitive receptor leads to low-level expression of type I-stimulated genes and is necessary to maintain expression levels of STAT1. A functional role for STAT2 in this system has been demonstrated (85), although it is not clear if ISGF3 is involved in the constitutive autocrine loop. Type I signaling mainly functions through ISGF3 activation but can also directly activate STAT1 homodimers, which in turn could induce expression of the STAT1 gene. This primes cells for the secondary IFN-γ signal, increasing sensitivity for extracellular IFN-γ levels. The IFN-γ signal then induces the expression of several hundred target genes, establishing an antiviral state. Some of the induced genes, namely, MDA-5, TBK1, TANK, IRF1, IRF7, and IRF8, are components of IFN-β-inducing pathways, therefore probably facilitating the observed transient expression of IFN-β by IFN-γ. This transient induction provides an additional feedback loop in the system, temporarily amplifying the constitutive autocrine loop and probably further increasing the levels of STAT1. If the functionality of the constitutive type I autocrine loop is impaired, levels of STAT1 are reduced and the system will be less responsive to the secondary IFN-γ signal.
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