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

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-(cid:1) / (cid:2) ]) and type II interferon (IFN- (cid:3) ) 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- (cid:3) , inhibit DNA viruses such as cytomegalovirus (CMV) is still not fully under-stood. Here, we investigate the antiviral state developed in macrophages upon reversible inhibition of murine CMV by IFN- (cid:3) . 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- (cid:3) activation. Genetic knockout of the type I-signaling pathway, in the context of IFN- (cid:3) 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- (cid:2) upon macrophage activation with IFN- (cid:3) is also detectable. In dose and kinetic viral replication inhibition experiments with IFN- (cid:3) , 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- (cid:2) . 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- (cid:3) 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 dem- onstrated (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- (cid:1) signal, increasing sensitivity for extracellular IFN- (cid:1) levels. The IFN- (cid:1) 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- (cid:3) -inducing pathways, therefore probably facilitating the observed transient expression of IFN- (cid:3) by IFN- (cid:1) . 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- (cid:1) signal.