Tumor necrosis factor (TNF) and members of the interferon (IFN) family have been shown to independently inhibit the replication of a variety of viruses. In addition, previous reports have shown that treatment with various combinations of these antiviral cytokines induces a synergistic antiviral state that can be significantly more potent than addition of any of these cytokines alone. The mechanism of this cytokine synergy and its effects on global gene expression, however, are not well characterized. Here, we use DNA microarray analysis to demonstrate that treatment of uninfected primary human fibroblasts with TNF plus IFN- induces a distinct synergistic state characterized by significant perturbations of several hundred genes which are coinduced by the individual cytokines alone, as well as the induction of more than 850 novel host cell genes. This synergy is mediated directly by the two ligands, not by intermediate secreted factors, and is necessary and sufficient to completely block the productive replication and spread of myxoma virus in human fibroblasts. In contrast, the replication of two other poxviruses, vaccinia virus and tanapox virus, are only partially inhibited in these cells by the synergistic antiviral state, whereas the spread of both of these viruses to neighboring cells was efficiently blocked. Taken together, our data indicate that the combination of TNF and IFN- induces a novel synergistic antiviral state that is highly distinct from that induced by either cytokine alone.Poxviruses are large enveloped DNA viruses whose replication cycle occurs exclusively in the cytoplasm (27). Although many poxviruses are highly species specific, some members, such as monkeypox virus, tanapox virus (TPV), and cowpox virus, are able to readily leap species barriers and initiate productive zoonotic infections in humans (19,23,28,30). Myxoma virus (MV), an example of more restricted host species range, is a member of the Leporipoxvirus genus, which is uniquely restricted to rabbits (10,16,47). This host species restriction is so profound that even injection of live MV into any other vertebrate species, including humans, fails to produce a productive infection. The molecular mechanisms behind this host restriction remain largely unknown; however, there is evidence that some of the host barriers to MV permissiveness outside the rabbit are regulated by antiviral cytokines such as interferon (IFN) and tumor necrosis factor (TNF) (50, 51). The issue of restriction of MV replication in primary human cells is of particular interest because MV replicates productively in a wide variety of human cancer cells and is currently being developed as a novel oncolytic virotherapeutic to treat human cancer (46). Consequently, a better understanding of the mechanisms by which MV is restricted in primary human cells and tissues is critical before MV enters human trials.Previously, our lab has shown that primary mouse fibroblasts are nonpermissive for MV replication because they induce and secrete type I IFN in response to MV in...