The balance between the innate immunity of the host and the ability of a pathogen to evade it strongly influences pathogenesis and virulence. The two nonstructural (NS) proteins, NS1 and NS2, of respiratory syncytial virus (RSV) are critically required for RSV virulence. Together, they strongly suppress the type I interferon (IFN)-mediated innate immunity of the host cells by degrading or inhibiting multiple cellular factors required for either IFN induction or response pathways, including RIG-I, IRF3, IRF7, TBK1 and STAT2. Here, we provide evidence for the existence of a large and heterogeneous degradative complex assembled by the NS proteins, which we named "NS-degradasome" (NSD). The NSD is roughly ~300-750 kD in size, and its degradative activity was enhanced by the addition of purified mitochondria in vitro. Inside the cell, the majority of the NS proteins and the substrates of the NSD translocated to the mitochondria upon RSV infection. Genetic and pharmacological evidence shows that optimal suppression of innate immunity requires mitochondrial MAVS and mitochondrial motility. Together, we propose a novel paradigm in which the mitochondria, known to be important for the innate immune activation of the host, are also important for viral suppression of the innate immunity.

Innate immunity is the first line of defense against microbial insult. The transcription factor, IRF3, is needed by mammalian cells to mount innate immune responses against many microbes, especially viruses. IRF3 remains inactive in the cytoplasm of uninfected cells; upon virus infection, it gets phosphorylated and then translocates to the nucleus, where it binds to the promoters of antiviral genes and induces their expression. Such genes include type I interferons (IFNs) as well as Interferon Stimulated Genes (ISGs). IRF3-/- cells support enhanced replication of many viruses and therefore, the corresponding mice are highly susceptible to viral pathogenesis. Here, we provide evidence for an unexpected pro-microbial role of IRF3: the replication of the protozoan parasite, Toxoplasma gondii, was significantly impaired in IRF3-/- cells. In exploring whether the transcriptional activity of IRF3 was important for its pro-parasitic function, we found that ISGs induced by parasite-activated IRF3 were indeed essential, whereas type I interferons were not important. To delineate the signaling pathway that activates IRF3 in response to parasite infection, we used genetically modified human and mouse cells. The pro-parasitic signaling pathway, which we termed PISA (Parasite-IRF3 Signaling Activation), activated IRF3 without any involvement of the Toll-like receptor or RIG-I-like receptor pathways, thereby ruling out a role of parasite-derived RNA species in activating PISA. Instead, PISA needed the presence of cGAS, STING, TBK1 and IRF3, indicating the necessity of DNA-triggered signaling. To evaluate the physiological significance of our in vitro findings, IRF3-/- mice were challenged with parasite infection and their morbidity and mortality were measured. Unlike WT mice, the IRF3-/- mice did not support replication of the parasite and were resistant to pathogenesis caused by it. Our results revealed a new paradigm in which the antiviral host factor, IRF3, plays a cell-intrinsic pro-parasitic role.

2=-5=-Oligoadenylate synthetase-like protein (OASL) is an interferon-inducible antiviral protein.Here we describe differential inhibitory activities of human OASL and the two mouse OASL homologs against respiratory syncytial virus (RSV) replication. Interestingly, nonstructural protein 1 (NS1) of RSV promoted proteasome-dependent degradation of specific OASL isoforms. We conclude that OASL acts as a cellular antiviral protein and that RSV NS1 suppresses this function to evade cellular innate immunity and allow virus growth. C ellular innate immunity against virus infection is primarily mediated by type I interferons (IFNs). In turn, the IFNs exert their pleiotropic effects through the induction of a variety of IFNstimulated genes (ISGs) (1-4). Although the general antiviral roles of several ISGs have been demonstrated, the roles of individual ISGs and their effect on specific viruses have remained largely unidentified (5-9). On the other hand, the coevolution of the host and the virus has resulted in viral strategies to evade the host IFN response by targeting ISGs and other IFN pathway proteins (10). Oligoadenylate synthetases (OAS) are a family of ISGs characterized by their ability to synthesize 2=-5=-oligoadenylate (2-5A), which induces RNA degradation by activating RNase L (11, 12). Human oligoadenylate synthetase-like protein (OASL) is related to the OAS family by its N-terminal OAS-like domain but is devoid of 2-5A synthetase activity. Additionally, OASL contains two tandem ubiquitin-like (UBL) domains in the C terminus, which are absent in any of the other members of the OAS family (12-15). OASL is directly and rapidly induced by virus infection via interferon regulatory factor 3 (IRF3) as well as by IFN signaling (1,12,16,17). Unlike in humans, two OASL isoforms have been identified in the mouse, Oasl1 and Oasl2. We have recently described the (18), were grown in monolayers on coverslips and infected with RSV Long at a multiplicity of infection of 3. At 18 h postinfection, cells were fixed and immunostained with mouse anti-RSV nucleoprotein (N) antibody (Abnova clone B023), followed by Alex Fluor 610-conjugated donkey anti-mouse IgG (Life Technologies). Images were captured in a Nikon AIRSI spectral confocal microscope system. (B) (Top) The same cell lines were infected as described above, and the total cell lysates were analyzed by immunoblotting using the same primary antibody described above and horseradish peroxidase (HRP)-conjugated secondary antibody, followed by ECL (enhanced chemiluminescence) detection. Actin is the loading control. (Bottom) Total RNA isolated from parallel cultures was subjected to quantitative reverse transcription-PCR (qRT-PCR), as described previously (38). The primers, synthesized by Integrated DNA Technologies (Coralville, IA), were as follows. RSV N gene, forward 5=-TGCAGGGCAAGTGATGTTAC-3=, and reverse, 5=-TTCCATTTCTGCTTGCACAC-3=; actin, forward, 5=-AGAAAATCTGGCACCACACC-3=, and reverse, 5=-GGGGTGTTGAAGGTCTCAAA-3=. A portion of the PCR sample was analyzed on 1.5% agarose...