dsRNA-Mediated Nitric Oxide Response response in cells that received poly I:C treatment with and without Bay 11-7082; *, p,0.05, **, p,0.01, ***, p,0.0001. Data are expressed as the mean of three replicates 6 SEM. doi:10.1371/journal.pone.0031688.g005 was assessed by qRT-PCR (Figure 6A). IRF1 transcript was significantly reduced in IRF32/292/2 MEFs treated with IRF1- specific siRNA compared with cells treated with transfection reagent DharmaFECT (DF) alone. Although treatment with poly I:C increased levels of IRF1, these levels were significantly decreased upon addition of IRF1-targeting siRNA. Quantitative RT-PCR analysis indicated that inhibition of IRF1 by siRNA significantly reduced iNOS transcript accumulation in comparison to control siRNA (Figure 6B). Furthermore, initiation of HSV-1 replication was significantly increased in poly I:C treated IRF32/292/2 MEFs in which IRF1 levels were decreased following siRNA treatment (Figure 6C). Taken together, these data suggest that both NF-kB and IRF1 contribute to iNOS induction and subsequent nitric oxide production in poly I:C- treated IRF32/292/2MEFs. Moreover, it is likely that these transcription factors function in additional pathways that contrib- ute to antiviral activity, including production of type I IFN in WT MEFs. Discussion Fibroblasts are well established as important effectors of the early innate immune response to pathogens [2–4] due to their rapid production of IFNb and other cytokines [5]. Many pathogens, however, such as large DNA viruses, are particularly adept at subverting the type I IFN system, rendering their immediate environment IFN system defective. Thus, it is unclear how effector cells such as fibroblasts respond to specific pathogen triggers to protect themselves from a viral infection. DsRNA, a by- product of all viral infections, is a potent inducer of type I IFN, both as an intracellular and extracellular molecule [40]. Indeed, early recognition of DNA virus infection, including that of HSV-1, is mediated in part by dsRNA sensors [41,42]. Although we previously found that fibroblasts can respond to dsRNA and protect themselves from subsequent virus infection in the absence of a type I IFN system, the mechanism of protection was unknown, as we failed to detect the production of any cytokines or chemokines [16]. Nitric oxide is an important cellular messenger involved in diverse physiological and pathological processes. Among its many properties, nitric oxide has potent antiviral and antibacterial activity. While macrophages are primary producers of nitric oxide in response to pathogens, nitric oxide production by dermal skin fibroblasts and rat embryonic fibroblasts has previously been shown to play a role in wound healing and host defense in response to bacterial PAMPs and inflammatory cytokines [43,44]. With regards to the viral PAMP dsRNA, studies have shown induction of iNOS in human astroglia and bronchial epithelial cells [45,46]. However, to our knowledge, there have been no reports of dsRNA eliciting a nitric oxide-mediated antiviral response in fibroblasts in the absence of type I IFN responses. We set out to study the mechanism by which an antiviral response against HSV-1 occurred in the absence of IRF3, IFN and ISGs, particularly since this response was earlier and more robust than the cellular response against VSV, a small RNA virus that is exquisitely sensitive to the host IFN response [16]. We determined that the antiviral factor responsible for controlling HSV-1 replication was a soluble factor. Coincidentally, nitric oxide acts as a soluble antiviral factor that is more potent against DNA viruses in comparison to RNA viruses. We found that nitric oxide is rapidly produced by MEFs in response to the dsRNA mimetic poly I:C, and serves to inhibit HSV-1 replication. It is unclear why the by-products of nitric oxide production appear to decline over time, as the Griess assay measures nitrite, one of two stable break down products of nitric oxide. However, recent studies show that both nitrate and nitrite can be recycled back into the nitric oxide pathway [47], which may explain this observation. The antiviral activity of poly I:C-induced nitric oxide was confirmed by the addition of nitric oxide to MEFs using DETA-NO. While we cannot directly measure nitric oxide in cellular supernatants, we found that inhibition of nitric oxide production in MEFs reduced the antiviral activity within supernatants, suggesting that either nitric oxide, or a product of the nitric oxide pathway, serves as a soluble antiviral factor. Of interest, in WT fibroblasts, nitric oxide is a contributor, albeit minor, to the antiviral response; the ability to make and respond to type I IFN likely constitutes the major antiviral activity in these cells. However, in IRF32/292/2 MEFs, the nitric oxide pathway appears to be the dominant antiviral pathway. These data suggest that nitric oxide is an important antiviral molecule in the absence of IFN, ISGs and other cytokines. Previous studies have shown that HSV-1 is susceptible to the effects of nitric oxide in vivo in mice and rats [33–35]. Although nitric oxide is synthesized during the host response to pathogen invasion, its precise role remains unclear. Despite its antiviral activity, nitric oxide is not always beneficial, as it can promote the pathogenesis of HSV-1 by damaging cells in host tissues, thus aiding infection [29,48]. It is unknown at this time whether the antiviral protection provided by nitric oxide in vivo is exerted in the form of cytotoxicity as a result of nitrative stress or by an alternative mechanism. In vitro, treatment of primary fibroblasts with dsRNA or DETA-NO does not elicit noticeable cytotoxic effects (data not shown), suggesting that the level of nitric oxide production that is sufficient to block virus replication is not linked to cytotoxicity. While it is well known that NO has antiviral activity, particularly against DNA viruses such as HSV and vaccinia virus [34,49,50], the mode of action remains to be fully elucidated. Thiol modification and protein nitrosylation are likely involved in the antiviral activity of NO [51]. Indeed, viral enzymes are an important target for NO [52,53]. Although NO displays antiviral activity against several RNA viruses, including influenza virus [54], its capacity to induce DNA damage [55] likely explains its preferential targeting of DNA viruses. We have found in this study the importance of iNOS as the enzyme by which nitric oxide is synthesized. This is not surprising, as iNOS induction in response to virus infection, as well as viral components, is well known [56–58]. During viral infection, nitric oxide production by iNOS is induced by cytokines such as IFN-c; however, virus infection can up-regulate iNOS independently of such cytokines [29]. While MEFs can respond to IFN-c, they do not make it in response to poly I:C (data not shown), suggesting that in fibroblasts, iNOS is induced independent of IFN-c. The iNOS gene locus has low homology between human, rat and mouse sequences. As a result, the transcription factors involved in iNOS induction are species and cell type dependent [39,59]. For example, regulation of nitric oxide production by iNOS in humans has been shown to be dependent on AP-1, but a binding site for this transcription factor is not present on the mouse iNOS promoter [59]. NF-kB and IRF1 are most commonly published as regulators of iNOS expression in various species [60– PLoS ONE | www.plosone.org 8 February 2012 | Volume 7 | Issue 2 | e31688