production of, or responsiveness to, either of the three classes of IFN increase susceptibility to mycobacterial and viral infection [9]. Viral infection of mice deficient for IFNb or type I IFN signaling is typically a lethal event, even under low multiplicity infection conditions [10–13]. Similarly, in the absence of IRF3, mice are more susceptible to virus infection due to a 20–50 fold reduction in type I IFN expression [14]. However, infection of mouse embryonic fibroblasts (MEFs) deficient for IRF3 with Newcastle disease virus induced a number of IRF3-independent direct response genes, including several p200 family proteins [15]. We have also observed IFN and ISG induction and a subsequent antiviral response to long dsRNA molecules independent of IRF3 [16]. Alternatively, ISG induction and antiviral protection can be independent of IFN due to IRF3 binding directly to the promoter of a subset of ISGs [17]. Despite the presence of multiple pathways, the type I IFN system dominates the antiviral response in non-hematopoietic cells such as fibroblasts. To ensure their success, however, viruses such as the highly successful human herpes simplex virus (HSV)-1 inactivate IRF3 [18–21] and subvert the type I IFN response [22]. In fact, over 200 anti-IRF3 and anti-IFN mechanisms encoded by diverse viruses have been identified [23–25]. In general, DNA viruses are particularly adept at subverting the type I IFN system, particularly the large DNA viruses that encode multiple immune response modifiers. Given the importance of IRF3 and type I IFN in protection against virus infection, it is likely that all viruses encode mechanisms to disable these proteins. Accordingly, there are compensatory mechanisms to protect the host in the event that either of these crucial proteins is compromised. While there have been numerous studies examining either the IRF3-independent or the IFN-independent antiviral response, until recently, it was unknown if the host could be protected if both IRF3 and IFN were absent. We have previously observed a protective response against both DNA and RNA viruses in the absence of IRF3 and IRF9 in primary MEFs [16]. In response to dsRNA, MEFs deficient for IRF3 and IRF9 fail to induce other IRFs, IFN or ISGs, suggesting that the antiviral response observed in these cells is independent of the type I IFN system [16]. In the absence of IFN and ISGs, there is approximately 60–90% inhibition of vesicular stomatitis virus (VSV) and HSV-1 replication following poly I:C stimulation. Interestingly, the earlier and more potent response occurs against HSV-1 [16], which is well known for its ability to subvert the host response in comparison to VSV, which is highly susceptible to the effects of type I IFN [26–28]. Given the importance of the type I IFN system as the primary antiviral defence mechanism in non-hematopoietic cells, it is unknown how these cells protect themselves in the absence of this system. There are other innate molecules induced by viral infection that play a role in the host antiviral response. One such molecule is nitric oxide. Endogenously produced nitric oxide has many biological functions including smooth muscle relaxation and neurotransmission [29]. This molecule also plays an important antimicrobial role against numerous pathogens [30–32]. Although the antibacterial effects of nitric oxide are well appreciated, nitric oxide is also effective in the clearance of viruses, particularly DNA viruses [33–35]; these effects can be independent of IFN and ISGs. Nitric oxide is synthesized by nitric oxide synthase (NOS) of which there are three known isoforms, endothelial (e), neuronal (n) and inducible (i) [29]. In the context of protection against invading pathogens, the inducible nitric oxide (iNOS) is mainly associated with phagocytes and other immune cells following induction of tumor necrosis factor-alpha and IFN-c. Here, we have investigated the potential role played by nitric oxide in the antiviral response dsRNA-Mediated Nitric Oxide Response observed against HSV-1 in MEFs in the absence of IFN production and signaling. We found that in MEFs, nitric oxide is made by iNOS in response to dsRNA and plays an important role in the antiviral state observed in the absence of IRF3 and IFN production. Results Poly I:C induces production of nitric oxide in the absence of IRF3 and IRF9 We previously showed that IRF32/292/2MEFs, which fail to make or respond to type I IFN, induce an antiviral response against HSV-1 and VSV following treatment with poly I:C. The antiviral response against HSV-1 in these cells was found to occur earlier and was more potent than the response against VSV [16]. To determine whether this protection is conferred by a soluble factor, supernatants from poly I:C-treated monolayers were transferred to na¨ıve monolayers. The transferred supernatants were able to significantly limit initiation of HSV-1 replication on na¨ıve monolayers, as indicated by the decrease in GFP fluorescence following challenge of monolayers with HSV-1gfp (Figure 1A). We routinely monitor GFP fluorescence as an indicator of initiation of virus replication [16]; in IRF32/292/2MEFs, treatment with poly I:C subsequently decreases HSV-1 infectious virus production by ,15 fold (data not shown). To confirm that residual poly I:C was not responsible for the protective effects, we measured the level of poly I:C in the supernatants, relative to a standard curve of poly I:C in medium (Figure 1B). The absorbance of poly I:C-treated supernatants was only slightly higher than poly I:C deficient (mock) supernatants (Figure 1C); moreover, poly I:C concentrations within this low range do not confer resistance to HSV-1 infection in IRF32/292/ 2MEFs [16]. Thus, the soluble factor present within the supernatants was not residual poly I:C. As nitric oxide is a soluble factor and an important modulator of protection against DNA viruses such as HSV-1, we sought to determine if this molecule is involved in the antiviral response in the absence of IRF3 and IRF9. It was found via Griess assay, which measures the nitric oxide by-product nitrite, that these cells make nitric oxide within 2 hours of stimulation with poly I:C. Nitric oxide production in both wild type (WT) and IRF32/292/2MEFs peaked within 5 hours of treatment with poly I:C (Figure 2A). To confirm that nitric oxide production in IRF32/292/2 MEFs decreased initiation of replication of HSV-1, varying concentra- tions of nitric oxide were added to untreated cells with the use of the nitric oxide donor, diethylenetriamineNONOate (DETA-NO). DETA-NO and the control compound diethylenetriamine (DETA) were added to cells at concentrations ranging from 0– 200 mM. It was determined by Griess assay that DETA-NO used between 50–100 mM produced nitric oxide to levels similar to those observed in MEFs after treatment with 8.5 nM poly I:C for 5 hours (Figure 2B). A concentration-dependent effect on initiation of HSV-1 replication in these cells was observed in both WT (Figure 2C) and IRF32/292/2 (Figure 2D) MEFs. Between 50–200 mM DETA-NO was able to limit initiation of HSV-1 replication in WT and IRF32/292/2 MEFs. At the higher concentrations, a greater effect of DETA-NO was observed in IRF32/292/2MEFs. In each case, the control reagent DETA did not significantly contribute to nitric oxide production or affect initiation of HSV-1 replication. Induction of iNOS is important to the antiviral response in IRF32/292/2 MEFs Nitric oxide can be made by eNOS, nNOS, or iNOS depending on the stimulus and the cell type. Generally, synthesis has been PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e31688