supernatants can limit initiation of HSV-1 replication upon treatment of na¨ıve cells. To our knowledge, there is no assay that directly measures nitric oxide within supernatants, in vitro. Thus, to determine whether nitric oxide or a component(s) of the nitric oxide pathway is responsible for protection of na¨ıve monolayers following supernatant transfer, we transferred supernatants from cells treated with dsRNA in the presence or absence of the iNOS inhibitor L-NIL. We observed partial (Figure 4A) or complete (Figure 4B) restoration of initiation of HSV-1 replication following transfer of supernatants from cells treated with poly IC in the presence of L-NIL, relative to transfer of supernatants from cells treated with poly IC in the absence of an iNOS inhibitor. Consistent with the ability of WT MEFs to produce IFN and ISGs in response to dsRNA, only partial restoration of HSV-1 replication initiation was observed in these cells. NF-kB- and IRF1-mediated induction of iNOS contributes to protection against HSV-1 in the absence of IRF3 and IRF9 While the transcription factors NF-kB and IRF1 bind to the iNOS promoter to induce its transcription, these factors can signal dsRNA-Mediated Nitric Oxide Response independently of one another [39]. To confirm the role of NF-kB in the antiviral response observed in the absence of IRF3 and IFN, NF-kB was blocked using the inhibitor Bay 11-7082, which targets the phosphorylation of IkBa. Concentrations of Bay 11-7082 ranging from 0 mM–10 mM were tested and 5 mM was determined to be the optimal concentration to inhibit the nuclear translocation of NF-kB following treatment of MEFs with poly I:C (Figure 5A). Inhibition of NF-kB significantly decreased the fold change in iNOS transcript expression as determined by qRT-PCR in WT and IRF32/292/2 MEFs (Figure 5B, 5C). In an antiviral assay, inhibition of NF-kB with Bay 11-7082 resulted in increased initiation of HSV-1 replication in poly I:C treated WT and IRF32/292/2 MEFs (Figure 5D, 5E); the increase in initiation of virus replication was found to be significant in IRF32/292/2 MEFs. To assess the importance of IRF1 in iNOS induction and the antiviral response in the absence of IRF3 and IFN, an IRF1- targeting siRNA was generated. The control siRNA was a scrambled sequence of the IRF1 siRNA used to account for nonspecific effects. The efficacy of siRNA on IRF1 transcript levels Figure 4. A component of the nitric oxide pathway provides antiviral protection in supernatants from dsRNA-treated MEFs. WT (A) or IRF32/292/2 (B) MEFs were subjected to the indicated treatment, and initiation of HSV-1gfp replication was subsequently assessed 24 hours following virus challenge, as a measure of GFP fluorescence. *, p,0.05, **, p,0.01, ***, p,0.0001. Data are expressed as the mean of three replicates 6 SEM, relative to mock treated samples, which were set at 100% in each experiment. doi:10.1371/journal.pone.0031688.g004 PLoS ONE | www.plosone.org 6 February 2012 | Volume 7 | Issue 2 | e31688