dsRNA-Mediated Nitric Oxide Response Figure 3. Nitric oxide made by iNOS is involved in the IRF3- and IFN-independent antiviral response. The efficacy of the iNOS inhibitors AMG and L-NIL to block nitric oxide production was determined by Griess assay following a 5 hour poly I:C treatment of WT (A) and IRF32/292/2 (B) MEFs. Initiation of HSV-1gfp replication in WT (C) and IRF32/292/2 (D) MEFs following treatment with poly I:C in the presence or absence of the iNOS inhibitors was measured relative to untreated cells, where fluorescence levels were set to 100% in each experiment. WT MEFs, which maintain the capacity to respond to IFN, are completely protected against HSV challenge following 24 hr pre-treatment with IFNb. *, 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.g003 by Griess assay, upon treatment with poly I:C in the presence of the inhibitors. Consistent with our hypothesis that dsRNA- mediated induction of nitric oxide limits initiation of HSV-1 replication, inhibition of iNOS with either AMG or L-NIL partially (Figure 3C) or fully (Figure 3D) restored initiation of HSV-1 replication in WT or IRF32/292/2MEFs, respectively. The partial effect observed in WT MEFs is consistent with the ability of dsRNA to elicit IFN and ISG induction within these cells [16]. For reference purposes, the effect of IFNb pre-treatment on limiting initiation of HSV-1 replication is shown (Figure 3C). Data presented in Figures 1 and 2 show that MEFs treated with dsRNA produce nitric oxide and that a soluble factor found within PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e31688