dsRNA-Mediated Nitric Oxide Response Figure 1. A soluble factor confers resistance to HSV-1 replication in IRF32/292/2 MEFs. (A) MEFs were directly treated with 8.5 nM poly IC for 6 hours, or with supernatants from poly IC-treated cells, and subsequently challenged with HSV-1gfp. Initiation of HSV-1 replication, as measured by GFP fluorescence, is displayed relative to untreated (mock), infected cells, where fluorescence in each experiment was set to 100%. (B) A standard curve of poly I:C in culture medium was generated. (C) The absorbance of supernatants collected after 6 hours of mock and poly I:C treatment were collected and absorbance was compared to determine the amount of residual poly I:C within supernatants of treated cells. *, p,0.05, **, p,0.01, ***, p,0.0001. Data are expressed as the mean 6 SEM; n = 3. doi:10.1371/journal.pone.0031688.g001 shown to be a result of iNOS induction in response to viruses and viral components [29]. We used qRT-PCR to measure the levels of eNOS and iNOS in WT and IRF32/292/2 MEFs. nNOS was not determined as this isoform is relatively restricted to neuronal cells [29]. While eNOS was not detected in either cell type, iNOS was basally detected in untreated cells to levels of approximately 0.3-fold in comparison to the housekeeping gene, Gapdh (data not shown). Transcript levels of iNOS increased in both WT and IRF32/292/2MEFs upon treatment with poly I:C within 2 hours (Figure 2E). Induction further increased after 5 hours of poly I:C treatment, coincident with the timeframe in which protection against HSV-1 was observed. Levels of iNOS transcript were reduced to baseline measurements within 7.5 hours of poly I:C treatment (data not shown). The classic iNOS inhibitor aminoguanidine hydrochloride (AMG) was used to determine the involvement of iNOS in the antiviral response observed against HSV-1 in IRF32/292/ 2MEFs. Based on conflicting reports on the degree of isozyme selectivity of AMG [36,37], N6-(1-iminoethyl)-L-lysine dihydro- chloride (L-NIL), a compound more selective in iNOS inhibition [38], was also used. The efficacy of AMG and L-NIL as inhibitors of iNOS in poly I:C-treated WT (Figure 3A) and IRF32/292/2 (Figure 3B) MEFs was determined; there was a statistically significant reduction in nitric oxide production, as determined PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e31688