dsRNA-Mediated Nitric Oxide Response Figure 6. IRF1 contributes to iNOS induction in response to HSV-1 in MEFs. (A) Knockdown of IRF1 by siRNA in IRF32/292/2 MEFs in the presence or absence of poly I:C treatment. Fold change in iNOS mRNA accumulation (B) and initiation of HSV-1gfp replication (C) in IRF32/292/2 MEFs treated with poly I:C in the presence or absence of siRNA against IRF1. *, 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.g006 62]. Furthermore, virus infection leads to the activation of NF-kB and IRF1 [63,64]. Here we have shown that regulation of iNOS expression is, at least in part, dependent on both NF-kB and IRF1. These data are consistent with other reports indicating that both transcription factors are important for iNOS expression in mice in response to dsRNA [39,65]. Our data do not exclude, however, the involvement of other transcription factors or the likelihood that NF-kB and IRF1 function in contributing to the antiviral response in another capacity. Overall, our data show that components of the nitric oxide pathway serve as alternative antiviral factors in the absence of the IRF3- and IFN-mediated signaling pathway, and that they also contribute to the antiviral response in WT cells. While we conclude that the nitric oxide pathway is important, we cannot rule out involvement of other pathways or factors. Furthermore, the factors involved in the antiviral response against VSV after 24 hours of poly I:C pretreatment [16] are still unknown. As levels of nitric oxide began to decline within 7.5 hours of treatment with poly I:C, it is unlikely that this pathway is involved in the antiviral response observed against VSV at this timepoint. These data emphasize the intricacies of the host response to different pathogens, and underscore the requirement of the host to have multiple strategies to counteract the immune evasion properties of viruses. Materials and Methods Ethics statement Human ethics approval was not required as neither human subjects nor primary human tissues were utilized. Production of primary mouse embryonic fibroblasts was completed with full approval from the McMaster Animal Review Ethics Board. All work with viruses was completed with full approval from the McMaster Presidential Biosafety Advisory Committee. Cells and Viruses Primary MEFs derived from WT C57Bl/6 and IRF32/ 2IRF92/2 mice [14] were maintained in alpha-minimal essential medium (MEM) supplemented with 15% fetal bovine serum (FBS), 100 UNml21 penicillin, 100 mgNml21 streptomycin and 2 mM L-glutamine. Experiments were performed with cells at passages four to eight. All cells were incubated at 37uC in a humidified 5% CO2 incubator as previously described [16]. Vero cells (ATCC) were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 5% FBS. HSV-1 (KOS strain) expressing green fluorescent protein (HSV-1gfp) [66] was propagated on Vero cells. For viral infections, cells were split and seeded into dishes 24 hours prior to infection. Infections with HSV-1gfp utilized a multiplicity of infection of 0.1 particle forming units (PFU) per cell and occurred in serum-free alpha-MEM for 1 hour. This amount of virus is the maximal dose for which signal saturation in untreated cells does not occur. Following 1 hour of infection, the viral inoculum was removed and replaced with DMEM containing 1% methylcellulose. GFP fluorescence inten- sity was measured on a Typhoon Trio (GE Healthcare) 24 hours later and quantified using ImageQuant TL software. PLoS ONE | www.plosone.org 9 February 2012 | Volume 7 | Issue 2 | e31688