Hacia los Ayuntamientos y Asociaciones

Transcription specific suppression of translation can be mediated by the binding of miRNAs to the 3'UTRs of target mRNAs (Eulalio et al., 2008). As one means of investigating whether the suppression of IRF-1 translation during WNV infection is mediated by miRNAs, tC3H/He MEFs were transfected with nonspecific or dicer-specific siRNAs and 24 h later, the cells were infected with WNV Eg101 at a MOI of 5. In cells transfected with scrambled siRNA, dicer protein levels were almost undetectable in mock-treated cells (Fig. 4.5). An increase in dicer levels was observed in cells infected for 8 h. Although the levels remained above those in mock-infected cells throughout the infection, they decreased by 24 h after infection. In cells transfected with dicer specific siRNAs, efficient knock down of dicer was observed in infected cells. Analysis of IRF-1 protein levels in cells transfected with nonspecific siRNAs showed efficient IRF-1 protein production upon IFN stimulation and less efficient IRF-1 production in WNV infected cells. A small increase in IRF-1 protein levels in infected cells transfected with specific siRNAs was observed suggesting that miRNAs might be involved in regulating IRF-1 translational suppression. Interestingly, higher IRF-1 protein levels were observed in mock-infected cells that were transfected with dicer-specific siRNAs than in mock-treated cells that were transfected with nonspecific siRNAs. This result suggests that basal expression of IRF-1 in unstimulated cells may be regulated by miRNAs.

DISCUSSION

Viruses depend entirely on the host translational machinery for synthesis of the viral proteins required for viral replication and the assembly of viral particles. Many viruses have evolved

mechanisms to control host protein synthesis in order to suppress the production of antiviral proteins. Since IRF-1 has demonstrated antiviral activity against WNV (Schoggins et al., 2011), it would be beneficial for the virus to suppress IRF-1 production. In this study analysis of the production of IRF-1 protein in transformed C3H/He cells confirmed the previously published observation that IRF-1 levels in WNV Eg101-infected primary C3H/He cells remained similar to those in mock-treated cells even though the Irf1 mRNA levels increased significantly in the infected cells. Since IRF-1 has a short half-life (about 30 m) (Watanabe et al., 1991), the levels of this protein would be expected to increase in the presence of the proteasomal inhibitor MG132. The levels of IRF-1 in cells treated with IFN beta and MG132 increased indicating that IFN beta induced increased translation of IRF-1. However, the levels of IRF-1 in mock-treated or WNV Eg101-infected cells increased only slightly after treatment with the inhibitor indicating that IRF-1 is constantly translated at a low basal level in control cells and that this does not change with infection.

Transcript specific suppression of translation can occur due to the binding of proteins to cis- acting elements in the 3'UTRs of target mRNAs (Kuersten and Goodwin, 2003; Mazumder et al., 2003; Abaza and Gebauer, 2008). In many instances, translational control is mediated by AREs located in the 3'UTR of mRNAs (Chen and Shyu, 1995). Analysis of the Irf1 3'UTR sequence with the ARE database program showed the presence of AREs and suggested the possibility that 3'UTR mediated regulation may be occurring in WNV Eg101-infected cells. Transcript specific translational inhibition can also be mediated by binding of miRNAs to regions of the 3'UTR of a target mRNA (Lytle et al., 2007). However, the Irf1 3'UTR did not suppress the translation of a reporter gene. The insertion of the Irf1 3'UTR downstream of a heterologous gene could have

altered mRNA folding which prevented protein binding to Irf1 3'UTR. Alternatively, the endogenous factors (proteins and/or miRNAs) may not be expressed at sufficient levels to suppress the translation of the overexpressed 3'UTRs. Additional experiments need to be done before the involvement of the Irf1 3'UTR in translational suppression can be ruled out.

Analysis of ISG mRNAs by real time qRT-PCR showed that the basal Irf1 mRNA levels in untreated cells are more than 100 fold higher than the mRNA levels of other ISGs including Oas1a, Oas1b and Irf7 (data not shown). The observation that relatively high basal levels of Irf1 mRNA are induced in untreated cells but protein levels are very low suggests the presence of a mechanism that tightly controls the amount of IRF-1 protein produced at basal levels. In contrast, when the cells are stimulated with type I IFN, Irf1 mRNA levels increase by tenfold or more and the translational suppression mechanism is overcome resulting in high IRF-1 protein levels. However, in the case of a WNV Eg101 infection, a similar increase in Irf1 mRNA levels does not result in relief of the translational suppression and low IRF-1 protein levels are observed. It appears that the WNV infection induces an increased suppression of IRF-1 translation to accommodate the increased Irf1 mRNA levels. One possibility is that Irf1 mRNA may be sequestered in P-bodies. It was previously shown that mRNAs with miRNAs bound to them are sequestered in P-bodies (Parker and Sheth, 2007). The preliminary results suggest that IRF-1 protein levels might be regulated by miRNAs. The observations that Irf1 mRNA levels in untreated cells are higher than those of other ISGs and that knock-down of dicer resulted in an increase in IRF-1 protein levels in mock-treated cells suggested that miRNAs might be involved in maintaining low basal levels of IRF-1. Since the use of siRNAs does not result in a complete

knock-down of expression, the experiments need to be repeated in cells that have dicer depleted through homologous recombination (Tan et al., 2009).

Although, it was previously reported that translation initiation can be also blocked through the binding of RNA binding proteins to cis-elements in the 5'UTR of a target message (Garfinkel and Katze, 1993; Damgaard and Lykke-Andersen, 2011), the required sites for this type of regulation were not predicted in the 5'UTR of Irf1. It is also unlikely that the translational suppression is mediated by miRNAs binding to the Irf1 5'UTR or ORF. Binding of miRNAs to these sites were shown to produce less effective silencing of translation (Lytle et al., 2007).

MATERIALS AND METHODS

4.6 Cells and viruses.

SV40-transformed tC3H/He (tC3H/He) MEFs were grown in minimal essential medium (MEM) supplemented with 5% fetal bovine serum (FCS) and 10 µg/ml gentamicin. The cells were grown at 37°C in a 5% CO2 atmosphere.

To prepare a lineage I WNV strain Eg101 stock, BHK cells were infected at a MOI of 0.1 and culture fluid was collected 32 h after infection. The virus was titered by plaque assay. The titer of the stock virus was ~ 1 x 108 PFU/ml.