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Title: Depletion of elongation initiation factor 4E binding proteins by CRISPR/Cas9 genome editing enhances antiviral response in porcine cells

item RAMIREZ-CARVAJAL, LISBETH - Oak Ridge Institute For Science And Education (ORISE)
item SINGH, NEETU - Texas A&M University
item De Los Santos, Teresa
item Rodriguez, Luis
item Zhu, James
item LONG, CHARLES - Texas A&M University

Submitted to: Antiviral Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/9/2015
Publication Date: 11/22/2015
Citation: Ramirez-Carvajal, L., Singh, N., De Los Santos, T.B., Rodriguez, L.L., Zhu, J.J., Long, C.R. 2015. Depletion of elongation initiation factor 4E binding proteins by CRISPR/Cas9 genome editing enhances antiviral response in porcine cells. Antiviral Research. 125:8-13. doi: 10.1016/j.antiviral.2015.11.002.

Interpretive Summary: Interferons (IFNs) are proteins made by animals to fight viral infections. Interferon response in cells is regulated by numerous pathways that involve complex protein interactions. Studies in mice suggested that IFNs are regulated by proteins that bind elongation initiation factor 4E (4E-BPs) and control interferon regulatory factor 7 (IRF-7) translation. However, similar mechanisms have not been described in livestock species. Here, we studied the role of 4E-BPs in the regulation of the antiviral response of pigs. To do this we reduced or suppressed 4E-BP1 gene expression in pig cells and studied the effects on the antiviral response against two viruses of agricultural importance, vesicular stomatitis virus (VSV) and foot-and-mouth disease virus (FMDV). We found that pig cells lacking of 4E-BP1 gene had higher interferon activity and better response to viral infection. Our results verified that 4E-BPs contribute to the regulation of the response against viruses in pigs and future application of this knowledge will help to develop better antiviral strategies required to mitigate the adverse effects of viral diseases on animal health.

Technical Abstract: Type I interferons (IFN) are key mediators of the innate antiviral response in mammalian cells. Elongation initiation factor 4E binding proteins (4E-BPs) are translational controllers of interferon regulatory factor 7 (IRF7), the master regulator of IFN transcription. The role of 4EBPs in the negative translational regulation of IRF7 through its 5’untranslated region (UTR) has been shown in the murine model. However, differences in IRF7 translational regulation mechanisms are unknown in other species. Researchers investigated whether 4E-BP1 depletion alters the innate antiviral response in porcine cells. We found that partial 4E-BP1 or 4E-BP2 knockdown induces no antiviral response against vesicular stomatitis virus (VSV) or foot-and mouth disease virus (FMDV). In contrast, porcine cells depleted of 4E-BP1 gene (4E-BP1-/- cells) by a CRISPR/Cas9 system showed increased expression of interferon (IFN a and ß), IFN stimulated genes (ISGs) and significant reduction in VSV titer as compare to 4E-BP1+/+ cells. In addition, the 5’UTR of porcine IRF7 reduced a reporter expression independently of 4E-BP1 depletion or 4E-BPs dephosphorylation. These studies demonstrate the enhancement of the innate antiviral response in pig cells after 4E-BP1 depletion and suggest differences in the regulatory role of the 5’UTR of porcine IRF7 as compared to murine ortholog.