Several families of  RNA viruses, such as Filoviridae, have evolved mechanisms that can inhibit normal immune surveillance and activation. These viruses pose a severe threat due to their recent outbreaks, possible use as biological weapons, and high mortality rate. The RIGI-like family of pattern-recognition receptors, MDA-5 and RIG-I, are key molecules which recognize and respond to 5'triphosphorylated viral RNA following infection. Recent evidence supports that the Filovirus Ebola can effectively evade immune detection through modified genomic RNA structure resulting in decreased mobilization of immune defenses, such as IFN-a/�. However, underlying mechanism by which Ebola is recognized via MDA-5 or RIG-I has yet to be fully defined. Given the essential role of these molecules in recognition of viral infection, we established shRNA knockdown cell lines to interrogate RIG-I family member signaling. The THP1 human monocytic cell line was chosen for immune modeling due to their undifferentiated state, viral infectivity and representation as a physiological relevant immune cell model for natural Ebola in-vivo infection.  Stable THP1 cell lines with either RIG-I or MDA-5 knockdown were generated through lentiviral transduction of shRNA targeting cassettes. qPCR analysis revealed a 60-80% reduction in RIG-I and MDA-5 expression. In accordance, immune activation in response to cytosolic dsRNA was impaired while exogenous TLR agonist signaling remained unaltered. Furthermore, RIG-I or MDA-5 deficient THP-1 monocytes maintained macrophage differentiation potential following stable selection. Collectively, these immune cell models may be utilized in not only Filovirus Ebola studies, but expanded on for analyzing Paramyxoviridae, Orthomyxoviridae, and Rhabdoviridae immune recognition in vivo.  The immune cell models may also lead to deeper understanding of the cross-talk that occurs between RIGI family receptors and agonist induced activation of immune response for viral drug treatment.

Generation of THP1 human monocytic cell lines with RIG-I or MDA-5 receptor knockdown for viral immune response modeling

247th National Meeting (2014)

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