Innate Immune Priming by cGAS as a Preparatory Countermeasure Against RNA Virus Infection

Michael T. Parker; Smita Gopinath; Corey E. Perez; Melissa M. Linehan; Jason M. Crawford; Akiko Iwasaki; Brett D. Lindenbach

In summary, we propose that cGAS may become activated in response to RNA 383 virus infection, such as by virus-induced mtDNA release, but also contributes to RNA 384 virus restriction via constitutive, low-level innate immune activation, likely via 385 recognition of endogenous DNA ligands (Fig. 8) . Pilot experiments showed that THP-1-derived macrophages were more 414 permissive for SINV-GFP than undifferentiated THP-1 monocytes. Differentiation of 415 . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/434027 doi: bioRxiv preprint THP-1 monocytes to macrophages was performed by plating cells at a concentration 416 of 5x10 5 cells/mL in fresh media and incubating for three days with 100 ng/mL 417 phorbol myristrate acetate (PMA; Invivogen). Adherent monolayers were washed 418 once with DPBS, dissociated with 0.05% trypsin/EDTA, resuspended in fresh media, 419 counted, and seeded for experimentation. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/434027 doi: bioRxiv preprint 4°C to remove insoluble material. Protein concentrations were quantified by using a 445 BCA protein assay kit (Thermo Scientific). Equal amounts of protein were separated 446 on 4-12% Bis-Tris Bolt SDS-PAGE gels (Thermo Scientific) and transferred to PVDF 447 membranes by using a Pierce Fast Semi-Dry Blotter. Immunoblotting was performed 448 by 30 minutes of blocking with either 5% milk (American Bio) or SuperBlock (Thermo 449 Scientific) followed by primary antibody and then secondary antibody (2 hours and 1 450 hour at room temperature, respectively), diluted in the same blocking solution. Blots 451