Cross-Species Genome-Wide Analysis Reveals Molecular and Functional Diversity of the Unconventional Interferon-? Subtype

Shields, Lauren E.; Jennings, Jordan; Liu, Qinfang; Lee, Jinhwa; Ma, Wenjun; Blecha, Frank; Miller, Laura C.; Sang, Yongming

IFN peptides were expressed using two eukaryotic systems, a HEK293-mammalian expression system (Invitrogen) and a yeast expression system through collaboration with Kingfisher Biotech (St. Paul, MN). The molecular authenticity of IFN peptides expressed by both systems was verified with the following aspects: (1) gene sequences; (2) protein band pattern on protein gels; and (3) potential antiviral function tested in different cell-virus systems. It is noteworthy that IFN peptides expressed by both systems have been comparatively determined multiple times to demonstrate the duplicity as shown in several publications (9, 13, 14, 23, 24) . The antiviral activity of IFN peptides was tested on the cell/virus systems including: MARC-145/PRRSV, porcine M /PRRSV, A549/pH1N1, PK-15/pH1N1, Mode-K/WSN, NIH3T3/WSN, Mode-K/KS07, and NIH3T3/KS07, respectively. Briefly, cells were seeded in flatbottom 96-well plates and grown to >95% confluence. Cells were infected with PRRSV or influenza viruses as indicated, and treated with 1:10 serially diluted IFN peptides at 20-2 × 10 −10 ng/ml. Viral infection was determined by staining the cytopathic effect on cell monolayer with 1% crystal violet or immuno-staining of the viruses, and quantified with a SpectraMax i3 (Molecular Devices) spectrometer. Antiviral activity of IFNs was calculated using the Reed-Muench Method to normalize TCID 50 and expressed as U/µg/ml. One unit (U) is the highest dilution that reduced cell number by 50% (9, 10, 13).