1. ORF1 polypeptide associates with host translation factors and assembles a multiprotein complex.
ORF1 polypeptide associates with host translation factors and assembles a multiprotein complex. (A) Western blot analysis of pGBKT7 and pGBKT7 ORF1 expressing Y2H gold whole-cell extract using anti-Myc antibody (upper panel). A shorter-exposure image is shown to clarify the band at the 75-kDa position (lower panel). Single asterisks (*) and double asterisks (**) denote processed ORF1 fragments. (B) Western blot analysis of pGBKT7 and pGBKT7 ORF1 expressing Y2H gold whole-cell extract using anti-Flag antibody. Single crosshatch symbols (#) and double crosshatch symbols (##) denote processed ORF1 fragments. (C) Coimmunoprecipitation of ORF1-Flag-associated proteins in Huh7 cells using Flag agarose beads (pUNO and pUNO ORF1, lanes 1 and 2 from left). GST-ORF4 was added to the lysate prior to addition of Flag agarose beads. Aliquots of IP samples were subjected to Western blotting using the indicated antibodies. Data represent results of a Flag-agarose pulldown assay of a mixture of in vitro-translated ORF1-Flag, GST-ORF4, and Huh7 whole-cell extracts (pGBKT7 and pGBKT7 ORF1, lanes 3 and 4 from left). Aliquots of eluates were subjected to Western blotting using the indicated antibodies. An uppercase Greek phi (Φ) denotes a processed ORF1 fragment. (D) Western blot analysis of pGBKT7 and pGBKT7 ORF1 synthesized by TNT using anti-Flag antibody. An uppercase Greek phi (Φ) denotes the processed ORF1 fragment.
Chandru Subramani et al. mSystems 2018; doi: /mSystems