Fig. 3

Porcine HnRNP F selectively binds PRRSV GRS. a Sequence of PRRSV negative RNA showing putative GRS regions. 10 released PRRSV complete genomes were retrieved from the NCBI website (http://www.ncbi.nlm.nih.gov) and aligned. b Wild-type or mutant GRS oligonucleotide was biotinylated and incubated with lysate from HnRNP F transfected HEK293T cells. Streptavidin beads were added to pulldown RNA-bound proteins. The pulldown complexes were eluted and analyzed by western blotting with an anti-Flag antibody. 5% of whole cell extracts were used in reactions as input. c HEK293T cells were transfected with Flag-HnRNP F, and cell extracts were incubated with biotinylated GRS along with increasing amounts of unlabeled GRS. Streptavidin beads were added to pulldown RNA-bound proteins, followed by western blotting with anti-Flag antibody. d MARC-145 cells were infected with PRRSV (1.0 MOI) for 12 h. The cell lysates were immunoprecipitated with an anti-HnRNP F antibody, or rabbit IgG. RNA was extracted from the immunoprecipitants by protein A + G and subjected to RT-PCR analysis. e HEK293T cells seeded in six-well plate were transfected with His-Nsp7α (1 μg), along with Flag-HnRNP F at increasing concentrations (0, 1, and 3 μg). The cell lysates were immunoblotted using anti-His and anti-Flag antibody