2.1. NS5A Interacts with eEF1A
In the yeast two-hybrid screen, only one protein, eEF1A, was identified to be a potential binding protein for NS5A (Figure 1A). To confirm the interaction between NS5A and eEF1A, glutathione S-transferase (GST) pulldown assay was performed with the GST-tagged NS5A protein expressed in Escherichia coli and the Myc-tagged eEF1A protein expressed in HEK293T cells. The results showed that GST-NS5A but not GST interacts with eEF1A (Figure 1B). To further verify the interaction between NS5A and eEF1A, coimmunoprecipitation (Co-IP) assay was performed with HEK293T cells overexpressing 3×Flag-tagged NS5A and Myc-tagged eEF1A. After incubation with anti-Flag monoclonal antibody (MAb) and Protein G-Agarose, the Myc-tagged eEF1A was found to coprecipitate with the 3×Flag-tagged NS5A (Figure 1C). Furthermore, the 3×Flag-tagged NS5A was shown to coprecipitate with Myc-eEF1A when the cell lysate was incubated with anti-Myc MAb and Protein G-Agarose (Figure 1D). Considering that both eEF1A and NS5A show a strong affinity for nucleic acids, we investigated whether the NS5A–eEF1A interaction might be mediated through a nonspecific RNA bridge. The results showed that the NS5A–eEF1A interaction was not influenced by RNase treatment, indicating that the interaction is not due to nonspecific RNA-mediated binding (Figure 1E). To examine the colocalization of NS5A protein with eEF1A, the subcellular localization of 3×Flag-NS5A and Myc-eEF1A was examined by confocal microscopy. Both 3×Flag-NS5A and Myc-eEF1A were colocalized in the cytoplasm (Figure 1F). Taken together, the results demonstrated that eEF1A is an interacting partner of the CSFV NS5A protein.