ILDR1 competes with NP in binding PLSCR1
We transfected either ILDR1-Cter or PLSCR1 together with NP protein in HEK 293T cells and examined their potential interaction by performing co-IP experiments. The results show that Myc-tagged PLSCR1 but not ILDR1-Cter is co-immunoprecipitated with EGFP-tagged NP protein (Figure 6A). Likewise, EGFP-tagged PLSCR1 but not ILDR1-Cter is co-immunoprecipitated with Myc-tagged NP (Figure 6B). We then moved on to examine whether ILDR1 and NP could interact with PLSCR1 simultaneously or compete with each other. We examined the interaction between NP and PLSCR1 in the presence of ILDR1 of increasing amounts. The results show that when the amount of ILDR1 is increased, the level of NP bound to PLSCR1 is decreased (Figures 6C and 6D). Similar results were obtained when increasing amounts of NP was used (Figures 6E and 6F).
In order to further verify that ILDR1 and PLSCR1 will affect the nuclear importation of NP, a cell fractionation experiment was conducted. Cells were transduced with ILDR1-GFP ,PLSCR1-Myc or empty retrovirus-transduced control for 24h, and then infected with SIV at an MOI of 0.1. At 6 h p.i., the cells were separated into nuclear (N) and cytoplasmic fractions (C) ,then subjected to western blotting. The results show that, GAPDH and LaminB1 were only detected in the cytoplasm and nucleus, respectively. Also we found that NP was primarily detected in the cytoplasm and was only weakly detected in the nucleus while PLSCR1 was overexpressed as reported previously[21]. In contrast, when we overexpressed ILDR1, it will promote the expression of NP protein into the nucleus. When ILDR1 and PLSCR1 were co-transfected, a considerable amount of NP was detected in both the nucleus and the cytoplasm (Figures 6G). Taken together, our present data suggest that ILDR1 and NP compete with each other in binding PLSCR1.