Fig 6.The viral PLSCR1-NP interaction is competitively by ILDR1. (A.B)No interaction of ILDR1 with viral NP as determined by the co-IP assays. The interaction between the ILDR1 Cter domain and the NP of SIV was validated using the co-IP assays.HEK293T cells were transfected with ILDR1and NP protein. The Myc-tagged PLSCR1 plasmids were used as positive controls. All cell lysates were prepared at 24 h post transfection and proteins were immunoprecipitated using an anti-Myc mouse MAb, or anti-EGFP rabbit MAb. The immunoprecipitated proteins were analyzed by Western blotting.(C) HEK293T cells were transfected with Myc-NP (0.5μg) , PLSCR1-pmCherry (0.5μg) and different concentrations of EGFP-ILDR1 cytoplasmic fragment (0μg,0.5μg,1μg,1.5μg,2μg) .The cell lysates were prepared, and proteins were immunoprecipitated using an anti-PLSCR1 rabbit PAb. The expression levels of the ILDR1 and NP protein after immunoprecipitation relative to that of GAPDH were analyzed by densitometry(D). (E) HEK293T cells were transfected with EGFP-ILDR1 cytoplasmic fragment (0.5μg) , PLSCR1-pmCherry (0.5μg) and different concentrations of Myc-NP (0μg,0.5μg,1μg,1.5μg,2μg) .The cell lysates were prepared, and proteins were immunoprecipitated using an anti-PLSCR1 rabbit PAb. The expression levels of the ILDR1 and NP protein after immunoprecipitation relative to that of GAPDH were analyzed by densitometry(F).(G) 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). Each fraction was subjected to western blotting with corresponding antibody for protein detection.(H) Model of PLSCR1-NP interaction is competitively by ILDR1.ILDR1 located in the cytoplasm, when in the presence of PLSCR1, ILDR1 and PLSCR1 co-translocates into the nuclei . PLSCR1 could prevent the nuclear import of NP protein and ILDR1 could bind to PLSCR1competitively with NP. So in the nuclei ,PLSCR1 inhibit the expression of ILDR1 to reduce NP protein synthesis to inhibit virus replication .