Co-affinity precipitation studies were conducted with use of Strep-tagged protein purification to know if over-expressed truncated NSs can interact with MP-12 NSs in infected cells. 293 cells were infected with moi 3 of rMP-12-NSs-SF (recombinant virus tagged with tandem Strep-Tag and Flag) [19] and were then transfected using the in vitro synthesized capped RNA encoding each of the truncated NSs mutants. After 6 hours, newly synthesized host and viral proteins were labeled with [ 35 S] methionine/cysteine for 4 hours. Whole cell lysates were mixed with Strep-Tactin beads, and SF-tagged MP-12 NSs and bound host and viral proteins were precipitated. Presence of NSs bands were visualized with autoradiography ( Fig. 6 ). As expected, MP-12 NSs was co-precipitated with NSs-SF, and MP-12 NSs was migrating slightly faster than that of NSs-SF. The expression of truncated NSs was lower than that of MP-12 NSs (see input), while the expression of NSsD206-230 was not detectable. The same phenomenon was observed in a repeated experiment, suggesting the instability of NSsD206-230 expression in this experiment. On the other hand, we could not detect co-precipitation of any truncated NSs with NSs-SF. Collectively, our results suggest that those truncated NSs accumulates in cells at low level, mislocalizes, and do not interrupt the MP-12 NSs functions by co-expression.