The plasmid encoding anti-viral-sense of MP-12 S-segment at the downstream of the T7 promoter, pProT7-S(+), was described previously [18] . Serial deletion of 75 bp (25 aa.) was introduced into the NSs open reading frame (ORF) of pProT7-S(+) by sitedirected mutagenesis with Pfu Turbo DNA polymerase (Stratagene), designated as pProT7-S(+)-NSD6-30, NSD31-55, NSD56-80, NSD81-105, NSD106-130, NSD131-155, NSD156-180, NSD181-205, NSD206-230 or NSD231-248, respectively. For C-terminus mutant, the PCR fragment encoding NSs ORF with C-terminus 51 bp (17 aa.) deletion was amplified, and cloned between HpaI and SpeI of pProT7-S(+) [18] , designated as NSD249-265. The alanine substitutions of NSs-E253-255A/ D257-259A, NSs-E253-255A or NSsD257-259A were made onto pProT7-S(+) plasmid by site-directed mutagenesis with Pfu Ultra (Agilent Technologies), and designated as pProT7-NSs-E253-255A/D257-259A, NSs-E253-255A or NSsD257-259A, respectively. NSs ORF of those NSs mutants were amplified by PCR with Phusion High Fidelity DNA polymerase (New England Biolab), and cloned into pcDNA3.1mycHisA (Invitrogen) between KpnI and XhoI, and designated as pcDNA3.1mycHisA-NSD6- were transfected with those plasmids as described previously [18] .