Table II. Effects of NS gene products on NP localization and virus generation^a.

RNA polymerase I plasmid used	Protein(s) encoded by the RNA polymerase I plasmid	NP localizationb	Virus generationc
pPol-WSN-NS	NS1(wt), NS2(wt)	cytoplasm + nucleus	yes
No pPolI-WSN-NS	No NS1, No NS2	nucleus	no
pPolI-WSN-NSΔSplice	NS1(wt), No NS2	nucleus	no
pPolI-WSN-NS-STOP124	NS1(aa1–124)d, NS2(wt)	cytoplasm + nucleus	yes
pPolI-WSN-NS-STOPΔNES	NS1(aa1–124), NS2ΔNESe	nucleus	no

^aResults are from three independent experiments.

^b293T cells were transfected with protein expression plasmids for all viral structural proteins, the respective RNA polymerase I construct for NS vRNA synthesis and the remaining RNA polymerase I plasmids. Forty-eight hours after transfection of 293T cells, MDCK cells were infected with aliquots of supernatants from the former cells. Cells were fixed 24 h after infection and processed for indirect immunofluorescence assays, using antibodies against NP (see also Figure 4).

^c293T cells were transfected with the same set of plasmids as described above. At 48 h after transfection, virus in the culture supernatant was titrated in MDCK cells. ‘No’ indicates no virus generation, whereas ‘yes’ indicates ≥10⁴ TCID₅₀/ml.

^dNS1(aa1–124) indicates a truncated NS1 protein expressing only the N-terminal 124 amino acids.

^eNS2ΔNES indicates an altered NS2-NES (16-AxxAxA-21).