Fig 8.

Physical interaction between NS1 and NS4B. (A) Coimmunoprecipitation and Western blotting. BHK21-15 cells were mock infected or infected with WNV-WT at an MOI of 10. At 28 h after infection, cells were lysed and NS1 proteins were immunoprecipitated with anti-NS1 MAb and protein A-Sepharose. After extensive washing, beads were boiled in SDS sample buffer, and the eluate was electrophoresed and subjected to Western blotting with a polyclonal anti-NS4B antibody. (B to D) Mass spectrometry (MS) identifies an interaction between WNV NS1 and NS4B. (B) Workflow for the immunoaffinity isolation of NS1 protein complexes. (C) Summary of viral proteins identified by mass spectrometry (nLC-MS/MS using an LTQ Orbitrap Velos) as coisolated with NS1 in WNV-KUNV-infected cells. The number of unique peptides and identifications of boundary peptides that indicate mature forms are shown. Three viral proteins coisolated with NS1 and validated as mature proteins are shown in bold. M, molecular mass. (D) Representative collision-induced dissociation (CID) MS/MS spectrum of a tryptic peptide of NS4B demonstrating its association with WNV NS1.