Fig 7.

Engineering infectious FLAG-tagged murine noroviruses. (A) The FLAG epitope tag was inserted into sites in NS4 and VP2. (B) Infectious virus was recovered from NS4-FLAG and VP2-FLAG using the DNA-based reverse genetics system. Significance was tested using one-way analysis of variance and Dunnett's multiple-comparison posttest to compare each insertion virus to the WT control, n = 3. **, P < 0.01. (C) Detection of the FLAG-tagged proteins in infected cell lysates by Western blotting. Detection with an anti-FLAG antibody gave proteins at the expected molecular mass for NS4 and VP2. *, a nonspecific protein with the same molecular mass as NS7 is detected by the anti-NS7 antibody. (D) Immunoprecipitation of NS4 and VP2 via the FLAG tag. RAW264.7 cells were infected with either NS4-FLAG and VP2-FLAG at a low MOI, at 36 hpi cell lysates were prepared, and anti-FLAG agarose was used to immunoprecipitate NS4 and VP2, respectively, as confirmed by Western blot analysis. NS1-2 was coimmunoprecipitated with NS4 only. Lanes M, molecular mass markers.