Terminal V5 Tagging of All Four Newcastle Disease Virus Structural Proteins Is Compatible with Particle Formation in Sf9 Cells
TL;DR
Particles formed regardless of which structural protein carried the tag, including a variant in which all four proteins were tagged simultaneously, and under the conditions tested, 19-residue terminal insertions were compatible with NDVLP recovery at each of the four positions examined.
Abstract
Newcastle disease virus-like particles (NDVLPs) assemble from the matrix (M), nucleoprotein (NP), fusion (F) and hemagglutinin–neuraminidase (HN) proteins. Terminal epitope tags make these proteins easy to detect but may interfere with assembly, and it is not well-documented how much terminal modification the four proteins tolerate. Eight pFastBac Dual donor plasmids were used to generate recombinant baculoviruses, and twelve NDVLP variants were produced in Sf9 cells and purified by sucrose gradient ultracentrifugation. Particle-like structures were recovered from all twelve preparations. Western blotting confirmed production of the native and V5-tagged proteins and their co-purification in the particle fractions; transmission electron microscopy showed enveloped, roughly spherical particles with measured diameters of 55 to 150 nm (median 90 nm) in every preparation, similar in appearance to native NDV. Particles of a similar size were also present in the beta-glucuronidase control preparation, so the assignment of the particles rests on the protein composition of the fractions rather than on morphology alone. Particles formed regardless of which structural protein carried the tag, including a variant in which all four proteins were tagged simultaneously. Under the conditions tested, 19-residue terminal insertions were compatible with NDVLP recovery at each of the four positions examined.