MVA expression of NiV proteins results in protein release. (A) Vero cells were infected with rMVAs expressing NiV N, M, F, or G, along with MVAGKT7 and then metabolically labeled. Lysates were immunoprecipitated with either rabbit anti-NiV polyclonal serum (N, M, G) or rabbit anti-F polyclonal serum. (B) Vero cells were infected with rMVAs and MVAGKT7 and metabolically labeled. Released protein was purified by centrifugation through a 10% sucrose cushion followed by flotation in a sucrose gradient. Proteins derived from cell lysates (L) or culture supernatants (S) were immunoprecipitated and analyzed as described above and in Methods. Lysate bands represent 1/20 of total lysate. (C) Supernatant from radiolabeled cells expressing N, M, F, and G was clarified and released protein was loaded onto a 5–45% sucrose gradient followed by centrifugation for 16 h as described in Methods. Fractions were obtained and proteins were analyzed by immunoprecipitation. A portion of each fraction was used to determine density.
Patch et al. Virology Journal 2007 4:1 doi:10.1186/1743-422X-4-1