FIG. 1.

Mammalian expression of the PRRSV GP₃ protein and analysis of its glycosylated nature. (A) Human 293 cells (10⁷) were infected by the recombinant Ad AdCMV5/ORF3 carrying the coding sequence of the PRRSV IAF-Klop strain ORF3 gene and pulse-labelled for 30 min with [³⁵S]methionine at 17 to 17.5 h p.i. Solubilized labelled proteins were subjected to radioimmunoprecipitation with α3 antiserum (anti-ORF3 product) and analyzed by SDS–12% PAGE. As a negative control, mock-infected 293 cells were processed similarly. For comparison, IAF-Klop-infected or mock-infected MARC-145 cells (10⁷) labelled for 30 min at 24 h p.i. were processed in parallel. The α3 monospecific antiserum immunoprecipitated a 42-kDa protein from both PRRSV-infected (lane 1) and AdCMV5/ORF3-infected (lane 3) cells. In the latter case, coprecipitation of the Ad hexon protein (110 kDa) was also observed. (B) α3 immunoprecipitates from IAF-Klop-infected MARC-145 cells (subjected to 30 min of labelling) were either treated with Endo H (lane 2) or Glyco F (lane 3) or left untreated (lane 1) and were then analyzed by SDS–12% PAGE. In lane M, the positions of [¹⁴C]Rainbow Marker standards (Amersham) are indicated (in kilodaltons). P3 refers to the unglycosylated precursor of GP₃.