FIG. 5.

The expression cassettes designed for expression of secreted forms of RVFV Gn and analysis of the Gn expression. (A) The hydrophobicity profile (Kyte-Doolitle) of the Gn protein. Fragments cloned into the VEEV structural polyprotein are indicated by black lines. (B) The schematic representation of the recombinant VEEV genomes, containing the insertions of the Gn-encoding sequences in the subgenomic RNA. The numbers correspond to the positions of the first and the last a.a. of the insertion in the polyprotein encoded by the RVFV M segment. The triangles indicate positions of engineered and natural furin-specific cleavage sites. Indicated viral titers and plaque size were derived from one of multiple, highly reproducible experiments. (C) Analysis of the stability of VEEV/C/spGn(opt)td⁻ during serial passaging in tissue culture. Virus was passaged three times in BHK-21 by infecting the monolayer of BHK-21 cells in 100-mm dishes with 100 μl of the stock harvested at the previous passage. For analysis of protein expression, BHK-21 cells were infected with harvested viruses and VEEV TC-83 at an MOI of 20 PFU/cell. At 16 h post infection, cells were labeled with [³⁵S]methionine, as described in Materials and Methods, and equal aliquots of proteins were analyzed by electrophoresis in SDS-10% polyacrylamide gels. The positions of VEEV-specific proteins and Gn-2A are indicated. Asterisks indicate the positions of incompletely processed products. (D) and (E) Analysis of proteins expressed by the recombinant viruses and secreted to the medium. Metabolic labeling of the proteins was performed as described in Materials and Methods. Viral particles (V) were pelleted by ultracentrifugation and the RVFV Gn proteins were isolated by immunoprecipitation. Samples were analyzed by electrophoresis in SDS-10% polyacrylamide gels, followed by autoradiography. The positions of proteins and molecular weight markers are indicated. The molecular weights of the expressed Gn protein fragments and its fusion forms, predicted based on the a.a. sequence, are indicated in the brackets. Gn239 and Gn318 contain one potential glucosylation site, Gn340-2A-E3, and Gn340-E3 have two potential sites.