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. 2006 Aug;80(16):8089–8099. doi: 10.1128/JVI.00579-06

FIG. 4.

FIG. 4.

Effect of internal deletions in NSm on virus assembly, protein processing, and intracellular transport. (A) Production of virus-like particles. BSR-T7/5 cells were transfected with minigenome-component plasmids and either wt BUNV M segment cDNA or mutated NSm-containing plasmids as indicated. Controls included pTM1 instead of pTM1-BUNM, substitution of wt BUNV L segment cDNA with an inactive L cDNA mutant, and mock-infected cells. Supernatants from these cells were taken 24 h posttransfection and used to infect fresh BSR-T7/5 cells that had been transfected with BUNV L and N protein-expressing plasmids 5 h previously. In one case, the supernatant was reacted with anti-BUN antibody prior to infection. Renilla luciferase activity in all extracts of these cells was measured after 24 h and is shown in arbitrary light units. 1, pTM1 vector control; 2, pTM1-BUNM (wt control); 3, anti-BUNV serum-neutralized supernatant from pTM1-BUN-M-transfected cells; 4, pTM1-BUNM-NSmΔ1; 5, pTM1-BUNM-NSmΔ2; 6, pTM1-BUNM-NSmΔ3; 7, pTM1-BUNM-NSmΔ4; 8, pTM1-BUNM-NSmΔ5; 9, pTM1-BUNM-NSmΔ6; 10, pTM1-BUNMΔNSm; 11, substitution with inactive L mutant; 12, mock-transfected cells. (B) Processing of BUNV glycoproteins from Vero E6 cells transfected with wt or NSm deletion mutant cDNA clones. Vero E6 cells were infected with recombinant vaccinia virus vTF7-3, followed by transfection with pTM1-BUNM (wt) or NSm mutant cDNA constructs. Cells were labeled with [35S]methionine for 15 h, extracts were prepared, and viral proteins were immunoprecipitated with anti-BUN serum. The labeled glycoproteins were subjected to endo H (H) or mock digestion (C) and analyzed by SDS-10% PAGE under reducing conditions. The relevant portions of the gels are shown. (C) Intracellular localization of Gc expressed from wt and mutated NSm-containing M segment cDNAs. BSR-T7/5 cells were transfected cDNA constructs as indicated and were stained with a mixture of anti-Gc MAb 742 and anti-GM130 antibodies and 4′,6′-diamidino-2-phenylindole (DAPI). Cells were examined using the DeltaVision microscopy system (AppliedPrecision), and triple-stained images are shown. Gc proteins stain red, the Golgi stains green, and cell nuclei, stained with DAPI, are shown in blue.