4)

4). replicons demonstrate higher degrees of heterologous protein synthesis, which correlates with the increase of SG RNA production. Infected cells were metabolically labeled with [35S]methionine at 7 h postinfection (for details). Fragments of the same gel are presented. Radioactivity in the GFP- and Luc-containing bands was measured on Storm phosphorimager. The data were normalized to the radioactivity in the Isatoribine GFP and Luc bands detected in the VEErep/GFP and VEErep/Luc/GFP samples, respectively. There were noticeable differences in the cytopathogenicity of the designed replicons in all of the cell types that were used. Replicons expressing mutated capsid protein were poorly cytopathic and demonstrated only a cell growth arrest, LAP18 whereas the VEErep/DI-Ubi-GFP and VEErep/DI-IRES-GFP replicons induced very strong morphological changes of the cells within 12C16 h postinfection. For the VEErep/DI-Ubi-GFP replicon, the abnormally high intracellular concentration of Ubi most likely had a deleterious effect on cell biology. We also noticed that the level of heterologous protein expression mediated by the designed constructs was specific to the cell type. Most of the constructs produced less GFP in HEK293, Vero, and National Institutes of Health (NIH) 3T3 cells than they did in BHK-21 cells (Fig. S2). Isatoribine However, the VEErep/DI-Cm-GFP was always more productive than a standard VEErep/GFP, suggesting that in the previous studies, the entire potential of VEEV replicons in terms of expression of heterologous proteins was not entirely exploited. Higher Expression Level of Heterologous Genes Is Not Specific to GFP. To rule out the possibility that the increased level of protein expression is specific to GFP, we also designed DI replicons expressing a firefly luciferase (Fig. 2and Fig. S3). The designed replicons demonstrated an even more profound increase in heterologous protein (Luc) expression. At 20 h postinfection, the DI replicons, but not the control VEErep/Luc/GFP, produced quantities of luciferase that were readily detectable on Coomassie blue-stained gels (Fig. S3). Accordingly, at 24 h postinfection, the activity of luciferase in VEErep/GFP/DI-Cm-LucCinfected cells was 50-fold higher than in cells infected with VEErep/GFP/Luc (Fig. S3). DI Replicons Are Capable of Replication of Subgenomic RNA. Next we attempted to directly demonstrate that presence of RNA replication promoter elements had a positive impact on subgenomic RNA synthesis. In all cases, the DI RNA-encoding replicons produced their SG RNAs at levels higher than those found in the cells infected with replicons with a standard SG RNA design (Figs. 2and ?and3),3), (see control VEErep/GFP and VEErep/Luc/GFP). The stimulatory effect of the new 5 promoter sequences on DI SG RNA synthesis was especially evident in the case of Luc-encoding constructs; the DI SG RNAs were synthesized 14- to 24-fold more efficiently than those Isatoribine transcribed from another SG promoter and having natural 5 UTR. Because both SG RNA promoters were identical, the detected differences in SG RNA synthesis were most likely the result of additional replication of the SG DI RNAs. Moreover, in the Isatoribine cells infected with capsid-expressing replicons VEErep/DI-Cm-GFP and VEErep/DI-Cm-Luc/GFP (Fig. 3), the [3H]-labeled replicon genomic and SG RNAs were found at higher concentrations. Intracellular Accumulation of Heterologous Proteins Is Not Exclusively Determined by Translation Efficiency. The final levels of heterologous protein accumulation could be dependent not only on the efficiency of their synthesis, but also on the cytopathic effect (CPE)-associated changes in the intracellular environment, the degradation rates of replicon RNAs, and individual proteins and other parameters. To directly compare the rates of GFP and Luc translation, we metabolically labeled cells infected with different DI replicons with [35S]methionine at 7 h postinfection. The results presented in Fig. 4, demonstrate that GFP and Luc syntheses were particularly efficient in case of Ubi- and FMDV 2A-dependent constructs. At 7 h postinfection, these replicons produced heterologous proteins at rates 30-fold higher than standard VEErep/GFP and VEErep/GFP/Luc (Fig. 4). The Cm-dependent cassettes also synthesized 10- to-20-fold higher levels of the protein of interest. As shown in Fig. 2 and Figs. S2 and S3, the latter constructs were very productive in long-term expression, suggesting that high levels.