HIV-1 Rev/RRE system and cis elements in the 5'UTR augment vector titers. A. Full-length MLV/HIV chimeric vector RNAs are expressed from a CMV (cytomegalovirus) promoter in transfected 293T cells. MLV and HIV cis elements can be distinguished by black underscore. Chimeric vector names are represented as MLV/HIV followed by corresponding HIV cis elements incorporated: RRE (Rev Response Element), R (repeat), U5 (unique region 5), PS (packaging signal comprised of ψ [canonical packaging signal and into 5' Gag region]), cPPT (central polypurine tract), PBS (primer binding site). Also incorporated are the WPRE (woodchuck hepatitis virus post-transcriptional regulatory element), FLuc (firefly luciferase gene), and GFP (green fluorescent protein gene). HIV-1 Gag-Pol 4X CTE helper construct was used to express structural and enzymatic proteins to generate viral particles independent of HIV-1 Rev protein. B. Vector titers normalized to p24 are shown in the absence (white bars) and presence (black bars) of Rev. The influence of adding HIV-1 cis elements to the MLV vector is indicated by fold increases in the presence of Rev relative to the standard MLV vector. Fold increases for MLV/HIV RRE + PS (38 fold) and MLV/HIV RRE + RU5 (5 fold) are not indicated on the graph. C. Luciferase levels normalized to total protein are shown for each vector. D. Titers expressed as a ratio to luciferase are shown as arbitrary units (AU). Fold increases for MLV/HIV RRE + PS (22 fold) and MLV/HIV RRE + RU5 (4 fold) are not indicated on the graph. Error for all bar graphs is expressed as ±S.D. All experiments were performed in triplicate.