Figure 3

HIV-1 Rev/RRE and cis elements in the 5'UTR cooperatively enhance RNA encapsidation into HIV-1 viral particles. A. Vector RNA was measured by qRT-PCR and expressed in arbitrary units (AU). RNA levels for all graphs 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 in vector RNA for MLV/HIV RU5PS (1.9 fold), MLV/HIV RRE + PS (17 fold) and MLV/HIV RRE + RU5 (7.7 fold) are not indicated on the graph. B. Cytoplasmic RNA was isolated from vector producer 293T cells at the time of vector harvesting. Relative RNA levels were obtained and recorded as done for vector RNA in part A. C. Efficiency of encapsidating RNA into HIV-1 viral particles is expressed as a ratio of vector RNA in viral particles to cytoplasmic RNA available for encapsidation. Relative levels are expressed like vector RNA in part A. Fold increases for MLV/HIV RU5PS (1.2 fold), MLV/HIV RRE + PS (6.7 fold) and MLV/HIV RRE + RU5 (3.9 fold) are not indicated on the graph. D. Northern blot analysis of cytoplasmic and vector RNA isolated from MLV and MLV/HIV RRE in the absence (-) and presence (+) of Rev. Vector length RNA species were detected with a GFP labeled probe, as well as an additional RNA species (labeled GFP) generated from the internal CMV promoter. E. Northern blot analysis of cytoplasmic and vector RNA isolated from MLV/HIV RU5PS and MLV/HIV RRE + RU5PS in the absence (-) and presence (+) of Rev. Vector length RNA species were detected with a probe to a region in the 5' end of the vector, as well as an additional RNA species (labeled 'partial vector RNA'). Cytoplasmic and vector RNAs are shown at different exposures of the same blot. Last lane (far right) is a shorter exposure of adjacent left lane. Error for all bar graphs is expressed as ±S.D. All experiments were performed in triplicate.