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Figure 2 | Retrovirology

Figure 2

From: Balanced splicing at the Tat-specific HIV-1 3′ss A3 is critical for HIV-1 replication

Figure 2

ESE tat is required for activation of Tat-specific 3′ss A3. (A) 2.5 × 105 HEK293T cells were transiently transfected with each of the proviral plasmids. 48 h post transfection, total RNA was isolated from the cells and analyzed by RT-PCR using different sets of primer pairs (primer positions are shown in Figure 1A). HIV-1 mRNA species are indicated to the right of the gel images according to the nomenclature published previously [6]. (B) Real-time PCR assays to specifically quantitate the relative levels of tat-mRNAs (top) and all viral mRNAs (bottom). For normalization we monitored the total amount of cellular GAPDH present in every sample. Data represent expression ratios relative to that of wild-type pNL4-3 (bar 1). Values and error bars show the average ± standard deviation of three independent transfection experiments. (C) Left: Northern blot analysis of total RNA isolated from the same RNA preparation as in (A). A hybridization probe was used specifically detecting HIV-1 exon 7. Right: Quantification of Northern blot using RNAs from three independently performed transfection experiments. Data represent expression ratios relative to that of wild-type pNL4-3 (bar 1), which was set to 1. For normalization the ribosomal RNA amount of each sample was calculated. Values and error bars show the average ± standard deviation of three independent transfection experiments. (D) Western blot analysis of viral Gag and Tat expressed by wild-type reference and mutant provirus. Supernatants and lysates were probed with a primary antibody against HIV-1 p24gag or HIV-1 tat. Equal amounts of cell lysates were controlled by the detection of α-actin. (E) 2.5 × 105 HEK293T cells were transfected with 1 μg of proviral plasmids and 0.5 μg of pcDNA3.1(+) or SVctat expressing viral Tat protein from a cDNA. Western blot analysis was performed as described in (D).

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