Fig. 1. HIV-1 Rev inhibits the nuclear export of IFN-1 mRNA in a NES-dependent manner. (A) Rev NES-dependent inhibition of IFN-1 gene expression. HeLa cells were cotransfected with various amounts of either pCG-HA-Rev (open circles), pCG-HA-Rev^27-29A (closed circles) or pCG-HA-RevM10 (open triangles), and 1 µg of phuIFN-1 for 16 hours at 37°C. pCG-HA, the empty parental vector, was included to normalise the amounts of the vector-based expression plasmids. pUC12 was included to give a total DNA amount of 20 µg. 32 hours after the DNA addition, the culture supernatants were collected to quantify the IFN-1 secreted from the transfected cells as described in the Materials and Methods. Values are presented as the percentage IFN-1 concentration relative to that secreted from phuIFN-1 alone transfected HeLa cells. Values of a representative experiment of three independent transfection experiments are shown; mean±s.e.m. of triplicate samples. (Bars cannot be seen when they are smaller than the graph symbols.) (B) The top western blot shows the plasmid dosage-dependent increase in HIV-1 Rev protein expression. The bottom western blot indicates the comparable amounts of Rev and Rev mutant proteins expressed in HeLa cells transfected with the corresponding expression plasmids (16 µg each). (C) Coexpression of Rev results in a NES-dependent reduction in the cytoplasmic IFN-1 mRNA levels, but an increase in the nuclear IFN-1 mRNA levels. HeLa cells were cotransfected with pCG-HA-Rev, pCG-HA-Rev^27-29A or pCG-HA-RevM10, and phuIFN-1. Cytoplasmic and nuclear total RNAs were collected at 32 hours after the DNA addition and subjected to RT-PCR analysis as described in the Materials and Methods. The cDNA synthesis reaction mixtures were appropriately diluted to ensure linear amplification of the cDNAs in the PCR reactions. The ratio of each IFN-1 transcript level normalised to the corresponding GAPDH transcript level in the presence of Rev proteins, to that in the absence of Rev proteins, is shown below each lane. C, mock-transfected control. The sizes of the amplicons of the IFN-1 and GAPDH transcripts are indicated. The autoradiographs shown were obtained after 3 hours of exposure at –70°C. The results are from a representative experiment of three independent experiments.