FIG. 9.

Quantification of nuclear and cytoplasmic levels of HIV-1 reporter RNA by RPA. HIV-1 Gag reporter plasmids were transfected into 293 cells. At 48 h posttransfection, nuclear and cytoplasmic RNAs were isolated and treated with DNase, and 15 μg of nuclear or 25 μg of cytoplasmic RNA was subjected to RPA with the HIV-1 5′ UTR and a GAPDH RNA probe. Transcripts were subjected to polyacrylamide gel electrophoresis, and RNA signals were quantified by phosphorimager analysis. Undigested probes are labeled in italics, lanes are labeled with the reporter RNA, and RNase protection products are indicated on the right. (A) RPA of nuclear (N) and cytoplasmic (C) RNA from 293 cells transfected with pSVgagpolrre in the absence (−) or presence (+) of Rev. (B) RPA of nuclear (N) and cytoplasmic (C) RNA from 293 cells transfected with wild-type SNV RU5 (ABC) and selected mutants (6). (C) Summary of cytoplasmic gag RNA and Gag protein expressed from SNV RU5 and selected mutants. The RNA signals were quantified from the RPA by ImageQuant version 4.2 software (Molecular Dynamics) and normalized to GAPDH RNA levels. P.I., phosphorimager units. Gag levels were measured by ELISA. <MD, less than minimum detectable levels.