Determination of Rev–RRE functional activity

All plasmids utilized in this study were given the notation pHRXXXX for easy identification. To measure Rev–RRE function, we utilized an HIV vector system that has been previously described.^20,38,39 This vector produces a genomic RNA whose nucleocytoplasmic export and packaging are dependent on Rev–RRE function. Thus the titer of the virus-vector particles produced from the system is a measure of Rev–RRE functional activity. Vector titer can be readily measured since the vector transduces target cells with a hygromycin resistance cassette.²⁰

The system utilized 293T cells transfected with five plasmids and rev and RRE sequences that were chemically synthesized (Integrated DNA Technologies). For testing the function of the different RREs, the original vector (pTR167 pHR1266)³⁸ was modified to contain an inactive RRE in the native position (pHR5096)⁴⁰ and the different RRE sequences to be tested were cloned into the Nef region.²⁰ The rev sequences were cloned into a CMV expression plasmid.¹⁹ HIV structural proteins were provided by the GPV-4xCTE (pHR3296) construct, which produces Gag and Pol in a Rev-independent manner.^41,42 Vesicular stomatitis virus G protein was provided by pMD-G (pHR2004)⁴³ and Tat was provided by pCMV–Tat (pHR136). A detailed list of the plasmids utilized in these assays is given in the Supplementary Table S1 (Supplementary Data are available online at www.liebertpub.com/aid).

The day before transfection, 3 × 10⁶ 293T/17 cells were seeded onto a 100 mm plate in medium containing Iscove's modified Dulbecco's medium, 10% bovine calf serum, and gentamicin. Using the calcium phosphate method,⁴⁴ the cells were transfected using 20 μg pTR167 (RRE−) (nef−) (selected RRE+), 15 μg GPV-4xCTE, 5 μg pMD-G, 1 μg CMV–Tat, and 1 μg CMV–Rev such that each producer cell culture would receive the appropriate combination of Rev and RRE sequences to be tested. Cell-free supernatant was harvested from the producer culture 72 h after transfection and serial dilutions of viral stocks were used to infect Hela cells using DEAE dextran. Beginning 2 days after infection, Hela cell cultures were maintained in hygromycin-containing medium (200 μg/ml) until background cells were killed and colonies were clearly visible. The hygromycin-resistant colonies were fixed and stained with crystal violet, and colonies were counted to determine titer.

The cell-free supernatant from the producer cell culture was also used to determine p24 production using an ELISA.⁴⁵ To calculate the relative Rev–RRE functional activity, the titer of vector as determined by hygromycin-resistant colony counting was normalized to the p24 production of the producer cell culture to account for variation in transfection efficiency. When necessary to compare functional activity levels between assays, colony counts were also normalized to the NL4-3 Rev–RRE cognate pair functional activity as measured by an assay performed on the same day.