Construction of hRIP derivatives. Schematic representation of N- and C-terminally truncated and fluorescent hRIP derivatives. (ZnF) C₄H₂-type zinc finger domain; (S/TRR) serine/threonine-rich region; (FGM) phenylalanine-glycine motifs; (arrowhead) asparagine-proline-phenylalanine (NPF) motifs; (EGFP) enhanced green fluorescent protein; (WT) hRIP; (aa) amino acids.

A dominant-negative hRIP mutant interferes with Rev function. (A) A transient transfection assay for Rev function. The CAT reporter plasmid pCMV128 and pCMV β-gal were cotransfected with the Rev expression plasmid pcRev or pcRev and the indicated hRIP expression plasmid into Cos-1 cells. CAT and β-galactosidase enzyme activities were assayed as described in Zapp et al. (1993). (–) Absence; (+) presence; (WT) hRIP. A dominant-negative hRIP mutant interferes with Rev function. (B) The hRIPΔN360 mutant inhibits Rev function in a dose-dependent manner. Cos-1 cells were cotransfected with pCMV128, pcRev, pCMV β-gal, and increasing amounts of phRIPΔN360. Enzyme activities were assayed as in (A). (–) Absence; (+) presence.

The hRIPΔN360 mutant causes Rev-directed RNAs to mislocalize and aberrantly accumulate at the nuclear periphery. (A) The subgenomic HIV-1 tat expression plasmid (pgTAT) was cotransfected with pcRev or pcRev and the indicated hRIP expression plasmid into Cos-1 cells. Control transfections contained pgTAT alone, pgTAT and pCMV, pgTAT and pRevM10, or pgTAT and pΔCAN. The intracellular distribution of tat RNA was analyzed by in situ hybridization using a Cy3-labeled RRE-specific probe. (B) The U6-RRE expression plasmid (pU6RRE) was cotransfected with pcRev, or pcRev and phRIPΔN360 into Cos-1 cells. Control transfections contained pCMV alone or pU6RRE and pCMV. The intracellular distribution of U6RRE RNA was analyzed as described in A.

Rev-directed RNAs accumulate on the outside of the nucleus in the presence of the hRIPΔN360 mutant. Cos-1 cells were transfected with the indicated plasmids and RNA localization analyzed by fluorescent in situ hybridization as in the previous experiment (Fluorescence, left column) and differential interference contrast (DIC) microscopy (Nomarski, right panel). (Control panels) No DAPI, no probe; (DAPI) (+) DAPI; (Cy3) (+) probe; (merge) (+) DAPI and probe.

The hRIPΔN360 mutant specifically inhibits Rev-directed RNA export. (A) The hRIPΔN360 mutant does not affect the intracellular distribution of NLS- or NES-containing proteins or total poly(A)⁺ mRNA in mammalian cells. Cos-1 cells were transfected with the indicated EGFP-fusion reporter plasmid in the absence or presence of phRIPΔN360 (first and second rows, left and center panels). HeLa cells were transfected with phRIPΔN360 and the intracellular distribution of poly(A)⁺ mRNA was analyzed by in situ hybridization using an Alexa Green-labeled oligo dT₅₂ probe (third row, left and center panels). The intracellular distribution of hRIPΔN360 was analyzed by indirect immunofluorescence microscopy using anti-HA (primary) and Cy3-labeled anti-mouse (secondary) antibodies (right panels). (B) The hRIPΔN360 mutant is localized at the nuclear periphery in mammalian cells. Cos-1 cells were transfected with phRIPΔN360 and analyzed by indirect immunofluorescence as in A (Fluorescence, left panel) and DIC microscopy (Nomarski, center and right panels). (C) The hRIPΔN360 mutant does not affect the intracellular distribution of MLV envelope glycoproteins in mammalian cells. HeLa cells were transfected with an MLV-envelope expression plasmid (pSV-A-MLV-env) alone (first row) or cotransfected with phRIPΔN360-EGFP (second row). The intracellular distribution of nascent MLV envelope glycoproteins was analyzed by indirect immunofluorescence microscopy using anti-MLV (primary) and anti-rat Alexa Red (secondary) antibodies. (D) The hRIPΔN360 mutant does not affect constitutive secretion of SEAP in mammalian cells. 293-T cells were cotransfected with the indicated plasmid in the presence of pCMV β-gal. Untransfected cells were incubated with Brefeldin A (BFA) as a positive control for SEAP inhibition. Endogenous SEAP activity (left panel) and β-galactosidase expression (right panel) were monitored using standard enzyme assays. SEAP secretion levels were quantified as the ratio of SEAP activity in culture supernatants to the sum of SEAP activity in both the culture supernatants and cell lysates.

Ablation of hRIP activity inhibits HIV Rev function by mislocalizing Rev-directed RNAs to the nuclear periphery. (A) Depletion of cellular hRIP using RNA interference. HeLa cells were transfected with hRIP-specific (H1 or H2) or mutant siRNA duplexes (M1 or M2) using Oligofectamine. Control cells were transfected with Oligofectamine alone (no siRNA). hRIP and Lamin A/C levels in cell lysates prepared from the bulk culture were analyzed by Western blotting using goat anti-hRIP or anti-Lamin A/C polyclonal antibodies, respectively, and visualized by ECL. (B) Ablation of hRIP activity inhibits Rev function in mammalian cells. HeLa cells treated with the indicated siRNA duplexes were cotransfected with pCMV128, pcREV, and pCMV β-gal. CAT and β-galactosidase enzyme activities were assayed as in Figure 2,Figure 2. (1X) 50 nM; (2X) 100 nM; (–) absence; (+) presence. (C) Rev-directed RNAs mislocalize and aberrantly accumulate at the nuclear periphery of hRIP-depleted cells. HeLa cells treated with siRNA M1 or H1 were cotransfected with pgTAT and pcRev and RNA localization analyzed as in Figure 3,Figure 3.