Analysis of IAP Gag expression. (A) Schematic diagram of plasmid pL-MIA4 encoding the gag and pol open reading frames of MIA14 (57) and plasmids 3-MIA4ΔN without and 3-MIA4ΔN-CTE with CTE. The relative positions of the restriction sites used for the deletion constructs pL-MIA4ΔS and pL-MIA4ΔN, as well as the positions of the RNA elements, are indicated. (B) Immunoblot analysis of Cos-7 cells transiently transfected with plasmids pL-MIA4, pL-MIA4ΔS, and pL-MIA4ΔN (lanes 2, 3, and 4) or mock transfected (lane 1) (left blot) and with plasmids 3-MIA4ΔN (lane 5)and 3-MIA4ΔN-CTE (lane 6) (right blot). The cell lysates were analyzed with a polyclonal rabbit anti-IAP Gag serum, and the IAP Gag polyprotein is marked with arrowheads. The additional proteins migrating with higher mobility than the Gag polyprotein correspond to Gag cleavage products (lane 2) (57). (C) Northern blot analysis of Cos cells mock transfected (lane 1) or transfected with pL-MIA4 or pL-MIA4ΔN (lanes 2 and 3). Total RNA was subjected to Northern blot analysis, and IAP Gag-specific RNA was detected with a ³²P-labeled probe directed against the gag coding region, as depicted in panel A. The gag RNA is indicated on the right, and RNA standards (in nucleotides) are given on the left.

Sequence and predicted secondary structure of the IAPE. The sequence mediating Rev-independent HIV-1 Gag expression was subjected to computer prediction of folding potential using the Mulfold program. This analysis yielded a stable secondary structure corresponding to nt 5444 to 5810 of the MIA14 sequence (shown schematically at the upper right) originally published by Mietz et al. (39). Resequencing of this region showed seven deletions, one insertion, and one nucleotide exchange compared to the original sequence. The corrected sequence was subsequently subjected to folding analysis, yielding the depicted secondary-structure element of 360 nt (IAPE). The calculated free energy value for the proposed RNA structure of the IAPE is −95.8 kcal. A predicted splice donor (SD) within the element is marked. In addition, the BamHI and the SalI site within the element are shown (compare to Fig. 3A). Putative single-stranded regions that have sequence identity with the CTE of D-type retroviruses are circled (compare to inset), as well as a purine-rich single-stranded region. The inset depicts a schematic representation of the mapped secondary structure of the M-PMV CTE and the sequences of its single-stranded regions.

Analysis of Crm-1 dependence of Gag expression. (A) Schematic representation of a competitor protein for NES-dependent nuclear export (30, 49). The competitor corresponds to a fusion protein containing the NLS of the SV40 large T antigen and the NES of the human T-cell leukemia virus Rex protein lacking the RNA binding domain. This molecule is expected to continuously shuttle between the nucleus and cytoplasm and titrates limiting factors of the NES export pathway. A variant containing an inactivating mutation in the NES domain of the fusion protein (M90) was used as a control. (B) HeLa cells were transiently transfected with plasmid 3-RRE in the absence (−; lane 1) or in the presence (+; lanes 2 to 4) of Rev, with plasmid 3-CTE (lanes 5 to 7), or with plasmid 3-IAPE (lanes 8 to 10). Transfections were performed with or without cotransfected expression plasmids for the NLS-NES competitor (NES) or the M90 variant thereof, as indicated above each lane. Gag expression was determined by immunoblot analysis as described in the legend to Fig. 1. (C) Analysis of Crm-1 dependence of Gag expression using leptomycin B. Cos-7 cells were transiently transfected with plasmid 3-RRE in the absence (lanes 2 and 5) or in the presence (lanes 3 and 6) of Rev or with plasmid 3-IAPE (lanes 1 and 4). Transfection solution was left on the cells for 3 h and then replaced with fresh medium (lanes 1 to 3) or with medium containing 100 nM leptomycin B (lanes 4 to 6). The cells were harvested 16 h after addition of the drug and subsequently analyzed for HIV-1 Gag expression as described in the legend to Fig. 1.

Schematic representation of expression plasmids and analysis of HIV-1 Gag expression. (A) All expression plasmids contain part of the 5′ untranslated region of HIV-1 as well as the gag and PR coding regions. RNA elements derived from HIV-1 (RRE; pK-R-gpII) or from the IAP MIA14 (pK-M-gpII) were inserted 3′ of the coding region and are depicted as shaded (RRE) or solid (MIA14) boxes. At the top, the relative positions of the gag-PR region and of the RRE, as well as the coding exons for Rev, are highlighted in the HIV-1 genome. At the bottom, the relative position of the murine element is highlighted in the IAP genome. (B) Immunoblot analysis of transfected HeLa cells using antiserum against HIV-1 CA. Cells were transfected with plasmid pK-R-gpII (in the absence or presence of Rev [lanes 1 and 2]) or pK-gpII-M (containing the IAPE in sense or antisense [as.] orientation [lanes 3 and 4]). The cell lysates were normalized for transfection efficiency. The Gag polyprotein (Pr55) and CA are marked on the right; molecular mass standards (in kilodaltons) are given on the left. The additional proteins migrating between Pr55 and CA correspond to the Gag intermediate cleavage products MA-CA-NC and MA-CA.

Mapping of the MIA14 expression element. (A) Schematic representation depicting the results of a functional analysis of various subfragments derived from the MIA14 element. The relevant restriction sites used for cloning of the subfragments are indicated. All subfragments were inserted into pK-gpII, and transiently transfected cells were analyzed for HIV-1 Gag expression. At least three independent experiments were performed for each construct. The relative (rel.) fluorescence intensity of each fragment is scored on the right. Subregions mediating Rev-independent Gag expression are shown as a solid line; those yielding negative results are depicted as a dashed line. Insertion of a subfragment in reverse orientation is termed antisense (a.s.). The central darkly shaded region corresponds to the region defined by structure prediction analysis (Fig. 4) and termed the IAPE. The lightly shaded region corresponds to a nonoverlapping sequence mediating weak Rev-independent Gag expression. (B) Western blot analysis of HeLa cells transfected with pK-R-gpII, with (+) or without Rev (lanes 1 and 2), pK-gpII-M (lane 3), or pK-gpII-IAPE (lane 4). HIV-1-specific products and marker protein were detected as described in the legend to Fig. 1. (C) Northern blot analysis of nuclear (nuc.; lanes 1 to 3) and cytoplasmic (cyt.; lanes 4 to 6) RNA preparations from HeLa cells transfected with plasmids pK-R-gpII, without and with (+) Rev (lanes 1 and 2 and 4 and 5), or pK-gpII-IAPE (lanes 3 and 6). The blot was hybridized with a radiolabeled probe directed against the gag-PR region present in all constructs. The arrowheads point to specific gag RNAs. The difference in migration results from size differences of the corresponding elements. The additional RNAs of lower mobility correspond to products derived from transcriptional readthrough into the hygromycin resistance transcription unit located 3′ of the polyadenylation signal. The same blot was subsequently stripped and reanalyzed with a probe specific for the cellular housekeeping GAPDH gene (bottom).

Mutational analysis of IAPE function. (A) Schematic representation of the mutations introduced into the IAPE. The diagram on the left delineates the mutations introduced in order to generate mutant MI, which converts the predicted loops A1 and B2 into a (predicted) stable stem structure. The diagram on the right shows the mutations introduced to delete loop B1, which generated mutant DI. Folding prediction of mutant DI also displayed the disruption of loop A2 (data not shown). Compare to Fig. 4 for localization of the loops. wt, wild type. (B) HeLa cells were transiently transfected with pK-gpII derivatives containing the wild-type IAPE (lane 2) or deletion or substitution variants thereof. Variant DI (lane 3) is deleted in the B1 loop (compare Fig. 4), variant DII (lane 4) is deleted in the purine-rich loop (compare Fig. 4), and variant MI (lane 5) contains substitutions predicted to transform loops A1 and B2 (compare Fig. 4) into a stable RNA helix. Cell lysates normalized for transfection were analyzed by immunoblotting, as described in the legend to Fig. 1. (C) Northern blot analysis of RNA preparations derived from the same experiment shown in panel A. Total RNA was isolated from HeLa cells transiently transfected as indicated above each lane (+Rev, with Rev; a.s., antisense). HeLa cells transfected with pK-R-gpII and a Rev expression vector (lane 1) were analyzed as a control. The gag-specific RNA is marked on the right. Hybridization and detection were performed as described in the legend to Fig. 3C. The same blot was subsequently stripped and reanalyzed with a probe specific for GAPDH (bottom).

An expression element is present in the 3′ part of the pol open reading frame of primate foamy virus. (A) Schematic representation of the genome of foamy virus (top) showing the relative position of the region amplified by PCR (hatched box) and cloned in sense and antisense orientations into pK-gpII (bottom). LTR, long terminal repeat. (B) HeLa cells were mock transfected (lane 1) or transiently transfected with pK-gpII derivatives containing sequences derived from foamy virus (Fo; lanes 2 and 3) or the IAPE (lanes 4 and 5) either in sense (even lanes) or in antisense (as.) (odd lanes) orientation. HIV-1 Gag expression was determined by immunoblotting, as described in the legend to Fig. 1. (C) Northern blot analysis of RNA preparations derived from the same experiment shown in panel B. The numbering of lanes is identical to that in panel B. Total RNA was prepared from transfected cells, and gag-specific RNAs were detected using a radiolabeled probe hybridizing to the gag-PR region present in all constructs (depicted in panel A). The same blot was subsequently stripped and reanalyzed with a probe specific for GAPDH (bottom).