Steroid-receptor fusion of the human immunodeficiency virus type 1 Rev transactivator: mapping cryptic functions of the arginine-rich motif.

Abstract

The human immunodeficiency virus type 1 (HIV-1) transactivator Rev is a nuclear protein that regulates expression of certain HIV-1 transcripts by binding to an RNA target element (the RRIE) present in these transcripts. A short arginine-rich sequence in Rev contains the signals required to direct this protein into nuclei, where it associates preferentially with nucleoli. We created a steroid-inducible transactivator by fusing Rev with the steroid-binding domain of the glucocorticoid receptor (GR). This Rev/GR protein remains inactive in the cytoplasm when steroids are absent, but it enters the nucleus and initiates transactivatlon within minutes after exposure to dexamethasone. Although the GR moiety is sufficient to transport Rev/GR into nuclei, mutation of certain residues in the arginine-rich region blocks nucleolar localization and also inhibits transactivation. We find that other mutations in this region, however, can abolish the function of Rev/GR without affecting its localization; the latter phenotype may reflect a specific defect in binding of the RRE.The rev gene of human immunodeficiency virus type 1 (HIV-1) encodes a 116-amino acid regulatory protein that transactivates the expression of other essential HIV-1 genes (1, 2). Localized in the nuclei of infected cells, where it associates preferentially with nucleoli (3), Rev acts at the posttranscriptional level by inducing the export of incompletely spliced viral mRNAs to the cytoplasm (1, 2, 4-8). The mechanism ofthis transactivation is not fully understood, but it is thought to depend on the binding of Rev protein to an array of RNA hairpin loops (termed the Rev response element, RRE) situated within the target HIV-1 transcripts (9)(10)(11)(12)(13)(14).The signals that direct Rev protein into nuclei are contained in a short basic region of the protein (approximately amino acids 25-50) that is rich in arginine residues (15, 16). Mutations in this basic region inhibit the nuclear accumulation of Rev and produce a concomitant defect in transactivation, implying that Rev must enter the nucleus to exert its effects. Although recent reports suggest that this region also harbors sequences that target Rev to nucleoli (17, 18) or participate in binding of the RRE (19), it has been difficult to evaluate these activities by conventional mutagenic analysis in vivo because of the need to maintain efficient nuclear translocation. We now describe studies of a Rev fusion protein whose entry into the nucleus is controlled by sequences from the glucocorticoid receptor (GR). This Rev/GR protein exhibits steroid-dependent Rev activity and thus provides the basis for an inducible system in which to study the dynamics of Rev transactivation. Moreover, because the GR moiety is sufficient to direct this protein into nuclei, mutational analysis ofRev/GR has enabled us to evaluate the functions of the basic region independently of any effect on nuclear translocation. Our findings illuminate the critical and complex role of this region in the localization and function ...

Structural and functional analysis of the avian leukemia virus constitutive transport element

“…The CTE identified in DR2 of the PrC strain of ASV was previously localized to between nt 8770 and 8925 (Ogert et al+, 1996)+ To confirm that a functional CTE is also present in the ALV, which contain only a single DR, we subcloned the equivalent sequence from a previously described RAV-2 ALV isolate (Bizub et al+, 1984) into the indicator construct pDM128/PL (Fridell et al+, 1993)+ This 159-bp sequence extends 3 bp 39 and 156 bp 59 to the 59 boundary of the ALV LTR and is 68% identical to the equivalent DR2 sequence in the PrC strain of ASV+ As previously described (Hope et al+, 1990), the pDM128 indicator plasmid contains a 59 and a 39 splice site, derived from the HIV-1 env gene, flanking an intronic cat indicator gene+ pDM128/PL additionally contains a polylinker between the cat gene and the 39 splice site, thus permitting the introduction of sequences of interest, such as the ALV CTE or, as previously described, the MPMV CTE or the HIV-1 RRE (Fridell et al+, 1993;Bogerd et al+, 1998)+ Because the cat gene is located between functional 59 and 39 splice sites, CAT enzyme expression is dependent on the nuclear export and translation of an unspliced mRNA, a process that, for the reasons described above, is normally very inefficient+ However, the introduction of an active CTE or of the HIV-1 RRE in cis (in the latter case in the presence of Rev) results in the efficient nuclear export of this unspliced mRNA and, hence, in a marked induction in CAT expression+ We therefore asked whether introduction of the putative ALV CTE would exert a similar effect+ Initially, we transfected the quail cell line QCl-3, which is permissive for RAV-2 replication (Cullen et al+, 1983), FIGURE 1. Schematic representation of the genomic organization of ALV and ASV+ The ASVs, such as Rous Sarcoma Virus, arose because of the recombinational acquisition of the host-cell-derived src gene by ALVs+ This resulted in a duplication of the 39 noncoding sequence located between the env gene and the LTR in the ALVs and this duplicated sequence was hence referred to as the direct repeat+ SD: splice donor; SA: splice acceptor+ with the parental pDM128/PL construct, with derivatives containing the ALV CTE sequence in the sense or anti-sense orientation, or with a pDM128/PL derivative containing the MPMV CTE+ As shown in Figure 2, the sense orientation ALV CTE potently activated CAT expression from the pDM128 indicator plasmid, whereas the same ALV sequence in the anti-sense orientation had no detectable effect+ As previously described , the MPMV CTE is not active in quail cells and the pDM128 construct containing this CTE was therefore also essentially inactive+ Whereas the MPMV CTE is active in human but not quail cells, the PrC ASV CTE was initially reported to be active in avian cells but not primate cells (Ogert et al+, 1996)+ To examine the species tropism of the ALV CTE, we compared the activity of these same four indicator plasmids upon transfection into the human cell line 293T, the simian cell line COS, or the murine L cell line+ As may be observed (Fig+ 2), both the ALV CTE and the MPMV CTE proved to be equivalently active in all three of these distinct cell types+ Therefore, based on this limited analysis, the ALV CTE does not display any obvious species tropism+ In fact, recent unpublished experiments utilizing the PrC ASV CTE demonstrate that this CTE is also active in hu...…”

“…The following expression plasmids have been previously described: the mammalian expression plasmids pBC12/CMV, pBC12/CMV/⌬CAN, pBC12/CMV/b-Gal, and pcRev (Malim et al+, 1991;Bogerd et al+, 1998); a derivative of the pDM128/ CMV indicator plasmid containing an intronic polylinker (pDM128/PL) (Hope et al+, 1990;Fridell et al+, 1993); and derivatives containing the full-length HIV-1 RRE or MPMV CTE (Bogerd et al+, 1998)+ The 159-bp, full-length ALV CTE, as well as truncated derivatives, were PCR amplified from a RAV-2 ALV isolate (Bizub et al+, 1984) and inserted into pDM128/PL at the polylinker Bgl II and KpnI sites to generate pDM128/ALV-CTE and the pDM128/SLIϩII, pDM128SLIIϩIII and pDM128/SLII constructs+ Point mutations of the ALV CTE were generated by using the QuickChange site-directed mutagenesis kit (Stratagene) on the parental pDM128/ALV-CTE construct+ The SLII fragment of the ALV CTE was PCR amplified and inserted into pDM128/SLII at the KpnI site to generate pDM128/2XSLII+ A pBluescript/SLII construct was cut with XbaI and ClaI, and the resultant SLII DNA fragment was inserted into pDM128/2XSLII to generate pDM128/3XSLII+ The pBluescript/ALV-CTE plasmid was constructed by inserting the full-length ALV CTE into the BamHI site of the polylinker region+ All constructs were sequenced to verify the correct orientation and sequence+…”

HIV Rev-dependent binding of SF2/ASF to the Rev response element: Possible role in Rev-mediated inhibition of HIV RNA splicing

“…3A) (33). Transcripts produced by pDM128 contain a single intron containing the HIV-1 RRE and the CAT coding sequence, which is excised when it is spliced (33). Cells transfected with pDM128 alone express only spliced transcripts and yield minimal levels of CAT activity; however, when Rev is cotransfected with pDM128, high levels of CAT enzyme are expressed (Fig.…”

“…To test this hypothesis, the SF2͞ASF cDNA was cloned into the pCMV4 eukaryotic expression plasmid and cotransfected into CV1 cells together with the pcRev and the Rev-dependent CAT-reporter plasmid pDM128 derived from the env region of HIV (Fig. 3A) (33). Transcripts produced by pDM128 contain a single intron containing the HIV-1 RRE and the CAT coding sequence, which is excised when it is spliced (33).…”

Tamoxifen-regulated adenoviral E1A chimeras for the control of tumor selective oncolytic adenovirus replication in vitro and in vivo

“…27,28 Binding of a functional ligand leads to an alteration in the structure of the HBD, which allows release of the chimera from hsp90, refolding to an active form and stabilization by dimerization of the HBD. 28,29 Several intracellular proteins have been rendered functionally hormone-dependent by fusing them with steroid receptors, among them are several viral and cellular transcription factors like E1A, 26,30 HIV Rev, 31 c-Myc 32 and v-Myb. 33 Previously it has been shown that fusion proteins of a mutated HBD of murine estrogen receptor (Mer) 34 or its human analogue (ER) 35 enable very tight regulation of the corresponding effector proteins in vitro and in vivo.…”