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Proc. Nati. Acad. Sci. USA Vol. 91, pp. 8314-8318, August 1994 Biochemistry

A cis-acting repressive sequence that overlaps the Rev-responsive element of human immunodeficiency virus type 1 regulates nuclear retention of env mRNAs independently of known splice signals (AIDS/gpl6O/pre-mRNA

cedsng/Drosophila cels)

DAVID W. BRIGHTY AND MARTIN ROSENBERG Department of Gene Expression Sciences, SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406-0939

Communicated by Joan A. Steitz, March 29, 1994

While splice signals have been implicated as mediators of nuclear retention, nuclear sequestration and Rev responsiveness have also been observed for HIV-1-derived transcripts that lack functional splice sites (6-8, 15, 16); these observations challenge the notion that splice signals are the primary determinants of nuclear sequestration. Furthermore, a number of cis-acting sequences derived from various regions of HIV-1 have been shown to reduce expression of chimeric reporter genes (16-19), suggesting that such sequences may be responsible for the nuclear sequestration of HIV-1 mRNAs. These elements designated as cis-acting repressive sequences (CRS), or instability sequences, have been mapped to the gag, pol, and env regions of the HIV-1 genome. However, chimeric transcripts containing these negative elements retain some splice signal information, albeit nonfunctional, and consequently a role for splice signals in the nuclear retention mechanism cannot be ruled out. Expression of the HIV-1 envelope glycoprotein in transfected Drosophila melanogaster cells is also dependent upon coexpression of Rev, and this pattern of regulation directly reflects the Rev-mediated trans-activation observed in mammalian cells (20). Here we employ stably transfected Drosophila cells as a model system in which to examine the cis-acting sequences responsible for the nuclear retention of HIV-1 envelope mRNAs. We demonstrate that HIV-1 splice donor and acceptor sites are not required for nuclear retention of envelope-derived mRNAs or for Rev responsiveness. Furthermore, we provide evidence that a region of env that overlaps the RRE acts as a primary determinant of nuclear retention and restricted cytoplasmic accumulation for envelope mRNAs.

ABSTRACT The Rev protein of human immunodefidenc virus type 1 (HIV-1) binds to an RNA stuctre, the Revresponsive element (RRE), to enhance expression of the viral structural genes by relieving -the nuclear sequestration of incompletely spliced viral transcripts. It has been utd that nuclear retention of these mRNAs, in mam lian cells, Is due to the activity of either cis-acting repressive sequence elements or to inefficient splcig signals. Expression of the HIV-1 envelope gene in trnected Drosophila cells is also dependent upon Rev coexpression and, hence, the mechanism of nuclear retention and Rev regulation are highly conserved. Here we use the Drosophila system to identify a major cis-acting repressive sequence element that overlaps the RRE and is responsible for the nucear entrapment and Rev-dependent expression of IUV-1 env mRNAs. Moreover, the splice signals snning env are not required for nuclear retention or Revdependent trans-activation of env mRNAs. We suggest that the RRE and its assoclated RNA s ure are necesr for both the repressive and known trans-activation effects of Rev regulation. Human immunodeficiency virus type 1 (HIV-1) modulates viral gene expression such that early in infection only multiply spliced mRNA species accumulate within the cytoplasm of infected cells while unspliced and incompletely spliced viral mRNAs remain confined to the nucleus. Only late in infection do the incompletely spliced RNAs accumulate within the cytoplasm and express the viral structural proteins. This temporal regulation is mediated by the essential viral gene product Rev, a 116-aa nuclear phosphoprotein that demonstrates sequence-specific RNA-binding activity (1-5). Rev promotes the cytoplasmic accumulation and expression of incompletely spliced viral mRNAs and thus plays a pivotal role in the developmental pathway of HIV-1 (1, 6-10). The Rev-induced liberation of incompletely spliced transcripts from nuclear sequestration requires a cis-acting RNA target sequence known as the Rev-responsive element (RRE). The RRE adopts an extensive secondary structure and binds Rev both in vitro and in vivo (1, 5, 7, 11-13). It has been suggested that in the absence of Rev, HIV-1 splice donor and acceptor sequences function as important determinants of nuclear retention for viral mRNAs (14). It was proposed that binding of Rev to the RRE actively displaces splicing complexes from nascent transcripts, allowing export of the incompletely spliced mRNAs to the cytoplasm (14). Alternatively, binding of Rev to the RRE may direct viral transcripts through a nuclear export pathway, effectively removing these intron-containing mRNAs from the activity of the splicing machinery (8).

MATERIALS AND METHODS Plasmids. Vectors pMtRev, pMtl6OA32, and pMt120A32 have been described (20, 21). pDB160ASA was derived from pMt120A32 by replacing a Pvu II-Sac I env fragment with a Pvu IH-HindIII gp160 fragment from pMtl6OA32 and a HindIII-Sac I oligonucleotide linker introducing a stop codon immediately after nt 7721 (all numbering according to ref. 22). pDB41ASA was generated from pDB160ASA by replacing two gp120 Bgi II fragments with an oligonucleotide linker

(5'-GATCCGATACAGAGCAGGCCTCCGAGTCCGA-3';

and 5'-GATCTCGGACTCGGAGGCCTGCTCTGTATCG3'). An in-frame deletion of RRE sequences (deletion of nt 7383-7682 inclusive) was achieved by PCR overlap extension (ref. 23; details available upon request). The PCR product was used to replace the corresponding env region of pDB160ASA and pMt16OA32 to yield pDBARRE and pDB160ARRE, respectively.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviations: HIV-1, human immunodeficiency virus type 1- RRE, Rev-responsive element; CRS, cis-acting repressive sequence. 8314

Cell Culture. The stable transfection of D. melanogaster S2 cells has been described (24). Transfections contained 10 pig of the env expression vector, 5 pg ofpMtRev, and 1 pg of the hygromycin selection vector pCOhygro (pBR322 DNA was used to bring DNA to 20 pg and was substituted for pMtRev in Rev-deficient cell lines). The metallothionein promoter was induced as described (24). RNA Analysis. Nuclear and cytoplasmic RNA fractions were prepared as described (20). Subsequently, poly(A)+ mRNA was selected using oligo(dT)-cellulose chromatography. RNA samples from 1.5 x 106 cells were analyzed by Northern blotting, and RNAs were probed with a 32P-labeled Stu I-HindIII env fragment as described (20). Protein Analysis. Western blot analysis of HIV-1 proteins was as described (20, 25). Bound proteins were probed, using 1:3000 or 1:6000 dilutions of primary antisera, and detected by enhanced chemiluminescence (ECL; Amersham).

RESULTS Unspliced Transcripts Express gpl60 (gpl20/41), but Not gpl20, in a Rev-Dependent Fashion. Generation of stably transfected Drosophila cell lines expressing a variety of HIV-1 gpl20 constructs, including pMtl2OA32 (Fig. 1), resulted in production of gpl20 protein, which was secreted from the cells and accumulated in the culture medium (refs. 25 and 26; Fig. 2A). In contrast, an essentially identical construct, pMtl60A32, containing the gpl60 (gpl20/41) coding information (Fig. 1) resulted in no expression of envelope proteins (ref. 20; Fig. 2A). Expression of envelope (gp120/41) proteins from pMt160A32 was dependent upon coexpression of the viral rev gene product (Fig. 2 A and B). Examination of nuclear and cytoplasmic RNA fractions from these cell lines indicated that the inability of the gp160 construct to express envelope in the absence of Rev was due to the failure of these cells to accumulate env mRNA in the cytoplasm (Fig. 2C; ref. 20). While gpl60 mRNA could be found in the nuclear fractions, either in the presence or absence of Rev expression, accumulation within the cytoplasm required Rev. Northern blot analysis also shows a 1.4-kb RNA, which is derived from the 5' end of our envelope constructs due to HIV- 1 Genome

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Proc. Natl. Acad. Sci. USA 91 (1994)

Biochemistry: Brighty and Rosenberg

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FIG. 1. The HIV-1 genome is shown illustrating the major genes and the splice donor (S.D.) and acceptor (S.A.) sites; the plasmid constructs are shown below. LTR, long terminal repeat. PMtU, Drosophila metallothionein promoter. SV40 poly(A), simian virus 40 early polyadenylylation signal. t-PA (speckled rectangle), signal sequence from human tissue plasminogen activator. Open rectangle, env sequences; thin line, vector sequences. The bars indicate the regions of env present in each deletion construct; nucleotide coordinates (according to ref. 23) are shown.

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FIG. 2. Expression of HIV-1 gpl60, but not gpl20, is Revdependent in Drosophila cells. (A) Cells transfected with pMtl60A32 (gp160) or pMtl20A32 (gpl20) in the presence (+) or absence (-) of pMtRev were assayed for envelope protein expression by Western analysis of culture supernatants using anti-gpl20 polyclonal antisera; gpl20 is indicated (solid chevron); a band migrating below gpl20 is a nonspecific cross-reacting protein present in the fetal bovine serum used to supplement the growth medium (compare with Figs. 3 and 5). Molecular size markers (in kDa) are shown. (B) Cell lysates were examined for expression of cell-associated gpl60 (solid chevron) and gp4l (solid arrowhead) proteins by Western blot analysis using an anti-gp4l antibody. (C) Northern analysis of poly(A)+ nuclear (Nucl.) and cytoplasmic (Cyto.) mRNA fractions from these cells was performed with an env-specific probe. Full-length env mRNAs are indicated (solid chevron). A truncated 1.4-kb env mRNA (open arrow) is also produced (see text). Markers (in kb; BRL) are shown.

recognition and use of an inefficient polyadenylylation signal within the gpl20 coding sequences (D.W.B., unpublished data). This Rev-independent RNA serves as an internal control for monitoring both the Rev-dependent effects seen with the larger gpl60 transcript (Fig. 2C) and RNA loading. Since both the gp120 and truncated RNA locate to the cytoplasm independently of Rev function, we conclude that the gpl20 coding region does not contain any CRS elements recognized in Drosophila cells. Thus, the CRS element conferring selective nuclear retention upon our gpl60 construct must be located within the gp4l coding sequences of pMtl60A32. Moreover, the dramatic Rev dependence (>100fold; see Fig. 2A) that occurs in this system appears to function on a transcription unit that undergoes no splicing. Deletion of the Major Splice Acceptor Site in gp4l Does Not Overcome the Requirement for REV. Our data suggested that sequences within the gp4l coding region were responsible for the inhibitory effect on the cytoplasmic accumulation of gpl60 mRNA and, in turn, the Rev dependence of envelope expression. A previous report has suggested that the major HIV-1 splice acceptor site located within the gp4l region may play a role in Rev regulation and that this splice acceptor, when recognized by the splicing machinery, retains precursor mRNA within the nucleus (14). Although in our constructs we have no evidence that this splice acceptor is ever utilized (its normal donors, which occur upstream of the env coding region, have been deleted in all of our constructs), we cannot rule out that its mere presence may be responsible for preventing cytoplasmic accumulation of env mRNAs. To examine the role ofthis splice acceptor, we constructed a deletion mutant that removes information encoding the entire transmembrane spanning region and cytoplasmic domains of gp4l (deletion of nt 7722-8369 inclusive) and thereby also removes the splice acceptor sequences (Fig. 1). The resulting vector, pDB160ASA, should express a truncated form of gpl60, which includes the gp120 coding region,

Proc. Natl. Acad. Sci. USA 91

Biochemistry: Brighty and Rosenberg

8316

extends 387 bp through the 5' end of the gp4l coding sequence (inclusive of the RRE), and is terminated by a stop codon introduced at this position. This vector was stably introduced into Drosophila cells with and without the Rev expression vector, and envelope and Rev protein expression were monitored after induction by Western blot analysis. The results (Fig. 3A) indicate that cell lines carrying pDB160ASA alone did not express detectable levels of envelope protein. In contrast, envelope protein expression was readily observed in cell lines carrying both pDB160ASA and pMtRev. Cells expressing full-length gp160 (pMtl6OA32) were used as controls in these experiments, and cell extracts were examined for appropriate expression of Rev (Fig. 3B). The data show that efficient envelope protein expression from pDB160ASA retained its dependence on Rev in a manner identical to that observed with the full-length gpl60 construct (pMtl60A32). Also, we examined nuclear and cytoplasmic RNA fractions prepared from these cells by Northern blot analysis (Fig. 3C). As expected, the truncated gpl6OASA transcripts, like the control gpl60 transcripts, were found in the nuclear fractions either in the presence or absence of Rev. However, both sets of transcripts selectively accumulated in the cytoplasmic fraction only in the presence of Rev. Taken together, the protein and RNA expression analyses demonstrate that env constructs lacking the HIV-1 splice acceptor (as well as the donor) sites retain their ability to be restricted from cytoplasmic expression of env mRNA and exhibit full Rev regulation. Apparently, splice information is not a prerequisite for either nuclear retention of env transcripts or for Rev-mediated env gene expression. Rev-Dependent Expression of an env Minigene That Spans the RRE. Our results could not discount the possibility that Nucl.

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