INTRODUCTION

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HTDV/HERV-K is a human endogenous retrovirus present in about 50 copies in the human genome. In addition, about 10,000 solitary long terminal repeats (LTRs) exist. HTDV/HERV-K elements are genetic footprints of germ line infection with an exogenous predecessor almost 30 million years ago. Like all other retroviruses, HTDV/HERV-K needs to export partially spliced and unspliced mRNAs from the cell nucleus during its life cycle. Retroviruses have developed different systems to circumvent the splicing machinery of the cell by enhanced nuclear export of viral mRNA. Simply structured retroviruses, such as the D-type viruses, contain a constitutive transport element (4, 8), an RNA element that directly interacts with the cellular TAP protein (12), an export receptor involved in the nuclear export of cellular mRNA molecules. Recently, it was reported that Rous sarcoma virus, which contains an element like the constitutive transport element, uses a cellular export factor distinct from TAP (26).

Complex viruses, such as human immunodeficiency virus (HIV) and human T-cell leukemia virus, contain genes encoding a regulatory protein (Rev in HIV and Rex in human T-cell leukemia virus) translated from completely spliced mRNAs that are exported from the nucleus by the regular mRNA export pathway (for reviews, see references 5, 25, and 29). Rev and Rex shuttle into the nucleus and bind as multimers to their responsive elements (Rev-responsive element [RRE] and Rex-responsive element [RxRE]) present in the respective primary viral transcripts. In a second step, they recruit the cellular export receptor exportin 1 (10, 11, 24) to mediate the export of unspliced and incompletely spliced viral transcripts, thus competing with the splicing machinery.

The RRE and RxRE sequences form highly structured RNA elements with several stem-loop structures. Within these tightly folded structures, Rev and Rex bind to defined sites located in a specific stem with an internal bulge (1, 2, 3, 9, 14).

Recently, it was reported that HTDV/HERV-K also encodes a regulatory protein which is a functional homologue of Rev and Rex (18, 21, 31). This protein was previously termed Corf. The fact, however, that the Corf export domain cannot functionally replace the Rev export domain and in many respects rather resembles Rex (unpublished data) prompted us to rename it Rec (regulator of expression encoded by corf). Rec binds to an RNA element, the Rec-responsive element (RcRE), in the 3' LTR of HTDV/HERV-K transcripts. The RcRE is located in U3 and R and is approximately 430 nucleotides (nt) long (21). In this report, we demonstrate not only that Rec and Rev can use the RcRE, as previously reported (21), but also that Rex exports transcripts encoding the RcRE. Like the RRE and the RxRE, the RcRE seems to be a highly structured RNA element with several stem-loop structures (21, 32). However, little is known about the sequences and structural requirements which enable binding of the Rec protein to the RcRE. Further, it is unknown whether all HTDV/HERV-K copies encode an active RcRE.

Here, we demonstrate that a subset of these elements contains six clusters of mutations within the RcRE region compared to the biologically active RcRE sequence obtained from clone pcK30 (21). We show that the mutated sequences do not support Rec- and Rex-mediated RNA export, whereas Rev is inactive only with some of these sequences. By analyzing hybrid RcRE (HyRcRE) sequences consisting of active and inactive parts, we demonstrate that two of the six clusters abrogate Rec binding and function when mutated, while the others do not affect the functionality of the element. Rev and Rex function is inhibited or even enhanced by different sets of mutations. Computational RNA folding analyses predict that inactivating mutations evoke a very different structure for the RcRE, indicating strict conformational constraints for function. Deletion of single stem-loop structures present in the active folding structure of the RcRE enabled us to identify four stem-loop structures that are essential for Rec function and two stem-loop structures that influence Rev function on the RcRE, while Rex, in contrast, was only moderately influenced by these deletions.

	MATERIALS AND METHODS

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Plasmid constructions. Plasmids pHIVgagRRE (pNLcgagA2 in references 6 and 13), pHIVgagRcRE, pREC (formerly named pcORF), pREV (pBsrev in references 7 and 23), and p() have been described previously (21, 22). Plasmids pHIVgagRcRELTR3, pHIVgagRcRELTR10, pHIVgagRcRELTR21, pHIVgagRcRELTR23, pHIVgagRcRELTR26, and pHIVgagRcRELTR18 were generated by PCR amplification of different RcRE sequences from several HERV-K LTRs with the same primers as those used to amplify the original RcRE from the pcK30 LTR (21) and replacement of the RRE in pHIVgagRRE with the amplified RcRE sequences by use of SacII and XhoI restriction sites. The resulting RcRE sequences are depicted in Fig. 1. Plasmid pHIVgagRxRE was cloned by PCR amplification of the RxRE from clone pDM128/CMV/RxRE (15) and replacement of the RRE by the RxRE. Plasmids pHIVgagHyRcRE0 to pHIVgagHyRcRE12 were generated by assembly PCR to fuse fragments of the pcK30 LTR to LTR21 and to insert specific clusters of mutations from the pcK30 LTR into the background of LTR21 (see Fig. 3A). The vectors with internal deletions were again cloned by assembly PCR to delete the following nucleotides after ensuring that the predicted RNA secondary structure of the remaining part of the RcRE was not affected by the deletions: pHIVgagdel1, nt 125 to 179; pHIVgagdel2, nt 184 to 233; pHIVgagdel3, nt 235 to 252; pHIVgagdel4, nt 100 to 118; pHIVgagdel5, nt 74 to 91; pHIVgagdel6, nt 272 to 286; pHIVgagdel7, nt 303 to 339; pHIVgagdel11, nt 167 and 168 as well as 174 and 175; pHIVgagdel12, nt 211 and 212; pHIVgagdel13, nt 194 to 197; and pHIVgagdel14, nt 220 and 223. Accession numbers are as follows: RcRE, part of X82272; and Rec, X82271. Plasmid pcRex (27) was termed pREX. A ³²P cycle sequencing kit (Amersham) was used to confirm the introduced mutations or deletions. Plasmids were produced in DH5 cells (Gibco BRL). Plasmid DNA was prepared with an endotoxin-free plasmid preparation kit (Qiagen).

View larger version (42K): [in this window] [in a new window]   FIG. 1.   Alignment of RcRE sequences of different genomic LTRs. Mutational clusters M0 to M5 are depicted in bold letters. cons, consensus.

Transfections. Transfections into HLtat cells (28) were performed with six-well plates (Greiner; 0.5 million cells per well) for HIV p24 quantification; for immunofluorescence studies, the six-well plates contained coverslips. Transfections were carried out with Lipofectamine Plus (Gibco BRL) in accordance with the user manual. Transfectants were analyzed 24 h after transfection. Cell viability was controlled by measuring the total protein content of the cell lysates.

Quantification of HIV p24. Cells were lysed with 250 µl of cell culture lysis buffer (Promega) per well and used in an HIV AG-1 monoclonal antibody p24 capture assay (Abbott) in accordance with the manufacturer's instructions.

Protein expression and purification. Escherichia coli BL21(SI) (Invitrogen) was transformed with a pET15 (Novagen) expression plasmid containing the rec sequence. Rec protein expression was induced with 500 mM NaCl for 4 to 5 h. Bacterial pellets were lysed by sonication in 50 mM Capso (Sigma) pH 9.5. Rec protein was purified using SP-Sepharose beads (Amersham).

In vitro transcription and RNA gel shifts. For in vitro transcription, the RcRE sequences were subcloned into plasmid pBluescript (Stratagene). Linearized plasmids were transcribed with T7 RNA polymerase by use of a Riboprobe in vitro transcription system (Promega) in accordance with the manufacturer's instructions. Probes were labeled with 25 µCi of [-³²P]CTP (Amersham).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Recently, it has been demonstrated that Rec (formerly Corf) can mediate nuclear export of RNA by binding to its responsive element, RcRE, present in a transcript (21, 31). We mapped the RcRE to a fragment of 429 nt in the U3R region of the 3' LTR and reported that a smaller element (374 nt) deleted from the 3' end was partially active (21). Longer deletions at the 3' end or deletions of about 50 nt at the 5' end abrogated the function of the RcRE (21), a fact recently confirmed by others (32).

Characteristic clusters of mutations abrogated the RNA export function of RcRE sequences. To elucidate putative functional domains within the RcRE, we first analyzed whether or not different HTDV/HERV-K LTRs harbor an active RcRE. These sequences, cloned to perform promoter studies (unpublished data), were obtained by PCR using human genomic DNA and primers located in U3 and downstream of U5 to ensure that the sequences were not amplified from solitary LTRs. From the respective RcRE sequences, a consensus sequence was compiled (Fig. 1) thatwith the exception of nt 143was identical to the pcK30 sequence, the RcRE sequence previously used for our studies (21, 22). Comparison of the different RcRE sequences revealed that a subset of them had five or six specific clusters of mutations (M0 to M5) (Fig. 1). Throughout this publication, the mutational clusters are designated Mn, e.g., M1, and the respective consensus sequence at that position is designated Cn, e.g., C1. The RcRE sequence of LTR26 was unique in lacking the M0 mutation. Additionally, most LTR sequences differed from each other by scattered singular point mutations.

View larger version (32K): [in this window] [in a new window]   FIG. 2.   Biological activity of RcRE sequences from HTDV/HERV-K genomes. The HIV Gag precursor in cell lysates was determined with an HIV p24 capture assay after cotransfection of the indicated pHIVgagRcRE reporter vectors into HLtat cells with the effector plasmid pRec or p() (A), pRev (B), or pRex (C). Expression of pHIVgagRcRE pcK30 in the presence of pRec was set as 100%; all other data are relative to this value. Error bars indicate average deviations.

RcRE hybrids of pcK30 and LTR21 revealed sequence and structural requirements for Rec, Rev, and Rex functions. Aiming to analyze the mutational clusters in detail in order to identify a putative Rec, Rev, or Rex binding site, we cloned a set of HyRcRE sequences. They consisted of fusions of the pcK30 LTR and LTR21. A schematic representation of the sequence composition is depicted in Fig. 3A. The positions of mutational clusters (M) respective to the consensus sequence (C) are indicated. All HyRcRE sequences were tested for their ability to mediate the nuclear export of HIV Gag reporter RNA in cotransfections with Rec, Rev, and Rex. The controls mentioned above (visualization of the viral export adapter and the reporter protein by immunofluorescence as well as comparison of cytoplasmic and nuclear RNA levels) were included, but only the data obtained by measuring the level of HIV Gag p24 are shown.

View larger version (70K): [in this window] [in a new window]   FIG. 3.   Activity of RcRE hybrids containing pcK30 and LTR21 sequences. (A) Schematic representation of hybrid sequences. pcK30 sequences are depicted as hatched bars; LTR21 sequences are depicted as white bars; 0 to 5 indicate the positions of mutations or consensus sequences. (B to D) Determination of the HIV Gag precursor in cell lysates after cotransfection of the indicated pHIVgagHyRcRE vectors into HLtat cells with the effector plasmid pRec or p() (B), pRev (C), or pRex (D). Error bars indicate average deviations.

Predicted secondary structures of the RcREpcK30 LTR, RcRELTR21, and the hybrids differed significantly. It has been previously shown that Rev and Rex bind to a discrete site in their respective responsive elements that is exposed in a larger, tightly folded region, as predicted by computer analyses (1, 3, 9, 14, 16). Computational approaches have repeatedly allowed the definition of the responsive elements of retroviral export adapters and were recently used to generate a candidate structure for the RcRE (reference 32 and references therein). We applied comparable computer softwarethe DNASIS RNA folding software (Hitachi) based on algorithms described by Zuker (33)to compare the predicted structures of the active (pcK30) and inactive (LTR21) RcREs as well as their hybrids. As shown in Fig. 4, the predicted structures of the RcREpcK30 and RcRELTR21 were both rather complex. Due to the six clusters of nucleotide exchanges, they differed significantly, especially in the shape and the arrangement of the stem-loop formations. In both structures, however, several major stem-loop formations (SL1 to SL7) were discerned in the inner core segment from nt 60 to nt 340, including C1 to C4 (M1 to M4, respectively). The 5' and 3' ends of the element folded back into a single long stem-loop stretch in the RcREpcK30 and a set of several smaller stem-loop formations in RcRELTR21.

View larger version (19K): [in this window] [in a new window]   FIG. 4.   Predicted folding structures of active and inactive RcREs. (A) pcK30. (B) LTR21. The structures were predicted with DNASIS software using algorithms defined by Zuker (33).

Deletion of individual predicted stem-loop structures in the RcRE and their influence on Rec, Rev, and Rex function. To elucidate the putative role of the diverse stem-loop structures in the biological functions of Rec and the other retroviral export adapters, a further set of mutated RcRE sequences was cloned. In each of these constructs, a single stem-loop was deleted (del1 to del7) (Fig. 4A) in a way that did not alter the formation of the predicted remaining stem-loop structures, which was controlled by folding of each of the deletion RcREs (data not shown). In addition to these large deletion mutants, four mutants (del11 to del14) (Fig. 4A) that each eliminated one or two (del11) of the small bulges in SL1 and SL2 were cloned. del11 affected a sequence with homology to the Rex binding site on the RxRE (21).

View larger version (40K): [in this window] [in a new window]   FIG. 5.   Activity of RcRE deletion mutants of pcK30. The HIV Gag precursor in cell lysates was determined after cotransfection of the indicated pHIVgagdel vectors (for exact positions of deletions, see Materials and Methods) into HLtat cells with the effector plasmid pRec or p() (A), pRev (B), or pRex (C). Error bars indicate average deviations.

In vitro binding studies. To confirm that the in vivo function of Rec was mediated by direct binding of Rec to the RcRE and to elucidate the sequence and/or structural requirements for this binding, we analyzed the ability of purified recombinant Rec protein to bind to the different RcRE RNAs transcribed in vitro in RNA gel shift assays (Fig. 6). Rec specifically bound to the biologically active pcK30 RcRE RNA, and no binding to biologically inactive RcRE LTR21 was observed (Fig. 6A). Binding of Rec to the HyRcRE sequences was measured as the ability of excess unlabeled HyRcRE RNA transcripts to compete for binding of Rec to a ³²P-labeled pcK30 RcRE RNA probe. The results are summarized in Fig. 6A and C. Only the biologically active RcRE transcripts (HyRcRE3, HyRcRE7, and HyRcRE10 as well as pcK30) were able to compete for Rec binding to the labeled pcK30 RcRE RNA probe. These in vitro data confirmed the in vivo data obtained with the LTR and HyRcRE sequences and demonstrated that direct binding of Rec to these elements was a prerequisite for the biological activity measured, that is, for the RcRE-dependent nuclear export of HIV Gag reporter transcripts.

View larger version (50K): [in this window] [in a new window]   FIG. 6.   In vitro binding of recombinant purified Rec to RcRE sequences transcribed in vitro. RNA gel shift assays were performed as described in Materials and Methods. (A) Titration of Rec on biologically active pcK30 RcRE and on inactive RcRE LTR21 (six lanes at left). Competition for Rec binding to pcK30 RcRE with active (pcK30, Hy3, Hy7, and Hy10) and inactive (LTR21, Hy0, Hy1, and Hy2) RcREs (eight lanes at right). (B) Titration of Rec on active pcK30 RcRE and on inactive RcREdel2 (six lanes at left). Competition for Rec binding to pcK30 RcRE with active (pcK30 and del14) and inactive (LTR21 and del2) RcREs (four lanes at right). (C) Summary of the competition experiments.

	DISCUSSION

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In recent years, it has become evident that many proteins are involved in RNA processing and export from the nucleus. Among the first factors identified in this scenario were the retroviral export adapters Rev and Rex, which mediate export of viral transcripts by binding to their respective responsive elements, RRE and RxRE, as multimers and by subsequent recruitment of cellular export receptor exportin 1 to the export complex (5, 20, 25, 29). Although the retroviral export adapters Rev and Rex bind to discrete RNA sequences, these sequences are located in stem-loop structures with internal bulges in the context of a larger RNA segment which is tightly folded into a very complex specific structure and which is essential for function (1, 2, 3, 9, 14). The human endogenous retrovirus HTDV/HERV-K encodes a functional equivalent to Rev and Rex. Its responsive element, RcRE, is located in the U3R region of 3' LTRs (21). However, the sequence and structural requirements for Rec binding to the RcRE were not yet elucidated, although a recent publication, in which Rec binding to the RcRE was analyzed using deletion mutants, gave some insights (32). Yang et al. (32) applied a wide variety of different experimental test systems (in vitro binding and yeast two- and three-hybrid systems, two rather different tests for biological activity in mammalian cells); however, these systems sometimes produced contradicting results. We therefore mainly relied on testing for biological activity, that is, for export activity from the cell nucleus. We used a mammalian transient transfection assay (21) in which the export activity of the retroviral export adapters was measured with an RcRE-dependent HIV Gag reporter construct that did not contain splice sites (6, 13). The assay was controlled by monitoring transfection efficiencies and by confirming that the level of reporter protein measured reflected the level of reporter RNA exported from the nucleus. In previous publications, this test system and these controls were instrumental in defining the RcRE and in identifying relevant domains in the Rec protein (21, 22). In this publication, data obtained with these controls are mentioned but not shown.

Since more than 30 copies of HTDV/HERV-K elements are present in the human genome, we analyzed the ability of diverse RcRE sequences to support Rec-mediated RNA export. Not surprisingly, some RcRE sequences were inactive. HTDV/HERV-K arose from germ line infection of Old World monkey predecessors with an exogenous retrovirus 30 million years ago. Vertical transmission of retroviruses no longer depends on the presence of replication-competent genomes and, since retroviruses may have a pathogenic potential, selection against intact genomes could be expected (17, 19). Indeed, most human endogenous retrovirus sequences known so far are rather defective, and none of them is infectious. In contrast, it is quite surprising that HTDV/HERV-K elements have retained open reading frames for the expression of viral genes (19) and that this expression is regulated by a viral export adapter and a responsive element. The conservation of these regulatory sequences is rather fascinating, because their presence could influence the regulation of cellular genes. It will be interesting to identify the exact positions of active RcREs in the genome and neighboring genes.

Inactive and active RcRE sequences differed by five or six clusters of mutations (M0 to M5) (Fig. 1 and 2A). Rec activity was entirely restricted to its own responsive element, and Rec was unable to use the RRE, as already reported (21), or the RxRE (Fig. 2A). Rev and Rex, however, could use RcRE sequences to mediate HIV Gag RNA export, as reported before (21), although they were less effective than in cotransfections with their genuine responsive elements (Fig. 2B and C). Interestingly, the presence of mutational clusters M0 to M5 led to a reduction in Rex-mediated export activity similar to that observed with Rec (Fig. 1 and 2C), while Rev activity was undisturbed by the presence of M1 to M5 in RcRE LTR26 (Fig. 1 and 2B).

Hybrid analyses revealed that Rec function depended on the presence of a rather large sequence comprising C1 to C3 (Fig. 3A and B). Mutations in C1 or C3 led to biologically inactive RcRE sequences which were not able to bind Rec (Fig. 3B and Fig. 6A and C). The hybrid mutants also demonstrated that the presence of either cluster M0 or cluster M2 led to a slight reduction in Rec-mediated export activity and that clusters M4 and M5 had no effect.

Hybrid analyses suggested the existence of two Rev binding sites, as the introduction of either C0 or C3 fully restored Rev-mediated export activity in an inactive backbone. None of these sites showed sequence homology to the Rev binding site in the RRE. Overall, however, none of the mutations introduced into the active RNA backbone had a severe abrogating effect on Rev function.

Interestingly, when C3 was present in the otherwise inactive RcRE LTR21 backbone (HyRcRE8) (Fig. 3A and D), Rex export function considerably exceeded that observed with the RxRE. With the RNA folding prediction algorithm, HyRcRE8 displayed a unique stem-loop which folded only in this hybrid and contained a sequence with 75% homology to the experimentally defined Rex binding site in the RxRE (Fig. 7). The observation that Rex was also active when C0, C1, and C2 were present in HyRcRE4 hinted at a second Rex interaction site. Again, similar to Rev function, Rex function was not completely abrogated by any of the hybrids. The observations that Rev and Rex probably had two independent binding sites and that the most efficient mutant probably created a new sequence-specific Rex binding site in an otherwise inactive structural context might indicate that only Rec was completely adapted to its genuine responsive element and that optimized structures could be generated for Rex and probably also for Rev. One could even imagine that the RxRE evolved from the ancient RcREs present in the human genome.

View larger version (16K): [in this window] [in a new window]   FIG. 7.   Predicted folding structures of single stem-loop formations in HyRcRE8 and RxRE. The structures were predicted with DNASIS, the RxRE secondary structure being identical to that described by Van Brussel et al. (30). The hypothetical Rex binding site in HyRcRE8 and the defined Rex binding site in the RxRE are indicated by the parallel lines.

Computational analyses predicted considerably divergent secondary structures for the active and inactive RcRE sequences as well as for the hybrid sequences (Table 1 and Fig. 4). The main differences affected an inner core segment comprising SL1 to SL7. Deletion of single stem-loop structures revealed that SL2, SL4, SL5, and SL6 were all important for in vivo Rec function (Fig. 5A), although RcRE deletion mutants missing only one of these four stem-loop structures still bound Rec in vitro (Fig. 6D). Several explanations can be given for this controversial result. Formation of a biologically active export complex may depend on interaction of Rec with more than a single contact point within a complex, folded RNA structure, and deletion of one contact point may not abrogate in vitro binding. Alternatively, more than one Rec molecule may be necessary to form an active export complex in vivo, and individual molecules of this oligomeric complex may bind to different but adjacent stem-loop structures. From in vivo competition experiments with Rec mutants, we could estimate that oligomeric complexes were formed containing either dimers or tetramers (22; unpublished data). In addition, cellular export factors may influence the formation, stability, affinity, and kinetics of active export complexes in vivo. Most likely the Rec-RcRE complexes formed in vitro did not completely resemble the functional export complexes formed in vivo.

Some of these results were also obtained in a recent study (32). The authors used an RNA folding algorithm that produced a slightly different active structure than the one presented here, a result which may also have been caused by the fact that the sequence depicted by Yang et al. (32) had eight nucleotide exchanges and one insertion compared to pcK30. Nevertheless, the stem-loop formations identified in their structures (SLIIa to SLIIf and SLIII) resembled SL1 to SL7. The main difference came from the fact that their stem-loop formations were arranged around a large central loop, whereas SL1 to SL7 formed a more complex core in which loops of SL3, SL4, SL5, and SL6 clustered in a center surrounded by SL1, SL2, and SL7. Yang et al. (32) identified two Rec binding sites with a test system in which a Tat-Rec fusion protein activated an HIV LTR that carried either SLIIb (equivalent to SL4) or SLIIf (equivalent to SL6) instead of the Tat-responsive element. SLIIb would be influenced by M1, and SLIIf might be influenced by M3. This information implies that if two Rec binding sites truly exist, either C1 or C3 alone should have rescued Rec activity, a result not obtained in our hybrid analysis. Unfortunately, the authors did not include SLIIc (equivalent to SL1), SLIId (almost identical to SL2), SLIIe (almost identical to SL3), SLIIa (equivalent to SL5), or SLIII (very similar to SL7) in their assay. In the study of Yang et al. (32), all seven stem-loop deletion mutants completely lacked biological activity, whereas our data showed that deletion of SL1, SL3, and SL7 reduced but did not completely abrogate Rec-mediated export activity (Fig. 5A). This major discrepancy may be explained either by differences in the extent of the sequence deleted or by different properties of the test systems used. In summary, the results reported by Yang et al. (32) do not contradict the results presented here.

The data presented in this publication, especially the hybrid analysis, revealed that the biological activity of Rec correlated with a large sequence in the RcRE containing clusters C1 and C3. Introduction of a single C cluster into an inactive backbone did not restore Rec activity. The biological activity of the hybrids also correlated with the in vitro RNA binding data. Rec in vivo function depended further on the presence of four putative stem-loop structures that were predicted by computational analysis only when the active sequence (C1 to C3) was present in the RcRE. A reasonable interpretation of the data is the hypothesis that Rec function required interaction with a complex, folded RNA structure within RcRE rather than binding to a discrete specific binding site.