The RNA transport element RTE is essential for IAP LTR-retrotransposon mobility

Virology 377 (2008) 88–99

Contents lists available at ScienceDirect

Virology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y v i r o

The RNA transport element RTE is essential for IAP LTR-retrotransposon mobility Andrei S. Zolotukhin a, Ralf Schneider b, Hiroaki Uranishi c, Jenifer Bear a, Irina Tretyakova a, Daniel Michalowski a, Sergey Smulevitch a, Sean O'Keeffe b, George N. Pavlakis c, Barbara K. Felber a,⁎ a b c

Human Retrovirus Pathogenesis Section, Vaccine Branch, Center for Cancer Research, National Cancer Institute-Frederick, Frederick, MD 21702-1201, USA Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany Human Retrovirus Section, Vaccine Branch, Center for Cancer Research, National Cancer Institute-Frederick, Frederick, MD 21702-1201, USA

A R T I C L E

I N F O

Article history: Received 6 February 2008 Returned to author for revision 17 March 2008 Accepted 3 April 2008 Available online 15 May 2008 Keywords: Retrotransposition Retroelement mRNA transport Nuclear export CTE Retrovirus HIV SRV NXF1 RBM15

A B S T R A C T We previously identified an RNA transport element (RTE) present at a high copy number in the mouse genome. Here, we show that a related element, RTE-D, is part of a mobile LTR-retrotransposon, which belongs to a family of intracisternal A-particle related elements (IAP). We demonstrate that RTE-D is essential for the mobility of the retrotransposon and it can be substituted by other known RNA export signals. RTE-deficient IAP transcripts are retained in the nucleus, while the RTE-containing transcripts accumulate in the cytoplasm allowing Gag protein expression. RTE-D acts as a posttranscriptional control element in a heterologous reporter mRNA and is activated by the cellular RNA binding protein 15 (RBM15), as reported for the previously described RTE. We identified a complex family of RTE-containing IAPs in mouse and mapped the active RTE-D-containing IAPs to the Mmr10 group of LTR-retrotransposons. These data reveal that, despite a complex evolutionary history, retroelements and retroviruses share the dependency on posttranscriptional regulation. Published by Elsevier Inc.

Introduction The retroviral full-length mRNA has to be exported to the cytoplasm in its unspliced form, since this transcript encodes the Gag-Pol polyprotein and also serves as genomic RNA. Such a transport mechanism has been first described for HIV, where the viral nuclear Rev protein binds to the cis-acting Rev-responsive element (RRE) and promotes the export and expression of these mRNAs. Rev and the Rev-like proteins from the other complex retroviruses bind to the nuclear receptor CRM1 that enables interaction with components of the nuclear pore complex resulting in export of their RNAs prior to splicing (Cochrane, 2004; Cochrane, McNally, and Mouland, 2006; Cullen, 2003; Felber, Zolotukhin, and Pavlakis, 2007; Kutay and Guttinger, 2005). In contrast to HIV-1 and other complex retroviruses, the export of unspliced mRNAs of simple retroviruses {e.g. the simian type D retroviruses (SRV/D) such as Mason–Pfizer Monkey virus (MPMV), simian retrovirus 1 (SRV-1) and 2 (SRV-2) (Bray et al., 1994; Tabernero et al., 1996)} is mediated solely by cellular factors. The export of CTE-containing RNAs is mediated by NXF1, which binds directly to CTE (Grüter et al., 1998). NXF1 is a conserved cellular mRNA export receptor (Grüter et al., 1998;

⁎ Corresponding author. Fax: +1 301 846 7146. E-mail address: [email protected] (B.K. Felber). 0042-6822/$ – see front matter. Published by Elsevier Inc. doi:10.1016/j.virol.2008.04.002

Herold, Klymenko, and Izaurralde, 2001; Segref et al., 1997; Tan et al., 2000; Wilkie et al., 2001), which directly interacts with the nuclear pore complex and mediates export of the CTE-containing mRNAs to the cytoplasm. Thus, retroviruses have adapted to interact with the nuclear export receptors either directly (via NXF1) or indirectly (via CRM1binding viral proteins). We have previously identified a potent RNA transport element (RTE) in the mouse genome, by using a Rev–RRE deficient HIV clone as molecular trap (Nappi et al., 2001). RTE is able to replace the lentiviral Rev–RRE regulatory system generating infectious HIV and SIV (Nappi et al., 2001; Smulevitch et al., 2006). RTE belongs to a family of elements that are present abundantly in the mouse genome and are likely associated with intracisternal A-particle retroelements (IAP) (Nappi et al., 2001). The RTE is functionally analogous to CTE, yet structurally unrelated, and both elements utilize the cellular mRNA export machinery (Nappi et al., 2001; Smulevitch et al., 2005). We recently showed that the RNA binding motif 15 protein (RBM15) binds to RTE directly and tethers the RTE-containing mRNAs to the NXF1 export machinery (Lindtner et al., 2006). The discovery of retrotransposition-competent IAP LTR-retroelements (Dewannieux et al., 2004) led us to investigate their posttranscriptional regulation. Here, we report that such IAPs contain a conserved element, termed RTE-D, which is closely related to the originally identified RTE (Nappi et al., 2001). We show that RTE-D is

A.S. Zolotukhin et al. / Virology 377 (2008) 88–99

fully functional and is essential for the life cycle of the IAP LTRretroelements. Thus, retroelements, like simple and complex retroviruses, depend on posttranscriptional regulation for the nucleocytoplasmic transport of their full-length RNA, which is essential to counteract the negative-acting determinants located within the IAP retroelement transcript. Results Mobile LTR-retrotransposons contain the RNA transport element RTE The identification of autonomously mobile IAP LTR-retrotransposons RP23-231P12, RP23-324C9, RP23-440N1 and RP23-92L23 (Dewannieux et al., 2004) prompted us to investigate whether they contain RNA export elements. Sequence alignment of the originally identified RTE (termed RTE-A) (Smulevitch et al., 2005) and these four active IAP sequences revealed the presence of RTE-like elements located between the pol open reading frame (ORF) and the polypurine tract (ppt) upstream of the 3′ LTR in all these retroelements. Fig. 1A shows the structure of the LTR-retrotransposon RP23-92L23 as representative example, with the RTE-like element (RTE-92L23) located between nt 6446–6687. This RTE-like element shares a relatively low sequence homology of 69.8% with RTE-A (Fig. 1B). We modeled RTE-92L23 (Fig. 1A, bottom panel) onto the experimentally identified secondary structure of RTE-A reported previously (Smulevitch et al., 2005). Despite the sequence diversity (nucleotide changes shown in red in Fig. 1B), the predicted structure of RTE-92L23 shares similarities with that of RTE-A. We noticed a high occurrence of compensatory changes, leading to the preservation of the overall RTE structure (indicated by asterisks; Fig.1A). Based on the variation in structural features (stem–loops SL I through SL IV), the RTEs were previously classified into 4 groups A, B, C, and D (Smulevitch et al., 2005) (see also Fig. 6A, top panel). In accord with the above classification, we placed the RTE-92L23 into the RTE group D, and hence, RTE-92L23 is referred to as RTE-D in this report. The differences between the RTEs of group A (RTE-A) and group D mostly affect the hairpin loop IV and the bottom portion spanning SL I and SL II, and these structures were shown to be not essential for RTE-A function (Smulevitch et al., 2005). Comparison of RTE-92L23 to those identified within the other mobile IAP LTR-retrotransposons RP23-324C9, RP23-440N1 and RP2392L23 (Dewannieux et al., 2004) showed a high degree of conservation in RP23-324C9 (100%), RP23-231P12 (99.2%) and RP23-440N1 (97.5%) (Fig. 1C). We also found that the IAP LTR-retrotransposon MIA14 (Mietz et al., 1987) contains an RTE identical to that of RTE-92L23, while RTE3061 that harbors the prototype RTE-D (Smulevitch et al., 2005) shows 99.6% identity. We further identified RTEs in IAP LTR-retrotransposons isolated from C3H/He inbred mice with myeloid leukemia (Ishihara et al., 2004). The RTE in the intact clone Q14 (IAP type I) is 97.5% identical to RTE-92L23, while the RTEs in clones with large deletions in gag/pol (IAP types IΔ1 to IΔ4 (Ishihara et al., 2004)) show 94–99% identity. We noted that the nucleotide changes are located in predicted single-stranded regions and mostly in the bottom part of the element. Fig. 1D shows the phylogenetic relationship of the different group D RTEs. Taken together, group D RTEs are present in IAP LTR-retrotransposons and are conserved in both intact and deleted IAPs, suggesting a biological role in the IAP LTR-retrotransposons life cycle. RTE is essential for retrotransposition We next chose one of the autonomously mobile IAP LTR-retrotransposons, the molecular clone 92L23, to study the role of RTE in the IAP life cycle, using a previously described mobility assay in HeLa cells (Dewannieux et al., 2004). Briefly, 92L23neoTNF contains a neo gene encoded in the minus strand of LTR-IAP provirus, which is interrupted by an intron in the plus orientation. HeLa cells were transfected with the wild type molecular clone or the mutant clone 92L23neoTNFΔRTE, where the

89

RTE homologous region was replaced with a polylinker (Fig. 2A), respectively, and the cells were subjected to G418 selection. G418 resistance can be only established upon integration of reverse transcription product originating from spliced plus-strand RNA, and the number of G418 resistant colonies provides a count of retrotransposition events. We found that the wild type IAP clone was mobile as expected (Dewannieux et al., 2004), whereas the ΔRTE mutant produced only few G418-resistant colonies (Fig. 2A). Fig. 2B shows a quantitative analysis of a representative retrotransposition assay. To count the colonies, serial dilutions of transfected cells were seeded and subjected to G418 selection. The ΔRTE-D mutant generated b1% of colonies compared to the wild type 92L23neoTNF. Similar data were obtained in 5 independent experiments, and demonstrate that RTE is essential for retrotransposition. To test whether the function of the RTE-D can be complemented by homologous and heterologous RNA export signals, we inserted such signals into 92L23neoTNFΔRTE and measured the mobility of the resultant hybrids (Fig. 2B). We found that insertion of the originally identified RTE-A (Nappi et al., 2001; Smulevitch et al., 2005) in ΔRTE-D/ RTE-A(+) restored the mobility. We next asked whether a heterologous RNA export signal, the constitutive transport element CTE of SRV-1 (Bray et al.,1994; Zolotukhin et al.,1994) could also support retrotransposition. Fig. 2B shows that the presence of CTE in cis in ΔRTE-D/CTE(+) also restored the mobility of the ΔRTE mutant. Thus, these data demonstrate that the presence of an active RNA export element (RTE, CTE) can substitute for RTE-D and support the finding that posttranscriptional regulation is essential for retrotransposition. RTE-D promotes the cytoplasmic accumulation and expression of LTR-IAP primary transcript We next investigated the mechanisms leading to RTE-dependence of the LTR-retrotransposon transcript. By analogy to retroviruses, we speculated that IAP LTR-retrotransposons require posttranscriptional regulation in order to increase the cytoplasmic levels and expression of their primary transcripts. We first examined the effects of RTE-D on the gag/prt/pol expression using the full-length LTR-IAP molecular clone 92L23. For this purpose, the neo cassette was removed from both the original IAP 92L23neoTNF construct (Dewannieux et al., 2004) and its ΔRTE mutant, restoring the authentic LTR-IAP retrotransposon structure. We used RT-PCR and Northern blot analysis to analyze the mRNA in the nuclear and cytoplasmic extracts of cells transfected with the wild type or ΔRTE molecular clone (Fig. 3). Using a primer pair that detects the gag-encoding primary transcript (Fig. 3A, s1 and a1), we found that both clones produced similar levels of nuclear gag mRNAs. In contrast, the lack of RTE led to a dramatic reduction of the gag mRNA accumulation in the cytoplasm. Northern blot analysis (Fig. 3B) confirmed that the RTE deletion led to a strong reduction of cytoplasmic gag/prt/pol mRNA level. In further support, we found that the RTE-D deletion also led to about 5-fold lower levels of Gag protein production, when compared to the wild type clone (Fig. 3C). Thus, the impaired cytoplasmic accumulation of the primary transcript and the low Gag production are key to the loss of the mobility of the ΔRTE IAP. Interestingly, both the wild type and ΔRTE full-length transcripts were found mostly in the nucleus and the RTE deletion did not significantly affect their nuclear levels, suggesting the presence of a strong nuclear retention determinant that was partly counteracted by RTE. In further support, it was previously reported that expression of the gag gene from the related IAP M1A14 required the presence of the CTE (Wodrich et al., 2001). Similarly, we found that subgenomic transcripts containing the individual IAP 92L23 gag and pol genes were expressed only poorly, while the presence of the cis-acting RNA export elements CTE or RTE activated the protein expression (data not shown). These findings confirm the notion that IAP gag/pol primary transcript, like that of HIV gag/pol, is subject to analogous posttranscriptional restriction. We further found that a portion of both the wild type and ΔRTE primary transcript were subject to splicing, but the RTE deletion did not

90

A.S. Zolotukhin et al. / Virology 377 (2008) 88–99

affect the splicing efficiency (compare lanes 6 and 7, Fig. 3B). The spliced mRNAs were not retained in the nucleus, suggesting that splicing removed the responsible retention determinant. RT-PCR and sequencing of the spliced products revealed a splice donor and splice acceptor located at nt 546 and nt 5632, respectively, generating a non-coding small transcript lacking the gag/prt/pol region. In conclusion, RTE-D is required to promote cytoplasmic accumulation and expression of the LTR-IAP primary transcript, and thus, this finding provides the molecular basis for the absolute requirement of RTE for retrotransposition. RTE-D promotes heterologous reporter gene expression We next tested the function of RTE-D in heterologous reporter transcripts. We inserted RTE-D into different RNA export reporter plasmids that are otherwise expression-defective, since they lack a posttranscriptional control element. In the HIV reporter, pNLgag (Felber et al., 1989), the gag gene is flanked by the major HIV splice donor and a cryptic splice acceptor site, and protein can be only produced from the unspliced mRNA. In this or similar HIV-gag based reporter systems, the level of protein expression upon insertion of a cis-acting RNA export element (e.g. RRE plus Rev, CTE or RTE) provides a quantitative measure of the posttranscriptional regulatory potency of the cis">cis-acting element (Felber et al., 1989; Hammarskjold,

2001; Smulevitch et al., 2006; Smulevitch et al., 2005; Tabernero et al., 1997; Tabernero et al., 1996; Wodrich et al., 2001; Wodrich, Schambach, and Krausslich, 2000). We found that the presence of the RTE-containing fragment of 92L23 (nt 5860–6921) led to a ~10-fold activation of Gag expression compared to the ‘empty’ pNLgag reporter (Fig. 4). RTE-D is more potent than the related RTE-A in this assay (Fig. 4). Similar data were obtained upon insertion of the RTE-D fragment into cat reporter mRNA (DM138) (Huang et al., 1991), where the cat gene is inserted into a modified HIV env fragment and flanked by the env splice sites, and CAT protein can be only expressed from the unspliced mRNA, analogous to the pNLgag (data not shown). We also found the RTE-D is active in the splice site deficient gag reporter mRNA pNLCgag (Felber et al., 1989) (data not shown). Thus, RTE-D, similarly to the originally identified RTE-A (Nappi et al., 2001; Smulevitch et al., 2005) or the CTE (Bray et al., 1994; Tabernero et al., 1996), acts in heterologous settings as a transferable posttranscriptional control element. RBM15 activates expression of the RTE-D containing reporter mRNA Since we previously identified RBM15 as a trans-acting factor that facilitates RTE-A activity, we asked whether RBM15 also acts on the related RTE-D. Fig. 4 shows that cotransfection of RBM15 activated the

Fig. 1. LTR-retrotransposon IAP RP23-92L23 contains an RTE-D element. (A) Genomic structure of LTR-IAP RP23-92L23. The long terminal repeat (LTR), gag, protease (prt) and polymerase (pol) genes, RTE element (RTE-D) and the polypurine tract (ppt) are indicated. The structure of the RTE-A RNA from (Smulevitch et al., 2005) is shown and its key structural features (stem–loop (SL) I through SL IV) are indicated (bottom panel left). RTE-92L23 folds into a structure that is typical of the group D RTE (Smulevitch et al., 2005) (bottom panel right). Letters in red indicate divergent nucleotides in RTE-92L23 compared to RTE-A, and asterisks depict compensatory changes preserving the shared structure elements. (B) Nucleotide sequence alignment of RTE-A and RTE-92L23. Divergent nucleotides in RTE-92L23 are shown in red. Regions spanning the hairpin loops I through IV are indicated. (C) Multiple nucleotide sequence alignment shows conservation of RTE elements belonging to the subgroup D from intact IAP LTR-retroelements. Grey shaded areas indicate the hairpin loop sequences. Black shading indicates the nucleotides that are divergent from the consensus. RTE-92L23 is underlined. Asterisks denote the RTEs from the mobile LTR-IAPs (Dewannieux et al., 2004). (D) Neighbor-joining, unrooted phylogram showing nucleotide sequence conservation of RTE-D elements in LTR-IAPs. Percent divergence values are indicated. RTE-3061 corresponds to the prototype RTE-D sequence reported in Smulevitch et al. (2005).

A.S. Zolotukhin et al. / Virology 377 (2008) 88–99

Fig. 1 (continued ).

91

92

A.S. Zolotukhin et al. / Virology 377 (2008) 88–99

Fig. 2. Retrotransposition depends on posttranscriptional regulation. (A) RTE is essential for retrotransposition. IAP LTR-retrotransposons 92L23 is shown on top. For the mobility assays, the wild type 92L23 reporter construct (92L23neoTNF) (Dewannieux et al., 2004) that contained the G418 selection cassettes inserted at nt 5673 used. We generated the ΔRTE mutant (92L23neoTNFΔRTE) as indicated. To the right, the resultant G418 selection from plates seeded with 106 cells are shown, and the number of clones provides a mobility count. A typical experiment is presented, and similar results were obtained in several independent experiments. (B) RTE-A and CTE substitute for RTE-D in mobility assay. Insertion of RTE-A or CTESRV-1 into 92L23neoTNFΔRTE-D generated ΔRTE/RTE-A(+) and ΔRTE/CTE(+), respectively, and the resulting plasmids were tested for their retrotransposition ability as described in panel A. Serial dilutions of the transfected cells were seeded into plates, subjected to selection. The G418-resistant colonies were counted in triplicate plates and the average retrotransposition frequencies per 105 cells including standard deviations are presented. Also shown are the average values in percent to those obtained with the wild type construct. A typical experiment is shown, and similar data were obtained in three independent experiments.

expression of pNLgag transcripts that contained RTE-A as expected (Lindtner et al., 2006). Similarly, we found that gag expression of the RTE-D containing reporter is also increased by RBM15. We noted that the RTE-D fragment spanning nt 5860–6921 showed ~ 3 fold activation by RBM15, while the fragment spanning nt 6097–7324, that has lower basal level activity, showed ~ 10-fold activation. We concluded that RTE-D and RTE-A share the trans-acting activator RBM15 as well as the underlying mechanism of function as predicted from their conserved primary and secondary structure.

Classification of active RTE-containing LTR-retroelements We next studied the relationship of RTE-containing LTR-IAPs to the known transposon categories. LTR-retrotransposons of M. musculus were previously categorized into 21 distinct families that form three classes (McCarthy and McDonald, 2004), based on amino acid homologies between reverse transcriptase proteins (Fig. 5). One such family, Mmr10_IAP, belongs to a clade comprising of seven additional families that are closely related (Fig. 5, asterisk), and members of any

A.S. Zolotukhin et al. / Virology 377 (2008) 88–99

93

Fig. 3. RTE-D is essential for the IAP gag/pol mRNA export and expression. (A) Nuclear and cytoplasmic mRNA was extracted from 293 cells transfected with 4 µg of the IAP plasmids plus 1 µg of the GFP plasmid FRED25 and analyzed by RT-PCR using primers located in IAP gag (primers s1 and a1 shown in the cartoon below) and with primers in GFP to amplify the gag and GFP mRNAs, respectively. As control, the PCR reactions were run with samples to which the reverse transcriptase (RT) was not added, as shown to the left. Both the regular (25 cycles) and the extended (32 cycles) RT-PCR analyses were performed as indicated. Nuc and cyto indicate nuclear and cytoplasmic enriched subcellular fractions. (B) Northern blot analysis of cells transfected with 1 µg of the IAP plasmids plus 0.2 µg of the GFP plasmid FRED25. The blots were hybridized with a probe spanning the 3′ end of the transcript (see panel A). Short and long exposures of the portion of the blot containing the cytoplasmic fractions are shown. The location of the unspliced and the spliced transcripts are indicated. The blot was rehybridized with a probe for GFP. (C) Human 293 cells were transfected with 10 µg of the plasmids expressing the authentic 92L23 (wt) or its ΔRTE mutant, or mock transfected as indicated. The GFP expression plasmid pFRED25 (0.3 µg) was cotransfected as internal control. Two days later, the cells were harvested and the IAP-Gag proteins were analyzed on immunoblot using IAP-Gag antiserum (αMIA-gag), and the same blots were subsequently reprobed with GFP antibodies as indicated. Arrowheads show the positions of processed IAP-Gag proteins. On the graph to the right, the quantificated signals from western blots are plotted on the X-axis, as raw pixel intensities (arbitrary units). Bold numbers show the gag expression values that were normalized to those of GFP.

of the eight families have been described as IAP by various authors (McCarthy and McDonald, 2004). We compared the sequences of the mobile clones RP23-92L23, RP23231P12, RP23-324C9 and RP23-440N1 (Dewannieux et al., 2004) to those of representative eight IAP families and found that they belong exclusively to Mmr10_IAP (Fig. 5). Comparison of the pol proteins of these retrotransposons to those of the prototype Mmr10_IAP (McCarthy and McDonald, 2004) showed a compelling ~99% homology (Fig. 5), whereas those of other Mmr families were far more distant (data not shown). In further support of this assignment, we found that the prototype Mmr10_IAP (McCarthy and McDonald, 2004) also contains an RTE that shares 95–96% sequence identity with the RTEs of the known mobile LTR-IAPs (Fig. 1C). Together, these data place the four mobile IAP LTR-retrotransposons RP23-92L23, RP23-231P12, RP23-324C9 and RP23-440N1 unambiguously into the Mmr10_IAP family (Fig. 5). Sequences representative of other Mmr families (McCarthy and McDonald, 2004) did not show significant homology to RTE, indicating that RTE-D uniquely belongs to Mmr10_IAP LTR-retrotransposons.

Additional RTE-containing LTR-retrotransposons in the mouse genome The assignment of RTE-D containing LTR-retrotransposons to the Mmr10_IAP family (Fig. 5) was based on the phylogeny drawn from reverse transcriptase amino acid homology, and hence, it was limited to the species that possessed the RT coding sequence. However, a large number of LTR-retrotransposons and their derived sequences contain large deletions affecting the RT open reading frame, and thus were excluded from these analyses. Therefore, we undertook a comprehensive screen of the mouse genome for loci that had properties expected of recently integrated RTE-containing LTR-retroelements, but did not necessarily contain the recognizable RT coding sequences. We previously reported the presence of more than 100 elements identical to the originally described RTE-A and more than 3000 closely related elements (RTE belonging to groups B, C, and D, see Fig. 6A, top panel) with at least 70% sequence identity, distributed on all mouse chromosomes (Smulevitch et al., 2005). We compiled the representative samplings of RTEs of each group, which reflected the sequence

94

A.S. Zolotukhin et al. / Virology 377 (2008) 88–99

Fig. 4. RBM15 activates RTE-A and RTE-D. pNLgag reporter (0.2 µg) containing RTE-A or RTE-D were transfected into HLtat cells in the absence or presence of 0.25 µg RBM15 expression plasmid. Two days later, the cell extracts were analyzed for Gag and GFP production and the data are displayed with standard deviation (SD). The fold induction in the presence of RBM15 is indicated.

diversity within such groups (Fig. 6A, initial RTE homology sequences), and these samplings were then used to map the matching loci in the mouse genome (Fig. 6A, RTE matches). In order to identify the putative RTE-containing LTR-retrotransposons, our queries required that (i) the mapped RTE loci (Fig. 6A) must be flanked by two LTRs of the same type and in the same orientation, and (ii) these LTRs should be within a distance of up to 12 kb. Such queries identified about 250 RTEcontaining putative LTR-retrotransposons (Fig. 6A, retrotransposon candidates). Strikingly, we found that RTEs of the different groups were linked in an obviously nonrandom manner to distinct LTR types (Fig. 6A, LTR type, named according to their prototypic sequences in Repbase). Thus, the group A RTE associated exclusively with RLTR10, whereas 90–95% of the group C and group D RTE were preferentially associated with IAPLTR2-MM and IAPLTR1-MM, respectively. Interestingly, 92% of the identified RTE-D-containing loci were highly homologous to an LTR-IAP internal sequence of the type IAPLTR1a_MM-int located between their LTRs. We found that

the closest match of the IAPLTR1a_MM-int prototype (81% identity, 1.8% insertions and 0.7% deletions) was to Mmr10_IAP (Fig. 5), establishing that the identified candidate loci indeed represent the Mmr10_IAP retrotransposons, and further verifying the validity of our approach. Only one of the RTE-C-containing loci showed internal matches to retroelement-like sequences (data not shown), likely due to a high degree of truncation of these IAPs (see also Fig. 6B). We noticed that a large proportion of RTE-A-containing candidates were of complex descent, combining the retroelement-like integrase, LINE, RTE-A and CTE-like sequences in one cassette (corresponding to RLTR10-int in Repbase) that were flanked by RLTR10 LTRs (see also Nappi et al., 2001). We did not find any association of RTE-B with LTRretrotransposons, likely because of its overall much lower abundance. In summary, the RTE-containing LTR-retrotransposons fall into three groups, characterized by the presence of distinct RTEs associated with distinct LTRs, suggesting a complex evolutionary history. To obtain an independent validation of our findings, we backmapped the identified Repbase prototypes of RTE-containing candidate retrotransposons onto mouse genome. This analysis resulted in the recovery of the same number of hits (Fig. 6A, back-mapping) as obtained with our initial queries (Fig. 6A, retrotransposon candidates), strongly suggesting that our candidate loci are authentic retrotransposons. By studying their length distributions, we found that the RTE-A-containing retrotransposons peaked at ~ 3.0 kb, closely matching the length of the Repbase prototype LTR-retrotransposon (RLTR10-int plus two RLTR10 type LTRs), as expected (Fig. 6B). The RTE-C-containing loci also peaked at ~ 3.0 kb, and since they did not display significant internal matches to the known sequences (data not shown), we speculate that they represented highly truncated retrotransposon-derived elements. In contrast, the RTE-D-containing sequences showed two peaks at ~ 5.2 and ~ 7.0 kb, closely matching the length of the deleted IAP LTR-retrotransposon type IΔ1 to IΔ4 and the full-length Mmr10_IAP retrotransposons, respectively (Fig. 6B). To trace independently the full-length Mmr10_IAP, we backmapped the autonomously mobile family member 92L23 sequence (7084 nt) onto the mouse genome (Fig. 6C) and found 196 high-scoring sequences. Their length distribution exhibited a compound peak at ~6.5 to 7 kb, in agreement with the data shown in Fig. 6B, indicating a high incidence of full-length or nearly full-length Mmr10_IAPs. To address the question whether the identified RTE-containing loci had amplified via retrotransposition or gene duplication, we studied their chromosomal distribution. As shown in Fig. 6D, we found that these candidates, and hence the associated RTEs, are found in all chromosomes, except the Y chromosome. Thus, retrotransposition is

Fig. 5. Phylogenic analysis of the IAP LTR-retrotransposons containing RTE-D. Dendrogram based on reverse transcriptase amino acid sequence (modified from McCarthy and McDonald, 2004) is shown (left). The assignment of RTE-containing IAP LTR-retrotransposons to the Mmr10_IAP family is illustrated in a table (right), presenting the homologies of the four mobile IAP LTR-retrotransposons to the prototype Mmr10_IAP clone. Asterisk indicates the IAP clade.

A.S. Zolotukhin et al. / Virology 377 (2008) 88–99

the most likely mechanism that led to the spread of the RTE-containing LTR-retrotransposons. Together with our phylogenetic data, these results revealed a novel class of murine retrotransposons that are characterized by the presence of RTE-like posttranscriptional regulatory element. Discussion Mobile elements have a major impact on genome structure, evolution and expression, and their mobility contributes to the genetic diversity. LTR-retrotransposons and their related sequences account for about 10% of the mouse genome (Maksakova et al., 2006; Waterston et al., 2002), and also constitute a significant portion of the human genome (for recent reviews see Bannert and Kurth, 2004; Bannert and Kurth, 2006). To gain further insight into the biological role of retroelements, it is important to understand the mechanisms by which they regulate their own expression in order to support efficient propagation. RTE is essential for the mobility of IAP LTR-retrotransposons Here, we report that mouse IAP LTR-retrotransposons encode cisacting RNA export signals that are essential for their mobility. As a model, we studied the autonomously mobile IAP LTR-retrotransposon 92L23 (Dewannieux et al., 2004) and found that it contains a posttranscriptional regulatory element related to RTE-A (Nappi et al., 2001) and belongs to a previously described structural RTE subgroup D (Smulevitch et al., 2005). RTE-D is absolutely required for the LTR-IAP mobility, but can be replaced by CTE, a direct NXF1 binding ligand, or RTE-A, the ligand for RBM15 (Lindtner et al., 2006). Both CTE and RTE act via the NXF1 pathway (Lindtner et al., 2006) and serve as constitutive RNA export signals, allowing nuclear export prior to and independently of splicing. RBM15 protein is thought to provide a direct link between RTE-A and the nuclear export receptor NXF1 (Lindtner et al., 2006).In agreement with this, we found that RBM15 promotes RTE-D mediated reporter gene expression, as we have previously shown for RTE-A (Lindtner et al., 2006). RTE-D and IAPE are distinct elements Another posttranscriptional regulatory element, the “IAP expression element” (IAPE), was previously identified within the 3′ portion of the pol gene of the murine MIA14 LTR-IAP (Wodrich et al., 2001) and was shown to promote expression of HIV gag in a heterologous reporter assay. Sequence comparison revealed a matching region within 92L23 (nt 4566– 6267) with 98% identity compared to the IAPE of MIA14. Importantly, IAPE and RTE represent two distinct regions within the LTR-retrotransposon. While both elements display posttranscriptional regulatory function, they could contribute independently to retrotransposon expression. It is noteworthy, that the presence of the IAPE within the RTE-deleted 92L23neoTNFΔRTE was not sufficient to mediate retrotransposition (see Fig. 2). Thus, our data suggest that RTE-D is the major posttranscriptional regulatory element in the IAP LTR-retrotransposons. Retroviruses and retroelements depend on posttranscriptional regulation Taken together, (i) the absolute requirement of RTE for LTR-IAP mobility, (ii) the reduced cytoplasmic levels of the RTE-deleted IAP transcript, (iii) the ability of CTE and RTE to increase IAP LTRretrotransposon expression, and (iv) the inability of the IAP gag or pol gene to be expressed in the absence of a cis">cis-acting posttranscriptional control element, demonstrate that posttranscriptional regulation is essential for the LTR-IAP lifecycle. This finding further groups the LTR-IAP retrotransposons together with retroviruses such as HIV-1 (Feinberg et al., 1986; Felber et al., 1989; Hadzopoulou-Cladaras et al., 1989; Sodroski et al., 1986), the HTLV retroviruses (Hidaka et al., 1988; Inoue, Seiki, and Yoshida, 1986; Inoue, Yoshida, and Seiki, 1987; Seiki et al., 1988), the

95

human endogenous retrovirus HTDV/HERV-K (Bogerd et al., 2000; Lower et al., 1995; Magin et al., 2000; Magin, Lower, and Lower, 1999; Yang et al., 2000; Yang et al., 1999), the mouse mammary tumor virus (MMTV) (BarSinai et al., 2005; Indik et al., 2005; Mertz et al., 2005) as well as SRV/ MPMV (Ernst et al.,1997; Tabernero et al.,1997) for which dependency on posttranscriptional regulation for their life cycle has been demonstrated. In all cases, it has been demonstrated that export and expression of the unspliced transcript requires a positive-acting regulatory system. In HIV, this step is executed via the viral Rev–RRE system (Fig. 7). In other complex retroviruses, this step is executed by the viral factors such as Rex in HTLV, Rec in HTDV/HERV-K and Rem in MMTV. In contrast, in SRV-1/ MPMV/SRV-2 and in the IAP LTR-retroelements, this step is controlled by cellular factors interacting with viral cis">cis-acting RNA elements (NXF1CTE and RBM15-RTE, respectively) (Fig. 7). Our understanding of the reason for the dependency on posttranscriptional regulation is based on the findings that many retroviral transcripts contain RNA signals that negatively affect the trafficking of the transcript in the absence of the positive-acting regulatory systems. The presence of such signals is responsible for the posttranscriptional fate of the transcript in the absence of positive regulation, which can be manifested at different steps as poor export, oversplicing, low stability, and poor translation (Cochrane, McNally, and Mouland, 2006; Felber, Zolotukhin, and Pavlakis, 2007; Stoltzfus and Madsen, 2006). It is possible that the nuclear retention determinants of the IAP LTR are analogous to the previously described INS/CRS elements of HIV. In support of this idea, we found that these determinants are transferable and confer expression defects when present in heterologous reporter transcripts, acting in the same fashion as HIV INS elements (data not shown). Thus, the presence of negative-acting RNA signals is functionally conserved in widely divergent retroviruses from HIV and SRV1/MPMV to IAP LTR-retrotransposons, which further corroborates their biological importance. Therefore RNA elements like RRE, CTE, and RTE provide an essential function to the retroviral transcript. Interestingly, we also found that inactive retroelements that lack intact gag/pol genes preserved the presence of active RTE or CTE. We previously identified the CTEORG, a CTE-related element located within the ORGIAP retroelement (Tabernero et al., 1997) (Fig. 7) that shares with SRV1/MPMV CTE the secondary structure and NXF-1 binding sites, and is an active RNA export element (Tabernero et al., 1997; von Gegerfelt et al., 2006). In this report, we also found that the highly truncated IAP LTR-retroelements types IΔ1–IΔ4 (Ishihara et al., 2004) preserved RTED. Thus, these truncated elements contain the minimal set of cis">cisacting signals that support retrotransposition, enabling their mobility via protein trans-complementation (see references in Maksakova et al., 2006). Although the region responsible for nuclear retention of the full-length LTR-IAP RNA (nt 546–5632 of 92L23) is largely deleted in IΔ1–IΔ4, even the shortest transposon, IΔ4, still retains two stretches of homology (nt 564–981 and 1620–1956 of 92L23) and these regions together comprise 32% of IΔ4′s transcript length. It is therefore plausible that such sequences in IΔ4 are sufficient to confer nuclear retention, explaining the preservation of RTE. In conclusion, similarly to infectious retroviruses, the LTR-IAP retrotransposons can utilize distinct, structurally unrelated but functionally analogous RNA export elements (RTE and CTE). These findings illustrate the likely convergent evolution of retroelements towards acquisition of functionally analogous RNA regulatory signals, further supporting the view that the evolution of retrotransposons may have involved acquisition and exchange of pre-existing genetic modules (Coffin, Hughes, and Varmus, 1997). Materials and methods Plasmids The following full-length LTR-IAP clones have been described: RP23-231P12, RP23-324C9, RP23-4 40N1 and RP23-92L23

96

A.S. Zolotukhin et al. / Virology 377 (2008) 88–99

A.S. Zolotukhin et al. / Virology 377 (2008) 88–99

97

Fig. 7. Retrovirus and retroelement expression depend on posttranscriptional regulation. Structures of HIV-1, SRV-1/MPMV, ORG-IAP retroelement, IAP LTR-retroelement and its deleted mutant types IIΔ4 are shown. There is functional conservation of negative-acting RNA elements (filled boxes) embedded within gag/pol of all retroviruses and retroelements. Only complex retroviruses like HIV-1 contain such determinants within env. Because the ORG-IAP retroelement is severely mutated it was not further analyzed, and the indicated putative negative-acting sequences (indicated in grey) are shown by analogy to the other retroviruses and retroelements having active RNA export signals. These retroviruses/ retroelements depend on the presence of a positive-acting mechanism and contain RNA export elements located upstream of the ppt and 3′ LTR like RRE, CTE, CTEORG, RTE-A, RTE-D. The RNA export factor can be viral (Rev for RRE) or cellular (NXF1 of CTE and CTEORG; RBM15-NXF1 for RTE).

(Dewannieux et al., 2004), MIA14 (GenBank accession no. M17551), the IAP clone Q14 (GenBank accession no. AB09818) (Ishihara et al., 2004), and the IAP mutants type IΔ1 to IΔ4 (Ishihara et al., 2004). For RP2392L23 constructs, the numbering starts from the first nucleotide of its 5′ LTR (nt 161601; GenBank accession no. AC012382). The RTE-92L23 is located between nt 6446–6687. ΔRTE-92L23 has nt 6269–6689 replaced by a polylinker. The originally identified RTE-A spans nt 391–616 (GenBank accession no. AF250999). The RTE-D prototype RTE3061 has been identified between nt 34124–34365 in GenBank accession no. AC003061 (Smulevitch et al., 2005) and is located within an intact IAP (nt 27673–34761). RTE-MIA14 is located between nt 6460–6701 (GenBank accession no. M17551) isolated from clone pMIA60 (Mietz et al., 1987) (a gift from K. Lueders). We resequenced this region of MIA14 and noted the following changes: T6569 deletion; G6577C; C6580T; T6582C; insertion of G between 6602 and 6603; G6618 deletion; C6643 deletion; G6672 deletion. The reporter plasmids pNLgag and pNLCgag were described previously (Felber et al., 1989). Briefly, pNLgag consists of the HIV-1 5′ LTR and the 5′ untranslated region containing the major splice donor of HIV-1 followed by the gag gene (nt 1–2621 of NL4-3 starting with the first nt of U3 as +1) and the HIV-1 sequence from nt 8886 (XhoI) to end of the 3′ LTR (nt 9709) including a cryptic splice acceptor. This plasmid produces an unspliced gag-encoding and spliced non-coding mRNA (Felber et al., 1989). pNLCgag lacks the splice donor and produces only

unspliced gag-encoding mRNA (Felber et al., 1989). The GFP expression plasmids pFRED25 and pFRED143 (Stauber et al., 1998) were described previously. The IAP-92L23 gag and pol coding sequences were PCRamplified using primers that added a 3XFLAG tag at the C-terminus and were inserted into pCMVkan (Rosati et al., 2005) between the CMV promoter and the bovine growth hormone (BGH) polyadenylation signal. The inserted pol gene contains an optimized translation initiation signal. The CMV promoter (SpeI–SacII) was replaced by the HIV-1 LTR to generate LTR-IAP-92L23 gag and pol expression plasmids. The IAPE-containing regions of MIA14 (nt 4684–6195) (Wodrich et al., 2001) and the corresponding region from RP23-92L23 (nt 4566–6267) as well as the RTEs of 92L23, MIA14, 3061 were inserted into pNLgag between the HIV-1 gag gene and the cryptic splice acceptor, or into a similar location within pNLCgag plasmid. The RBM15 expression plasmid was described (Lindtner et al., 2006). Transfection and expression analysis For mobility assays, HeLa cells were grown in 60 mm plates and transfected with 4 µg of the IAP molecular clones as described (Dewannieux et al., 2004). The cells were seeded in 100 mm plates at several dilutions ranging from 5 × 105 to 104 followed by G418 selection. After 10 days the cells were fixed and the colonies counted using 5 × 105 plate for the inactive IAP and the 104 plate for the active IAP.

Fig. 6. RTE-containing LTR-retrotransposons identified in the mouse genome. (A) The secondary structures of the previously identified RTEs (group A, B, C, D; modified from Smulevitch et al., 2005) are presented on top. The table below indicates for each RTE group the number of representative sequences used for genomic mapping (Initial RTE homology sequences); the number of RTE homology loci mapped in the mouse genome (RTE matches); and the number of the RTE-containing putative LTR-retrotransposons (Retrotransposon candidates) which are further split into subtypes (Subtype) according to their associated LTR sequences (LTR type). Back-mapping, the number of hits recovered by back-mapping of the Repbase prototype sequences onto mouse genome. (B) The histogram shows the length distribution (plotted on X-axis, in nt) of the candidate RTE-containing LTRretrotransposons. Relative frequencies are plotted on Y-axis. (C) The histogram shows the length distribution of high-scoring matches to IAP 92L23 in the mouse genome, presented as in panel B. (D) Chromosomal distribution of the LTR-retrotransposons from panel A. The number of matches is plotted on the Y-axis.

98

A.S. Zolotukhin et al. / Virology 377 (2008) 88–99

The HeLa derived cell line HLtat that constitutively produces HIV tat, or human 293 cells, were transiently transfected with HIV gag reporter plasmids using Superfect (Qiagen) or calcium coprecipitation technique, respectively, as described previously (Lindtner et al., 2006). For 293 cells a tat expression plasmid pBstat was cotransfected. Two days later, the cells were harvested and analyzed for protein or RNA expression. HIV gag expression was monitored using a p24gag antigen capture assay (Zeptometrix). GFP was measured as described (Lindtner et al., 2006). IAP expression was monitored on Western immunoblot using a rabbit anti-MIA14 gag antiserum 3607.X (1:1000 dilution) (Mietz et al., 1987) (a gift from K. Lueders), followed by HRPconjugated anti-rabbit antiserum (dilution of 1:10,000) and detected by Enhanced Chemi-Luminescence (ECLplus; Amersham, GE Healthcare). FLAG-tagged gag and pol were detected using anti-FLAG antibody (dilution of 1:10,000) followed by HRP-conjugated anti-mouse antiserum (dilution of 1:20,000) and detected by ECLplus. Quantification of Western blots was performed by using NIH image software. GFP fluorescence was measured as in Bear et al. (1999). RNA was isolated from the nuclear and cytoplasmic fractions (Zolotukhin et al., 2002) and subjected to semi-quantitative RT-PCR using primers located within gag (s1, nt 1172–1191; a1, nt 1399–1418). Northern blot analysis was used to detect IAP retroelement transcripts using a probe spanning the 3′ end of the transcript (nt 5673–7096). The spliced transcript was identified by RT-PCR and sequencing. Bioinformatics analyses The mouse retroviral LTRs and the complete genomic mouse retroviral sequences were collected from RepeatMasker (genome. washington.edu; Repbase v 8.6) and Genbank (NCBI). They included 72 complete retroviral genomic sequences (musretrovir (n = 49) from RepBase and MouseRetroviruses (n = 23) from GenBank) and of 281 solitary retroviral LTRs (musltr (n = 215) from RepBase and MouseLTR (n = 66) from GenBank). For genomic back-mapping and the final extraction of the “retroviral candidate” retroviral candidate” sequences, we used the complete mouse genome sequence assembly from the !?tlsb-.05pt?>ENSEMBL database (ENSEMBL_golden-path: ENSEMBL_DB:mus_musculus_core_19_30, NCBIM30). The back-mapping of the previously described (Smulevitch et al., 2005) RTE of groups A, B, C and D and the mouse LTR/mouse retroviral genome sequences was performed with the Sequence Search and Alignment by Hashing Algorithm (SSAHA; Ning, Cox, and Mullikin, 2001) and a local installation of the ENSEMBL database (Birney et al., 2004). Multiple sequence alignments and phylogenetic analyses were performed using ClustalW tools at European Bioinformatics Institute (http://www.ebi.ac.uk). Back-mapping of Repbase retroelement prototypes and the known LTR-retrotransposon sequences onto mouse genome was performed using BLAT utility in UCSC Genome Browser. RNA foldings were performed as described (Zolotukhin et al., 2001). Acknowledgments We thank S. Lindtner for comments, our Werner H. Kirsten Student Intern program recipients L. Kotani, T. Hudzik, and S. Sadtler for their contributions, K. Lueders for reagents and discussions, T. Heidmann, H.G. Krausslich, V. Kulkarni, and M. Rosati for reagents, G-M. Zhang for technical assistance, and T. Jones for editorial assistance. References Bannert, N., Kurth, R., 2004. Retroelements and the human genome: new perspectives on an old relation. Proc. Natl. Acad. Sci. U. S. A. 101 (Suppl 2), 14572–14579. Bannert, N., Kurth, R., 2006. The evolutionary dynamics of human endogenous retroviral families. Annu. Rev. Genomics Hum. Genet. 7, 149–173. Bar-Sinai, A., Bassa, N., Fischette, M., Gottesman, M.M., Love, D.C., Hanover, J.A., Hochman, J., 2005. Mouse mammary tumor virus Env-derived peptide associates with nucleolar

targets in lymphoma, mammary carcinoma, and human breast cancer. Cancer Res. 65, 7223–7230. Bear, J., Tan, W., Zolotukhin, A.S., Tabernero, C., Hudson, E.A., Felber, B.K., 1999. Identification of novel import and export signals of human TAP, the protein that binds to the constitutive transport element of the type D retrovirus mRNAs. Mol. Cell Biol. 19, 6306–6317. Birney, E., Andrews, T.D., Bevan, P., Caccamo, M., Chen, Y., Clarke, L., Coates, G., Cuff, J., Curwen, V., Cutts, T., Down, T., Eyras, E., Fernandez-Suarez, X.M., Gane, P., Gibbins, B., Gilbert, J., Hammond, M., Hotz, H.R., Iyer, V., Jekosch, K., Kahari, A., Kasprzyk, A., Keefe, D., Keenan, S., Lehvaslaiho, H., McVicker, G., Melsopp, C., Meidl, P., Mongin, E., Pettett, R., Potter, S., Proctor, G., Rae, M., Searle, S., Slater, G., Smedley, D., Smith, J., Spooner, W., Stabenau, A., Stalker, J., Storey, R., Ureta-Vidal, A., Woodwark, K.C., Cameron, G., Durbin, R., Cox, A., Hubbard, T., Clamp, M., 2004. An overview of Ensembl. Genome Res. 14, 925–928. Bogerd, H.P., Wiegand, H.L., Yang, J., Cullen, B.R., 2000. Mutational definition of functional domains within the Rev homolog encoded by human endogenous retrovirus K. J. Virol. 74, 9353–9361. Bray, M., Prasad, S., Dubay, J.W., Hunter, E., Jeang, K.-T., Rekosh, D., Hammarskjold, M.-L., 1994. A small element from the Mason–Pfizer monkey virus genome makes human immunodeficiency virus type 1 expression and replication Rev-independent. Proc. Natl. Acad. Sci. U. S. A. 91, 1256–1260. Cochrane, A., 2004. Controlling HIV-1 Rev function. Curr. Drug Targets Immune Endocr. Metabol. Disord. 4, 287–295. Cochrane, A.W., McNally, M.T., Mouland, A.J., 2006. The retrovirus RNA trafficking granule: from birth to maturity. Retrovirology 3, 18–34. Coffin, J.M., Hughes, S.H., Varmus, H., 1997. “Retroviruses”. Cold Spring Harbor Laboratory Press, Plainview, N.Y. Cullen, B.R., 2003. Nuclear mRNA export: insights from virology. Trends Biochem. Sci. 28, 419–424. Dewannieux, M., Dupressoir, A., Harper, F., Pierron, G., Heidmann, T., 2004. Identification of autonomous IAP LTR retrotransposons mobile in mammalian cells. Nat. Genet. 36, 534–539. Ernst, R.K., Bray, M., Rekosh, D., Hammarskjold, M.-L., 1997. A structured retroviral RNA element that mediates nucleocytoplasmic export of intron-containing RNA. Mol. Cell Biol. 17, 135–144. Feinberg, M.B., Jarrett, R.F., Aldovini, A., Gallo, R.C., Wong-Staal, F., 1986. HTLV-III expression and production involve complex regulation at the levels of splicing and translation of viral RNA. Cell 46, 807–817. Felber, B.K., Hadzopoulou-Cladaras, M., Cladaras, C., Copeland, T., Pavlakis, G.N., 1989. rev protein of human immunodeficiency virus type 1 affects the stability and transport of the viral mRNA. Proc. Natl. Acad. Sci. U. S. A. 86, 1495–1499. Felber, B.K., Zolotukhin, A.S., Pavlakis, G.N., 2007. Posttranscriptional control of HIV-1 and other retroviruses and its practical applications. Adv. Pharmacol. 55, 161–197. Grüter, P., Tabernero, C., von Kobbe, C., Schmitt, C., Saavedra, C., Bachi, A., Wilm, M., Felber, B.K., Izaurralde, E., 1998. TAP, the human homolog of Mex67p, mediates CTEdependent RNA export from the nucleus. Mol. Cell 1, 649–659. Hadzopoulou-Cladaras, M., Felber, B.K., Cladaras, C., Athanassopoulos, A., Tse, A., Pavlakis, G.N., 1989. The rev (trs/art) protein of human immunodeficiency virus type 1 affects viral mRNA and protein expression via a cis">cis-acting sequence in the env region. J. Virol. 63, 1265–1274. Hammarskjold, M.L., 2001. Constitutive transport element-mediated nuclear export. Curr. Top. Microbiol. Immunol. 259, 77–93. Herold, A., Klymenko, T., Izaurralde, E., 2001. NXF1/p15 heterodimers are essential for mRNA nuclear export in Drosophila. RNA 7, 1768–1780. Hidaka, M., Inoue, J., Yoshida, M., Seiki, M., 1988. Post-transcriptional regulator (rex) of HTLV-1 initiates expression of viral structural proteins but suppresses expression of regulatory proteins. EMBO J. 7, 519–523. Huang, X.J., Hope, T.J., Bond, B.L., McDonald, D., Grahl, K., Parslow, T.G., 1991. Minimal Rev-response element for type 1 human immunodeficiency virus. J. Virol. 65, 2131–2134. Indik, S., Gunzburg, W.H., Salmons, B., Rouault, F., 2005. A novel, mouse mammary tumor virus encoded protein with Rev-like properties. Virology 337, 1–6. Inoue, J., Seiki, M., Yoshida, M., 1986. The second pX product p27 chi-III of HTLV-1 is required for gag gene expression. FEBS Lett. 209, 187–190. Inoue, J., Yoshida, M., Seiki, M., 1987. Transcriptional (p40X) and post-transcriptional (p27X-III) regulators are required for the expression and replication of human T-cell leukemia virus type I genes. Proc. Natl. Acad. Sci. U. S. A. 84, 3653–3657. Ishihara, H., Tanaka, I., Wan, H., Nojima, K., Yoshida, K., 2004. Retrotransposition of limited deletion type of intracisternal A-particle elements in the myeloid leukemia Clls of C3H/He mice. J. Radiat. Res. (Tokyo) 45 (1), 25–32. Kutay, U., Guttinger, S., 2005. Leucine-rich nuclear-export signals: born to be weak. Trends Cell Biol. 15, 121–124. Lindtner, S.L., Zolotukhin, A.S., Uranishi, H., Bear, J., Kulkarni, V., Smulevitch, S., Samiotaki, M., Panayotou, G., Pavlakis, G.N., Felber, B.K., 2006. RNA-binding motif protein 15 binds to the RNA transport element RTE and provides a direct link to the NXF1 export pathway. J. Biol. Chem. 281, 36915–36928. Lower, R., Tonjes, R.R., Korbmacher, C., Kurth, R., Lower, J., 1995. Identification of a Revrelated protein by analysis of spliced transcripts of the human endogenous retroviruses HTDV/HERV-K. J. Virol. 69, 141–149. Magin, C., Hesse, J., Lower, J., Lower, R., 2000. Corf, the Rev/Rex homologue of HTDV/ HERV-K, encodes an arginine-rich nuclear localization signal that exerts a transdominant phenotype when mutated. Virology 274, 11–16. Magin, C., Lower, R., Lower, J., 1999. cORF and RcRE, the Rev/Rex and RRE/RxRE homologues of the human endogenous retrovirus family HTDV/HERV-K. J. Virol. 73, 9496–9507.

A.S. Zolotukhin et al. / Virology 377 (2008) 88–99 Maksakova, I.A., Romanish, M.T., Gagnier, L., Dunn, C.A., van de Lagemaat, L.N., Mager, D.L., 2006. Retroviral elements and their hosts: insertional mutagenesis in the mouse germ line. PLoS Genet. 2, e2. McCarthy, E.M., McDonald, J.F., 2004. Long terminal repeat retrotransposons of Mus musculus. Genome Biol. 5, R14. Mertz, J.A., Simper, M.S., Lozano, M.M., Payne, S.M., Dudley, J.P., 2005. Mouse mammary tumor virus encodes a self-regulatory RNA export protein and is a complex retrovirus. J. Virol. 79, 14737–14747. Mietz, J.A., Grossman, Z., Lueders, K.K., Kuff, E.L., 1987. Nucleotide sequence of a complete mouse intracisternal A-particle genome: relationship to known aspects of particle assembly and function. J. Virol. 61, 3020–3029. Nappi, F., Schneider, R., Zolotukhin, A., Smulevitch, S., Michalowski, D., Bear, J., Felber, B., Pavlakis, G., 2001. Identification of a novel posttranscriptional regulatory element using a rev and RRE mutated HIV-1 DNA proviral clone as a molecular trap. J. Virol. 75, 4558–4569. Ning, Z., Cox, A.J., Mullikin, J.C., 2001. SSAHA: a fast search method for large DNA databases. Genome Res. 11, 1725–1729. Rosati, M., von Gegerfelt, A., Roth, P., Alicea, C., Valentin, A., Robert-Guroff, M., Venzon, D., Montefiori, D.C., Markham, P., Felber, B.K., Pavlakis, G.N., 2005. DNA vaccines expressing different forms of simian immunodeficiency virus antigens decrease viremia upon SIVmac251 challenge. J. Virol. 79, 8480–8492. Segref, A., Sharma, K., Doye, V., Hellwig, A., Huber, J., Luhrmann, R., Hurt, E., 1997. Mex67p, a novel factor for nuclear mRNA export, binds to both poly(A)+ RNA and nuclear pores. EMBO J. 16, 3256–3271. Seiki, M., Inoue, J., Hidaka, M., Yoshida, M., 1988. Two cis">cis-acting elements responsible for posttranscriptional trans-regulation of gene expression of human T-cell leukemia virus type I. Proc. Natl. Acad. Sci. U. S. A. 85, 7124–7128. Smulevitch, S., Michalowski, D., Zolotukhin, A.S., Schneider, R., Bear, J., Roth, P., Pavlakis, G.N., Felber, B.K., 2005. Structural and functional analysis of the RNA transport element, a member of an extensive family present in the mouse genome. J. Virol. 79, 2356–2365. Smulevitch, S., Bear, J., Alicea, C., Rosati, M., Jalah, R., Zolotukhin, A.S., von Gegerfelt, A.S., Michalowski, D., Moroni, C., Pavlakis, G.N., Felber, B.K., 2006. RTE and CTE mRNA export elements synergistically increase expression of unstable, Rev-dependent HIV and SIV mRNAs. Retrovirology 3, 6–14. Sodroski, J., Goh, W.C., Rosen, C., Dayton, A., Terwilliger, E., Haseltine, W., 1986. A second post-transcriptional trans-activator gene required for HTLV-III replication. Nature 321, 412–417. Stauber, R.H., Horie, K., Carney, P., Hudson, E.A., Tarasova, N.I., Gaitanaris, G.A., Pavlakis, G.N., 1998. Development and applications of enhanced green fluorescent protein mutants. Biotechniques 24, 462–466 468–471. Stoltzfus, C.M., Madsen, J.M., 2006. Role of viral splicing elements and cellular RNA binding proteins in regulation of HIV-1 alternative RNA splicing. Curr. HIV Res. 4, 43–55. Tabernero, C., Zolotukhin, A.S., Valentin, A., Pavlakis, G.N., Felber, B.K., 1996. The posttranscriptional control element of the simian retrovirus type 1 forms an extensive RNA secondary structure necessary for its function. J. Virol. 70, 5998–6011. Tabernero, C., Zolotukhin, A.S., Bear, J., Schneider, R., Karsenty, G., Felber, B.K., 1997. Identification of an RNA sequence within an intracisternal-A particle element able to replace Rev-mediated posttranscriptional regulation of human immunodeficiency virus type 1. J. Virol. 71, 95–101. Tan, W., Zolotukhin, A.S., Bear, J., Patenaude, D.J., Felber, B.K., 2000. The mRNA export in C. elegans is mediated by Ce-NXF-1, an ortholog of human TAP and S cerevisiae Mex67p. RNA 6, 1762–1772. von Gegerfelt, A.S., Alicea, C., Valentin, A., Morrow, M., van Rompay, K.K., AyashRashkovsky, M., Markham, P., Else, J.G., Marthas, M.L., Pavlakis, G.N., Ruprecht, R.M., Felber, B.K., 2006. Long lasting control and lack of pathogenicity of the attenuated Rev-independent SIV in rhesus macaques. AIDS Res. Hum. Retroviruses 22, 516–528.

99

Waterston, R.H., Lindblad-Toh, K., Birney, E., Rogers, J., Abril, J.F., Agarwal, P., Agarwala, R., Ainscough, R., Alexandersson, M., An, P., Antonarakis, S.E., Attwood, J., Baertsch, R., Bailey, J., Barlow, K., Beck, S., Berry, E., Birren, B., Bloom, T., Bork, P., Botcherby, M., Bray, N., Brent, M.R., Brown, D.G., Brown, S.D., Bult, C., Burton, J., Butler, J., Campbell, R.D., Carninci, P., Cawley, S., Chiaromonte, F., Chinwalla, A.T., Church, D.M., Clamp, M., Clee, C., Collins, F.S., Cook, L.L., Copley, R.R., Coulson, A., Couronne, O., Cuff, J., Curwen, V., Cutts, T., Daly, M., David, R., Davies, J., Delehaunty, K.D., Deri, J., Dermitzakis, E.T., Dewey, C., Dickens, N.J., Diekhans, M., Dodge, S., Dubchak, I., Dunn, D.M., Eddy, S.R., Elnitski, L., Emes, R.D., Eswara, P., Eyras, E., Felsenfeld, A., Fewell, G.A., Flicek, P., Foley, K., Frankel, W.N., Fulton, L.A., Fulton, R.S., Furey, T.S., Gage, D., Gibbs, R.A., Glusman, G., Gnerre, S., Goldman, N., Goodstadt, L., Grafham, D., Graves, T.A., Green, E.D., Gregory, S., Guigo, R., Guyer, M., Hardison, R.C., Haussler, D., Hayashizaki, Y., Hillier, L.W., Hinrichs, A., Hlavina, W., Holzer, T., Hsu, F., Hua, A., Hubbard, T., Hunt, A., Jackson, I., Jaffe, D.B., Johnson, L.S., Jones, M., Jones, T.A., Joy, A., Kamal, M., Karlsson, E.K., Karolchik, D., Kasprzyk, A., Kawai, J., Keibler, E., Kells, C., Kent, W.J., Kirby, A., Kolbe, D.L., Korf, I., Kucherlapati, R.S., Kulbokas, E.J., Kulp, D., Landers, T., Leger, J.P., Leonard, S., Letunic, I., Levine, R., Li, J., Li, M., Lloyd, C., Lucas, S., Ma, B., Maglott, D.R., Mardis, E.R., Matthews, L., Mauceli, E., Mayer, J.H., McCarthy, M., McCombie, W.R., McLaren, S., McLay, K., McPherson, J.D., Meldrim, J., Meredith, B., Mesirov, J.P., Miller, W., Miner, T.L., Mongin, E., Montgomery, K.T., Morgan, M., Mott, R., Mullikin, J.C., Muzny, D.M., Nash, W.E., Nelson, J.O., Nhan, M.N., Nicol, R., Ning, Z., Nusbaum, C., O'Connor, M.J., Okazaki, Y., Oliver, K., Overton-Larty, E., Pachter, L., Parra, G., Pepin, K.H., Peterson, J., Pevzner, P., Plumb, R., Pohl, C.S., Poliakov, A., Ponce, T.C., Ponting, C.P., Potter, S., Quail, M., Reymond, A., Roe, B.A., Roskin, K.M., Rubin, E.M., Rust, A.G., Santos, R., Sapojnikov, V., Schultz, B., Schultz, J., Schwartz, M.S., Schwartz, S., Scott, C., Seaman, S., Searle, S., Sharpe, T., Sheridan, A., Shownkeen, R., Sims, S., Singer, J.B., Slater, G., Smit, A., Smith, D.R., Spencer, B., Stabenau, A., Stange-Thomann, N., Sugnet, C., Suyama, M., Tesler, G., Thompson, J., Torrents, D., Trevaskis, E., Tromp, J., Ucla, C., Ureta-Vidal, A., Vinson, J.P., Von Niederhausern, A.C., Wade, C.M., Wall, M., Weber, R.J., Weiss, R.B., Wendl, M.C., West, A.P., Wetterstrand, K., Wheeler, R., Whelan, S., Wierzbowski, J., Willey, D., Williams, S., Wilson, R.K., Winter, E., Worley, K.C., Wyman, D., Yang, S., Yang, S.P., Zdobnov, E.M., Zody, M.C., Lander, E.S., 2002. Initial sequencing and comparative analysis of the mouse genome. Nature 420, 520–562. Wilkie, G.S., Zimyanin, V., Kirby, R., Korey, C., Francis-Lang, H., Van Vactor, D., Davis, I., 2001. Small bristles, the Drosophila ortholog of NXF-1, is essential for mRNA export throughout development. RNA 7, 1781–1792. Wodrich, H., Schambach, A., Krausslich, H.G., 2000. Multiple copies of the Mason–Pfizer monkey virus constitutive RNA transport element lead to enhanced HIV-1 Gag expression in a context-dependent manner. Nucleic Acids Res. 28, 901–910. Wodrich, H., Bohne, J., Gumz, E., Welker, R., Krausslich, H.G., 2001. A new RNA element located in the coding region of a murine endogenous retrovirus can functionally replace the Rev/Rev-responsive element system in human immunodeficiency virus type 1 Gag expression. J. Virol. 75, 10670–10682. Yang, J., Bogerd, H., Le, S.Y., Cullen, B.R., 2000. The human endogenous retrovirus K Rev response element coincides with a predicted RNA folding region. RNA 6, 1551–1564. Yang, J., Bogerd, H.P., Peng, S., Wiegand, H., Truant, R., Cullen, B.R., 1999. An ancient family of human endogenous retroviruses encodes a functional homolog of the HIV-1 Rev protein. Proc. Natl. Acad. Sci. U. S. A. 96, 13404–13408. Zolotukhin, A.S., Valentin, A., Pavlakis, G.N., Felber, B.K., 1994. Continuous propagation of RRE(−) and Rev(−)RRE(−) human immunodeficiency virus type 1 molecular clones containing a cis">cis-acting element of simian retrovirus type 1 in human peripheral blood lymphocytes. J. Virol. 68, 7944–7952. Zolotukhin, A.S., Michalowski, D., Smulevitch, S., Felber, B.K., 2001. Retroviral constitutive transport element evolved from cellular TAP(NXF1)-binding sequences. J. Virol. 75, 5567–5575. Zolotukhin, A.S., Tan, W., Bear, J., Smulevitch, S., Felber, B.K., 2002. U2AF participates in the binding of TAP (NXF1) to mRNA. J. Biol. Chem. 277, 3935–3942.