Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel1
Author for correspondence: Arnona Gazit. Fax +972 3 642 2275. e-mail micro1{at}post.tau.ac.il
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Abstract |
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Introduction |
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The Rev protein of equine infectious anaemia virus (EIAV), the activation domain of which is functionally interchangeable with the leucine-rich activation domains of other lentiviral Rev proteins (Fridell et al., 1993 ; Mancuso et al., 1994
; Meyer et al., 1996
), was also shown to augment the expression of transcripts containing introns (Martarano et al., 1994
). Moreover, although the EIAV Rev activation domain possesses an atypical nuclear export signal that differs significantly from that of human immunodeficiency virus (HIV) Rev, it is believed to travel the same nuclear export signal-dependent export pathway (Otero et al., 1998
). In primate lentiviruses (Malim et al., 1988
; Rosen et al., 1988
) and several of the ungulate lentiviruses (Schoborg & Clements, 1996
) such as visna-maedi virus and caprine arthritis encephalitis virus, the Rev protein acts through binding the RRE located within the env gene. In the EIAV genome, however, computer analysis could not identify a stable stemloop RNA secondary structure analogous to the lentiviral RRE (Rosin-Arbesfeld et al., 1993
; Martarano et al., 1994
). Previously, we (Rosin-Arbesfeld et al., 1993
) and others (Harris et al., 1998
) showed that the 3 half of the EIAV genome sequence conferred Rev dependence on CAT-encoding transcripts. In another study, it was suggested that the responsiveness to EIAV Rev is mediated via two elements located in the env gene (Martarano et al., 1994
). Thus, by analogy with other lentiviruses (Cullen, 1998
), it could be assumed that the 3 half of the EIAV RNA contains sequences that confer cis-repressing dependence on EIAV unspliced and partially spliced transcripts and that this repression might be relieved by Rev. The present study suggests that Rev dependence of EIAV is mediated by several of the suboptimal, centrally located viral splice sites.
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Methods |
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Results |
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We anticipated that, if the splice sites were involved in CRS activity, their elimination by in vitro mutagenesis would hinder the CRS inhibitory effect, resulting in Rev-independent CAT expression. We used mutagenizing oligonucleotides (Fig. 3 and Table 2
) to disrupt the intron border dinucleotides, which are critical for the splicing process. First, we showed that disruption of all the splice sites (SA 5212, SD 5276, SA 5437 and SD 5538) in pCAT-CRS*, which lacked SA5135, resulted in complete elimination of CRS activity (Fig. 3
). Next, in order to evaluate the contribution of particular splice junctions to the inhibitory activity, we mutated individual splice sites. pCAT/5212* and pCAT/5276*, in which the splice signals SA5212 and SD5276, respectively, were eliminated, showed moderate levels of CRS activity (33 and 49% CAT activity), suggesting that each of these sites contributed slightly to CRS activity. In contrast, mutations that eliminated SA5135 and SA5437 diminished CRS activity significantly (70% and 61% CAT levels, respectively). The most pronounced reduction in CRS activity was obtained when SD5538 was eliminated (85% CAT activity). These data suggested that mainly SD5538, but also SA5135 and SA5437, play a major role in CRS activity. Since disruption of SD5538 reduced CRS activity drastically, it might be suggested that the presence of this site is essential for exerting efficient CRS activity. However, because the individual elimination of each of the other splice junctions also reduced CRS activity partially, it might be suggested that SD5538 is essential, but not sufficient, for conferring full CRS activity. This notion was substantiated by further experimental data that showed that the region that spanned nt 51115394, containing SA5135, SA5212 and SD5276, as well as the region that spanned nt 53475700, containing SA5437 and SD5538, both exhibited only moderate CRS activity (5355% CAT activity) (Fig. 2
). Interestingly, the region that spanned nt 44795130 also exhibited CRS activity (Fig. 2
). We postulated that this activity could probably be attributed to the non-functional site that, according to sequence data, is present at nt 5095 (Noiman et al., 1990
). To investigate this possibility, an additional mutated construct was constructed, designated pCAT/5095*, in which SA5095 within pCAT/44795130 was mutated (Table 2
). The data showed that elimination of SA5095 reduced CRS activity slightly (Fig. 3
; 34% CAT activity exhibited by pCAT/5095* compared with 22% exhibited by pCAT/44795130). These data suggested that, although SA5095 contributed slightly to CRS, the major inhibitory effect exerted by this region was independent of the splicing machinery.
Since the region that spanned nt 44796162 responded to Rev (Fig. 1), experiments were conducted to localize the elements that can act as RRE within this region. The data showed (Fig. 2
) that all CAT reporter constructs that contained sequences spanning nt 51116162 (pCAT/51115700, pCAT/51685700, pCAT/53475700, pCAT/51115394 and pCAT/52216162) were responsive to the supply of Rev in trans, resulting in CAT levels of 7280%. This suggested that sequences residing within the region that spanned nt 51116162 can interact directly or indirectly with Rev and, thus, can be regarded as the RRE of EIAV. Interestingly, CAT reporter constructs that contained sequences that extended further upstream, up to nt 4479 (pCAT/44795700, pCAT/44795226, pCAT/44795173, pCAT/44795130 and pCAT/44796162), displayed much lower responsiveness to Rev (1943% CAT activity). These data suggested that sequences located within nt 44795111 might interfere with Rev responsiveness when present in cis adjacent to RRE.
We next investigated whether Rev responsiveness can be mediated by some of the suboptimal splice sites located within nt 51355538. The various CRS-mutated CAT reporter constructs were co-transfected into Cf2Th cells together with the Rev-expressing plasmid, pCEV176 (Rosin-Arbesfeld et al., 1993 ), or with pCEV as a control and CAT activity was measured (Fig. 3
). It was expected that, in constructs in which an eliminated splice junction resulted in partial reduction of CRS activity, complete elimination of Rev activity would indicate that this site is indispensable for mediating Rev activity. The data showed that elimination of SA5212, SD5276 and SA5437 decreased Rev activity slightly, suggesting that their role in Rev responsiveness is limited. On the other hand, mutation of SD5538 eliminated Rev activity completely (85% CAT activity in the absence or presence of Rev), suggesting that this site plays a major role in mediating Rev responsiveness. Disruption of SA5135 did not inhibit Rev activity (100% CAT levels), indicating that SA5135 does not function in mediating Rev responsiveness. Obviously, mutations of all splice sites in pCAT/CRS*, which resulted in complete elimination of CRS activity, prevented a functional Rev assay. Thus, whereas no conclusions could be drawn regarding the extent of the contribution of SA5212, SD5276 and SA5437 to Rev responsiveness, our data suggested that SD5538, but not SA5135, functions in mediating Rev responsiveness. Since Rev responsiveness exhibited by pCAT/51115394 was not lower than that exhibited by pCAT/53475700 (Fig. 2
), it is assumed that, although SD5538 might serve as a major element that mediates Rev responsiveness, the presence of the entire region is required to exert a full response to Rev.
In order to reveal the mechanism that mediates Rev dependence of CRS-containing transcripts, we investigated whether the inhibitory effect of CRS on CAT activity was also reflected at the mRNA level. Cf2Th cells were co-transfected with the Rev-expressing plasmid pCEV176 or pCEV empty vector and with the Rev-responsive plasmid pCAT/51115700 or the Rev-independent plasmid pCAT/56956162. After 48 h, total RNA or RNA extracted from the cytoplasm or nuclear fraction as described elsewhere (Cochrane et al., 1991 ) was subjected to the RNase protection assay. While the level of total cat mRNA was not affected by the presence of Rev, the level of cat mRNA in the nuclear fraction of pCAT/51115700 transfectants was higher in the absence of Rev than in its presence (Fig. 4a
). In cytoplasmic RNA, the opposite occurred. The level of cytoplasmic cat mRNA of pCAT/51115700 transfectants was higher in the presence of Rev than in its absence. In contrast, the presence of Rev did not affect the levels of total, nuclear or cytoplasmic cat mRNA in pCAT/56956162 transfectants (Fig. 4b
). These data suggested that the presence of the EIAV CRS in cis suppressed nucleocytoplasmic transport of CAT transcripts and that the supply of Rev in trans could relieve this suppression.
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Discussion |
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The region that spanned nt 44795111 exhibited an inhibitory effect on CAT expression, most of which was independent of splice junctions. Moreover, Rev could barely overcome this inhibitory effect. Although the molecular mechanism of this negative effect is still obscure, this observation is consistent with the absence of this region in env transcripts, which are Rev dependent. Since this suppressing region is present in the unspliced genomic RNA, it might be assumed that its effect is diminished by the presence of the unused major splice donor, SD459, which is Rev responsive (Tan et al., 1996 ).
In the present study, we showed that the centrally located suboptimal EIAV splice sites confer Rev dependence on the CRS-containing transcripts. These sequences presumably act in concert with the major 5 splice site (Tan et al., 1996 ) and with the 3 end-located elements (Belshan et al., 1998
; Mancuso et al., 1994
) to exert Rev responsiveness. Additional regulatory elements reside within the EIAV genome that control EIAV gene expression positively and negatively independently of Rev. Thus, it is noteworthy that the region spanning nt 44737247 enabled efficient expression of CAT activity in the presence or absence of Rev (Fig. 1
) due to stimulatory elements residing in the region nt 61627247 (our unpublished results). Also, the inhibitory effect conferred by elements located within nt 44796162 could not be overcome by Rev (Fig. 2
), presumably due to the presence of an inhibitory element the repression effect of which was independent of Rev.
In conclusion, although the use of a heterologous CAT reporter construct enabled the identification of EIAV suboptimal splice sites as conferring Rev dependence, their activity should be evaluated in the context of the complete EIAV genome. Moreover, the lentiviral Rev protein plays a role in the switch between the early and late phases of virus infection (reviewed in Pollard & Malim, 1998 ). Thus, elucidation of the biological relevance of the unused EIAV splice sites to virus replication, persistence and pathogenesis should await further studies in the context of EIAV infection in vivo.
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Acknowledgments |
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References |
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Received 29 October 1999;
accepted 20 January 2000.
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