Downloaded from www.microbiologyresearch.org by IP: 93.91.26.109 On: Mon, 11 Jan 2016 13:58:22 Journal of General Virology (2000), 81, 1265–1272. Printed in Great Britain ................................................................................................................................................................................................................................................................................... Suboptimal splice sites of equine infectious anaemia virus control Rev responsiveness Rina Rosin-Arbesfeld, Abraham Yaniv and Arnona Gazit Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel The Rev protein of equine infectious anaemia virus (EIAV) was shown previously to stimulate the expression of a heterologous CAT reporter gene when the 3 half of the EIAV genome was present downstream in cis. However, computer analysis could not reveal the existence of a stable RNA secondary structure that could be analogous to the Rev-responsive element of other lentiviruses. In the present study, the inhibitory RNA element designated the cis-acting repressing sequence (CRS) has been localized to the centre of the EIAV genome. The inhibition exerted by this element could be overcome by supplying Rev in trans. The ability of the EIAV CRS to function in a heterologous context suggests that it does not require interactions with other viral proteins. Site- directed mutagenesis showed that the various centrally located suboptimal splice sites of the EIAV genome function as CRS and confer Rev-dependence on the CRS-containing transcripts. In addition, the data suggest that in canine Cf2Th cells, which are highly permissive for EIAV replication, CRS prevents nuclear export of CRS-containing transcripts and the supply of Rev relieves this suppression. Introduction In primate lentiviruses, cis-acting repressing sequences (CRS) have been localized to various regions in the gag, pol, env, pro and vif genes and the Rev-responsive element (RRE) (Rosen et al., 1988; Maldarelli et al., 1991; Schwartz et al., 1992 a, b ; Huffman & Arrigo, 1997). These inhibitory sequences were suggested to function by preventing the transport of unspliced and partially spliced mRNAs into the cytoplasm. Rev, as a nuclear RNA export factor, was found to relieve this suppression (reviewed in Cullen, 1998). The role of functional splice sites in conferring Rev dependence remains a point of intense debate (reviewed in Kingsman & Kingsman, 1996). 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 Author for correspondence: Arnona Gazit. Fax 972 3 642 2275. e-mail micro1post.tau.ac.il 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 stem–loop RNA secondary structure anal- ogous 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. Methods  Plasmid construction. The Rev-encoding cDNA p176 (designated pCEV176), the vector pCEV21 (designated pCEV) and the CAT reporter plasmid (pCEVCAT), containing nt 4473–7247 of the EIAV genome 0001-6791  2000 SGM BCGF