Enhancement of nucleocytoplasmic export of HTLV‐1 Rex mRNA through cis and trans interactions of the mRNA with the complex of Rex protein and Rex‐responsive element

Abstract

p27 rex of HTLV-1 promotes nucleocytoplasmic t xport of viral mRNAs through binding of the Rex-response element (RexRE) present at the 3' end of the viral transcripts in ds with respect to the ORFs of the viral mRNAs. We have found that expression of the RexRE in trans, as a separate RNA, still allows Rex protein to promote export of viral mRNAs lacking the RexRE. The data suggest the formation of a ternary complex between Rex protein, RexRE and upstream elements of viral mRNA and hence the existence of secondary sites of interaction between Rex protein and viral RNAs.~ey words: mRNA export; HTLV-1; Retrovirus; p27rex; t,'~ex-responsive element; Fluorescence in situ hybridisation !. IntroductionHuman T-cell leukaemic virus type 1 (HTLV-1) is a retro-, irus trophic for CD4 + T-cells associated with an aggressive lorm of leukaemia, adult T-cell leukaemia [1,2], and with a ~eurological disorder, tropical spastic paraparesis, also due to infection of T-cells [3]. In order to co-ordinate the regulation ,,f the structural and viral proteins necessary for viral replication a nuclear protein, Rex, encoded in the viral genome, makes efficient use of variably spliced reading frames in the , iral genome (reviewed in [4]). In the presence of Rex incom-1,1etely spliced transcripts of viral RNA are expressed in the • ytoplasm, allowing expression of proteins from the unspliced :ag-pol transcript and the singly spliced env transcript. In the ,,bsence of Rex the incompletely spliced transcripts remain .equestered in the nucleus to be degraded or for completion ,,f splicing [5,6]. Completely spliced transcripts encode the ~egulatory proteins of the virus, Tax and Rex [5]. Rex works i~y binding directly to a highly structured element of RNA of ~kbout 250 bases present in the viral transcripts, the Rex-re-, ponsive element (RexRE) [7][8][9]. Details of the mechanism of ~tction of Rex, and its analogue Rev from human immunode-!tciency virus (HIV) type 1, are becoming apparent. Despite ~heir lack of sequence homology both proteins contain two orresponding functional domains. The N-terminal domain is ~ich in basic amino acid residues and serves as a nuclear i ocalisation signal, which also has nucleolar localising capabilty [10]. In addition the same domain is the site for multi-' Corresponding author. Fax: (44) (171) 955-4191. E-mail: k.white@umds.ac.ukIbbreviations. FITC, fluorescein isothiocyanate; HIV, human immunodeficiency virus; HTLV-1, human T-cell leukaemic virus type 1 ; NOS, nucleolar localisation signal; RexRE, Rex-responsive element merisation [11] and for binding to the responsive element on the viral RNA [8,12]. A C-terminal domain encodes a leucinerich effector domain which includes a recently characterised nuclear export signal [13,14]. The combination of nuclear import and export signals accounts for the demonstrated ability of Rev to shuttle between the nucleus and cytoplasm [15,16].Two models have been proposed to account for the retention of unspliced viral mRNAs in the nucleus and for the relief of the ...

Expression of recombinant human ceruloplasmin – an absolute requirement for splicing signals in the expression cassette

“…The patterns of Cp mRNA distribution (Fig. 2 ) were evaluated semi‐quantitatively [24]and the results substantiate the above finding that export of mRNA was enhanced when expressed from pNUT‐I‐Cp.…”

Involvement of the nucleolus in replication of human viruses

“…The nucleolus also plays a central role in the nuclear export of mRNAs encoding the structural and enzymatic proteins of the HTLV. HTLV Rex protein, in association with CRM1, activates viral mRNA export, while viral protein p30 is suspected to prevent it [41][42][43]. Both Rex and p30 proteins are found in the nucleolus [44][45][46].…”

Anchoring an extended HTLV-1 Rex peptide within an RNA major groove containing junctional base triples

“…The direct interaction between Rex and RxRE has been established by various in vitro binding assays and is essential for its in vivo activity. Within RxRE, a segment of 24 nucleotides on stem-loop IID that contains two bulges was identified as the primary binding site for Rex by sitedirected mutation, chemical protection, in vitro selection, nuclease protection and modification-interference studies [6][7][8][9][10][11][12]. Recent studies have redefined this binding site to include a larger segment of adjacent RNA [13].…”