Identification of a high-affinity RNA-binding site for the human immunodeficiency virus type 1 Rev protein.

Abstract

Expression of the structural proteins of human immunodeficiency virus type 1 requires the direct interaction of multiple copies of the viral Rev protein with its highly structured RNA target sequence, the Rev response element (RRE). Nucleotides critical for Rev monomer binding have been mapped by chemical interference to a single site flanking the base of an RNA helix (stem HB) located within the 234-nucleotide RRE. Binding of additional Rev molecules to an RRE probe did not require any RNA primary sequence information detectable by modification interference beyond that required for binding of a single Rev protein molecule. A synthetic 29-nucleotide RNA molecule designed to incorporate nucleotides identified as critical for Rev binding retained the ability to bind Rev specifically and, therefore, represents a minimal Rev-binding site. We propose that Rev binding to the RRE initiates with the direct interaction of a Rev monomer with a high-affinity binding site located at the base of the IIB stem of the RRE. The subsequent formation of Rev multimers on the RRE appears, in contrast, primarily driven by specific protein-protein interactions.The Rev gene product of human immunodeficiency virus type 1 (HIV-1) is an RNA-sequence-specific nuclear regulatory protein essential for HIV-1 replication in culture (1-6). The target sequence for Rev, the rev response element (RRE), was initially defined as a complex 234-nucleotide (nt) RNA stem-loop structure located within the viral env gene (7). At least two, and possibly as many as eight, Rev molecules have been shown to bind specifically to the full-length [8][9][10]. Footprinting experiments suggest that these Rev protein molecules occupy discrete sites on the RRE (10). Although we and others have argued that the formation of Rev multimers on the RRE reflects the sequential binding of multiple Rev monomers (8, 9), it has also been suggested that the formation of multimers may be a prerequisite for Rev binding (11).Mutational analysis of the 116-amino acid Rev protein has shown that an arginine-rich stretch of amino acids is critical for sequence-specific binding to the RRE, whereas flanking sequences are important for the formation of Rev multimers on the RRE (8,11,12). It has therefore been proposed that this Rev "multimerization" is mediated, at least in part, by protein-protein interactions (8,9,11,12). Rev mutants that have lost the ability to form multimers on the RRE in vitro display a recessive negative phenotype in vivo (8,11).Mutational analysis of the RRE has defined a 66-nt subdomain, termed stem-loop II (SLII) (Fig. 1A), that is both necessary and sufficient for high-affinity Rev binding in vitro (4,5) and that displays at least partial RRE function in vivo (16). This RRE subdomain can bind up to three Rev molecules in vitro (9, 10). More recently, a 40-nt sequence contained within SLII has been proposed as a minimal RNA substrate based on its ability to bind a single Rev monomer (9). Here, we identify several noncontiguous nucleotides located within S...

Nucleocytoplasmic transport of HTLV-1 RNA is regulated by two independent LTR encoded nuclear retention elements

“…Previous reports indicate that expression of unspliced or singly spliced HTLV-1 RNAs is completely dependent upon action of Rex, and that the mechanism of this dependence is essentially identical to that observed with HIV Rev (Hope et al, 1991;Inoue et al, 1986;Malim et al, 1990;Tiley et al, 1992). Rev dependence may be related to the presence of constitutive repressive elements in the viral transcripts encoding structural genes (Brighty and Rosenberg, 1994;Churchill et al, 1996;Cochrane et al, 1991;Felber et al, 1989;Mikaelian et al, 1996;Nasioulas et al, 1994;Schwartz et al, 1992a,b).…”

The VP16 transcription activation domain is functional when targeted to a promoter-proximal RNA sequence.

“…The stem-loop IIB (SLIIB) subdomain of the HIV-1 RRE is necessary and sufficent for Rev binding in vitro and is critical for RRE function in vivo (Malim et al 1990;Bartel et al 1991;Heaphy et al 1991;Tiley et al 1992; Kjems et al 1992). To test whether the SLIIB sequence is also sufficient for Rev binding in vivo, we substituted a 29-nucleotide SLIIB RNA sequence in place of the essential apical region of TAR (Fig.…”

The packaging signal of influenza viral RNA molecules

“…Incorporation of viral RNA molecules into progeny virions must rely on specific, discriminating structural feature(s) present in all of the RNA segments, but at the same time remain independent of most of their nucleotide sequences+ Recognition of the respective structural element, the packaging signal, during morphogenesis of enveloped viruses also is a central element in the controlled merger of two separately synthesized groups of constituents, the viral glycoproteins and the viral RNP complexes, at the appropriate cellular membrane, plasma membrane or other+ Packaging signals have been determined among RNA viruses so far only for coronaviruses (Fosmire et al+, 1992), Sindbis virus (Geigenmüller-Gnirke et al+, 1993), retroviruses (Tiley et al+, 1992;Ippolito & Steitz, 2000), and also for hepatitis B viruses (Pollack & Ganem, 1993), and are not known for segmented nucleoprotein-covered minus-strand viral RNAs including those of influenza virus+…”