Sensitive in vitro analysis of HIV-1 Rev multimerization

Abstract

Oligomerization of the Rev protein of human immuno-deficiency virus type 1 on its cognate response element is essential for export of the late viral mRNAs from the nucleus. Two regions of the protein, flanking the RNA binding site, have been defined as oligomerization sites after mutants (M4 and M7) had been reported to bind specifically to the response element but not to oligomerize in vivo or in vitro. These mutants are often used as paradigms for studies of Rev multimerization. We have re-examined the in vitro binding of these mutants to model Rev response elements, using improved gel mobility assays. We find that both mutants will form oligomers on the Rev response element, but have somewhat lower affinities for RNA than the wild-type protein. M7 has lower specific affinity, but shows little deficiency in oligomerization once binding starts. In contrast, M4 is multimerization deficient, as previously reported. Therefore, whilethe sites are correctly defined, it is inappropriate to employ the original M7 deletion mutant to study Rev oligomerization.

Thermodynamic Profiling of HIV RREIIB RNA–Zinc Finger Interactions

“…The nuclear export of the viral pre-mRNA in unspliced and singly spliced forms is facilitated by the interaction between the regulatory protein Rev and the Rev responsive element (RRE), a 234-nucleotide-long untranslated RNA structure located within the env gene of the viral RNA genome (3,9,13,14). Rev initially binds RRE at a high affinity-binding site, localized to a relatively small stem loop structure, RREIIB (10,11), followed by subsequent oligomerisation of up to a total of 8 Rev molecules through RNA-protein contacts (12). This forms the nuclear export signal, which utilizes the cellular nuclear export machinery to translocate to the cytoplasm.…”

Protein structure and oligomerization are important for the formation of export‐competent HIV‐1 Rev–RRE complexes

“…Understanding the essential contribution made by Rev oligomerization to influence the course of the viral infection (Malim and Cullen 1991; Madore et al 1994; Mann et al 1994) has been hampered by the resulting lack of detailed structural information on the oligomeric complexes. Many questions concerning the connection between Rev structure and oligomeric Rev–RRE assembly (Blanco et al 2001; Havlin et al 2007), the self‐association tendency of Rev in the absence of its cognate RRE (Wingfield et al 1991; Cole et al 1993; Havlin et al 2007), and the observation of Rev variants with similar assembly properties on the RNA but profoundly different levels of trans ‐activation activity (Thomas et al 1997; Brice et al 1999; Trikha and Brighty 2005; Churchill et al 2007) remain to be answered.…”

The molecular biology of bovine immunodeficiency virus: a comparison with other lentiviruses

“…Beside the tat gene, lentivirus genomes, as mentioned above, carry another important gene coding for the Rev protein. Rev acts at the post-transcriptional level, whereas the Tat protein regulates viral gene expression at the transcriptional level (Mikaelian et al, 1996;Brice et al, 1999). Rev regulates the expression of viral structural proteins in HIV, SIV and MVV by facilitating the transport of unspliced and singly spliced transcripts from the nucleus to the cytoplasm of infected cells (Felber et al, 1989;Cheng et al, 1990;Tiley et al, 1990).…”