Structural Model for the Cooperative Assembly of HIV-1 Rev Multimers on the RRE as Deduced from Analysis of Assembly-Defective Mutants

Abstract

The functional efficacy of the HIV-1 Rev protein is highly dependent on its ability to assemble onto its HIV-1 RNA target (the RRE) as a multimeric complex. To elucidate the mechanism of multimeric assembly, we have devised two rapid and broadly applicable strategies for examining cooperative interactions between proteins bound to RNA, one based on cooperative translational repression of a two-site reporter and the other on gel shift analysis with crude E. coli extracts. Using these strategies, we have identified two distinct surfaces of Rev (head and tail) that are critical for different steps in multimeric assembly. Our data indicate that Rev assembles cooperatively on the RRE via a series of symmetrical tail-to-tail and head-to-head protein-protein interactions. The insights into molecular architecture suggested by these findings have enabled us to derive a structural model for Rev and its multimerization on the RRE.

Effects of human T‐cell leukemia virus type 1 (HTLV‐1) p13 on mitochondrial K+ permeability: A new member of the viroporin family?

“…Biochemical fractionation studies showed that p13 is indeed inserted as integral membrane protein in the inner mitochondrial membrane (see below; [6]). The high flexibility score of the PPTSSRP motif (residues [42][43][44][45][46][47][48] suggests that this might represent a hinge region. The C-terminal region (residues 65-75) is predicted to fold as a b-sheet structure.…”

Phenotypic analysis of human immunodeficiency virus type 1 Rev trimerization-interface mutants in human cells

“…Rev binds to an elaborate RNA structure, the Rev-response element (RRE), and promotes nuclear export of RRE-containing mRNAs that encode gag/pol and env (Pollard & Malim, 1998). Regions required for RNA binding, trans-activation and multimerization have been mapped within Rev (Daly et al, 1989;Malim & Cullen, 1991;Malim et al, 1989 Malim et al, , 1990Olsen et al, 1990;Pollard & Malim, 1998), but the precise features that determine the specificity and stability of the Rev-multimerization process have yet to be defined fully.An elegant genetic selection was used to identify Rev mutants with deficiencies in the Rev multimeric-assembly pathway (Jain & Belasco, 2001). Three classes of multimerization defect were resolved.…”

“…Amino acid residues 11-32 of the amino-terminal helix constitute multimerization domain I (MD-I), whereas aa 52-67, which constitute multimerization domain II (MD-II), are part of a second, longer helical region that also includes the Rev RNA-binding domain. The helical regions of MD-I and -II are held together through hydrophobic interactions and amino acid residues comprising the dimerization and trimerization interfaces map to opposite, solvent-exposed sides of the antiparallel helices (Jain & Belasco, 2001). …”

“…The helical regions of MD-I and -II are held together through hydrophobic interactions and amino acid residues comprising the dimerization and trimerization interfaces map to opposite, solvent-exposed sides of the antiparallel helices (Jain & Belasco, 2001). The genetic screen used to resolve Rev-multimerization defects was based on Rev-dependent translational repression of a target RNA in bacteria (Jain & Belasco, 2001). To date, the phenotype of Rev trimerization-interface mutants has not been examined in a mammalian cell system capable of recapitulating Rev-dependent gene expression.…”

“…An elegant genetic selection was used to identify Rev mutants with deficiencies in the Rev multimeric-assembly pathway (Jain & Belasco, 2001). Three classes of multimerization defect were resolved.…”