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

Abstract

Nuclear export of unspliced and incompletely spliced human immunodeficiency virus type 1 mRNA is mediated by the viral Rev protein. Rev binds to a structured RNA motif known as the Rev-response element (RRE), which is present in all Rev-dependent transcripts, and thereby promotes entry of the ribonucleoprotein complex into the nuclear-export pathway. Recent evidence indicates that a dimerization interface and a genetically separable 'trimerization' interface are required for multimeric assembly of Rev on the RRE. In this report, the effect of mutations within the trimerization interface on Rev function was examined in mammalian cells. All trimerization-defective Rev molecules had profoundly compromised Rev function and a range of localization defects was observed. However, despite the potential for formation of heterodimers between functional and non-functional Rev proteins, trimerization-defective Rev mutants were unable to inhibit wild-type Rev function in a trans-dominant-negative manner.The human immunodeficiency virus (HIV) rev gene product is a prototypic example of a class of functionally diverse, arginine-rich, sequence-specific RNA-binding proteins. 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. Class one Rev mutants bind to the RRE as monomers, but are defective in their ability to form dimers; consequently, these mutants do not form multimers readily on the RRE. Moreover, class three mutants exhibit defects at all stages of RRE binding and Rev dimerization and multimerization and are probably structurally defective. In contrast, and perhaps most interestingly, class two mutants are competent for dimerization and RNA binding, but show greatly reduced multimerization properties. Thus, Jain & Belasco (2001) were able to genetically separate the process of dimerization from the subsequent process of multimeric Rev assembly. The refined molecular model (Jain & Belasco, 2001) suggests that there are two Rev-interaction surfaces; one surface is required for Rev-Rev dimerization, whereas the second is required for trimerization and higher-order assembly (Fig. 1a).Considerable evidence suggests that the amino-terminal half of Rev adopts a helix-loop-helix motif (Hope et al., 1990;Jain & Belasco, 2001;Madore et al., 1994;Pollard & Malim, 1998;Thomas et al., 1998;Zapp et al., 1991). Amino acid residues 11-32 of the amino-terminal helix cons...

Implications of the HIV-1 Rev dimer structure at 3.2 Å resolution for multimeric binding to the Rev response element

“…Additionally, the lateral position for L18 may explain its putative role in HIV-1 latency by slightly hindering Rev oligomerization, which in turn reduces Rev function, leading to less Gag expression and greater possibility for infected cells to escape anti-Gag cytotoxic T-cell responses. Mutations of the polar residues Y23, S25, and N26 disrupt oligomerization, resulting in an increased cytoplasmic concentration of Rev (2,24,25). Y23 makes significant contacts both within the Rev monomer and at the A-A interface, explaining the functional phenotype; S25 and N26 are on the B-surface and so support the imputed role of this surface in Rev oligomerization.…”

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

“…Shuttling Rev between the cytoplasm and the nucleus requires an interaction between unbound Rev and importins such as importin β, as well as an interaction between Rev–RNA complexes and the export receptor Crm‐1 (Pollard and Malim 1998). Both L60R and M4‐Rev with mutations to positions 23, 25, and 26 (Malim et al 1989b) show an increased presence in the cytoplasm relative to wild type (Szilvay et al 1997; Trikha and Brighty 2005). This pattern of cytoplasmic localization has been cited as evidence for an oligomerization requirement for nuclear import (Trikha and Brighty 2005).…”

“…Both L60R and M4‐Rev with mutations to positions 23, 25, and 26 (Malim et al 1989b) show an increased presence in the cytoplasm relative to wild type (Szilvay et al 1997; Trikha and Brighty 2005). This pattern of cytoplasmic localization has been cited as evidence for an oligomerization requirement for nuclear import (Trikha and Brighty 2005). Cytoplasmic accumulation of Rev also results from increasing the rate of Rev export (Soros and Cochrane 2001).…”

“…In the previous study by Jain and Belasco (2001), the behavior of Rev mutations that disrupt oligomeric binding on the RRE was used to support a model of oligomerization wherein a helix‐turn‐helix motif undergoes oligomerization into groups of four‐helix bundles. Their work was based on studies of Rev mutants that have established Rev oligomerization on the RRE as a prerequisite for export activity (Malim and Cullen 1991) and that have been used to map the Rev oligomerization interfaces (Malim and Cullen 1991; Thomas et al 1997, 1998; Trikha and Brighty 2005). However, because the relationship between oligomeric binding and RNA‐free Rev was untested and because equal changes in oligomerization have been related to different effects on trans ‐activation (Thomas et al 1997; Brice et al 1999; Trikha and Brighty 2005; Churchill et al 2007), further investigation of the Jain and Belasco (2001) model was warranted.…”

“…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.…”