The Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UK
Correspondence
David W. Brighty
brighty{at}cancer.org.uk
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ABSTRACT |
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A supplementary figure showing localization of RevFlag and mutant RevFlag proteins in 293T cells is available in JGV Online.
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MAIN TEXT |
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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 RevRev dimerization, whereas the second is required for trimerization and higher-order assembly (Fig. 1a
).
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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. Therefore, the effects of such mutations on Rev expression, localization and trans-activation of viral gene expression are unknown. Moreover, classically identified Rev-multimerization mutants have a uniformly recessive negative phenotype (Malim et al., 1989
; Malim & Cullen, 1991
). It is therefore surprising to note that the current Rev-assembly model appears to predict that novel Rev mutants that are competent for dimerization, but incompetent for trimerization, should inhibit or squelch the activity of native Rev through the formation of non-functional heterodimers (Fig. 1a
). To address these issues, the phenotype of novel Rev-trimerization mutants was examined in human cells.
Amino acid residues L12, V16 and L60 (Fig. 1b) appear to be important for trimerization and higher-order assembly of Rev (Jain & Belasco, 2001
). Sequence comparisons between diverse HIV isolates in the Los Alamos HIV database (http://hiv-web.lanl.gov/content/index) revealed that L12 was invariant among the isolates examined and only conservative substitutions were found at positions 16 and 60. These observations lend considerable support to the notion that L12, V16 and L60 are key functional residues within the trimerization interface of Rev.
To assess the impact of mutations within the assembly interface on Rev function, site-directed mutagenesis was used to generate a series of mutant Rev constructs. The single substitution RevL60R was constructed, as this particular substitution exhibits in vitro properties that are broadly typical of the trimerization-defective mutants and because it shows a strong tendency to bind to the RRE as a dimer (Jain & Belasco, 2001). As multiple hydrophobic contacts occur across the trimerization interface, it was suspected that single amino acid substitutions would exhibit a weak or leaky phenotype in mammalian cell-based assays, whereas combined mutations should provide a robust phenotype. Therefore, Rev constructs with multiple mutations were generated: RevL12E/V16D and Rev
H (L12E, V16D and L60R). For comparison and as a control, RevI52D was also generated, as defects at this particular residue are typical of class three mutants and affect all stages of Rev dimerization, RNA binding and higher-order assembly.
Under steady-state conditions in both infected CD4+ T cells and heterologous cell types, HIV Rev localizes primarily to the nucleus and nucleoli and this pattern of localization is essential to Rev function. To examine the subcellular localization of the novel Rev-multimerization mutants, vectors expressing each mutant, the prototypic trans-dominant mutant RevM10 (Malim et al., 1989) and the control vector RevFlag were transfected into HeLa cells. Twenty-four hours post-transfection, cells were fixed and Rev protein was detected by using a Rev-specific monoclonal antibody, mAb287, and fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Fig. 2
). As observed previously (Fasken et al., 2000
; Madore et al., 1994
; Pollard & Malim, 1998
), the control Rev protein and the RevM10 mutant (Malim et al., 1989
) were observed principally within the nucleoplasm and nucleoli of transfected cells and very little Rev was detected within the cytoplasmic compartment (Fig. 2af
). However, the Rev-multimerization mutants displayed a range of localization phenotypes. The RevI52D mutant localized to the nuclei and nucleoli in a pattern that was indistinguishable from that of control Rev (Fig. 2pr
), whereas RevL60R demonstrated a more diffuse pattern of staining. RevL60R could be detected in both the nucleoplasm and nucleoli, but also demonstrated a low-level diffuse staining throughout the cytoplasm of cells (Fig. 2gi
). In marked contrast, Rev
H and RevL12E/V16D had dramatically different patterns of staining. Both mutants were essentially restricted to the cytoplasmic compartment of cells and only exceedingly low levels of Rev
H and RevL12E/V16D were observed within nuclei (Fig. 2jo
). Thus, multiple mutations affecting the multimerization-assembly domains of Rev had a severe impact on the localization of Rev in human cells.
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Here, mammalian cell-based assays have been used to examine the phenotype of Rev mutants carrying amino acid substitutions within the trimerization interface of Rev. As expected, each of the trimerization mutants demonstrated dramatically reduced functional activity in Rev-dependent reporter assays; this is consistent with the lack of multimerization potential of these mutants. However, trimerization mutants had pleiotropic defects. Given that the arginine-rich nuclear-localization signal was intact, it was surprising to find that some trimerization mutants demonstrated localization defects. This suggested that these Rev variants may not interact efficiently with the cellular factors that promote nuclear import (Truant & Cullen, 1999). Moreover, the refined molecular model of Rev assembly (Jain & Belasco, 2001
) appears to predict that heterodimerization of functional and trimerization-deficient Rev proteins should inhibit the function of wild-type Rev by blocking multimeric assembly. However, contrary to expectation, trimerization-deficient Rev proteins did not exhibit a trans-dominant-negative effect on Rev function. Given the anomalous migration of the trimerization-deficient Rev variants by Western blotting, it is suspected that these Rev mutants adopt conformations that differ distinctly from that of native Rev. If this is indeed the case, the pleiotropic defects in Rev function and the lack of trans-dominant-negative activity may simply reflect an inability of these Rev mutants to dimerize efficiently with wild-type Rev or to interact with the cellular co-factors that support Rev function. Thus, from our observations, it is clear that the data derived from in vitro assays of Rev multimerization, whilst providing valuable information on the biochemistry of proteinRNA interactions, are not particularly informative of the events that occur in mammalian cells. Therefore, a full understanding of the multimeric assembly of Rev on the RRE, with a view to developing antiviral drugs targeting the multimerization interaction, awaits a high-resolution crystal structure for Rev and a holistic approach to the structural and functional analysis of Rev assembly.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 1 September 2004;
accepted 5 February 2005.
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