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

Roochi Trikha and David W. Brighty

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


   ABSTRACT
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
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.

A supplementary figure showing localization of RevFlag and mutant RevFlag proteins in 293T cells is available in JGV Online.


   MAIN TEXT
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
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, 1990; Olsen 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).



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 1. Structural features required for Rev multimerization. (a) Rev multimerizes along the RRE through a series of tail–tail and head–head interactions. A Rev monomer is represented as a shaded oval, where the dimerization ‘tail’ interface is shown in black and the trimerization ‘head’ interface in grey. Upper part: Rev monomer binds the high-affinity site on the RRE such that the ‘tail’ interface is available for dimerization. Trimerization occurs through subsequent head–head interactions, thereby promoting multimeric assembly of Rev on the RRE. Lower part: a model for trans-dominant inhibition by trimerization-deficient mutants, whereby Rev monomers containing mutations in the trimerization interface dimerize readily with wild-type Rev, but progression to a trimeric complex is blocked due to the defective trimerization interface (represented by X). (b) The locations of MD-I and MD-II within Rev are depicted. The amino acids mutated in this study are shown in bold. NES, Nuclear-export signal; NLS, nuclear-localization signal; RBD, RNA-binding domain.

 
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 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 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{Delta}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. 2a–f). 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. 2p–r), 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. 2g–i). In marked contrast, Rev{Delta}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{Delta}H and RevL12E/V16D were observed within nuclei (Fig. 2j–o). Thus, multiple mutations affecting the multimerization-assembly domains of Rev had a severe impact on the localization of Rev in human cells.



View larger version (83K):
[in this window]
[in a new window]
 
Fig. 2. Localization of RevFlag and mutant RevFlag proteins in HeLa cells. HeLa cells were transiently transfected with 1 µg wild-type or mutant RevFlag expression constructs as indicated. To localize these proteins, cells were fixed, permeabilized and stained with anti-Rev mAb287 primary antibody and FITC-conjugated goat anti-mouse IgG secondary antibody. Corresponding DAPI, phase-contrast and FITC-specific fluorescence images are shown. Identical results were obtained from human 293T cells (see Supplementary Figure in JGV Online) and primate Cos-1 cells (data not shown).

 
Multimerization of Rev is an essential aspect of Rev function (Jain & Belasco, 2001; Pollard & Malim, 1998). Consequently, it was anticipated that the novel trimerization mutants would exhibit profound defects in their ability to support Rev-dependent gene expression. The Rev-dependent reporter construct pCMV-160{Delta}SA was therefore used to assess the function of the Rev mutants. The vector pCMV-160{Delta}SA is similar to previously described Rev-dependent envelope constructs (Churchill et al., 1996) and carries an envelope-derived reporter gene under the transcriptional control of the cytomegalovirus immediate-early promoter and the transcription unit is terminated by an SV40 early polyadenylation signal. To test the functional activity of the Rev mutants, pCMV-160{Delta}SA was transfected into HeLa cells on its own, in combination with each of the mutant Rev constructs or, alternatively, with each of the control vectors expressing RevFlag or RevM10. Subsequently, the transfected cells were examined for envelope expression by Western blotting using polyclonal antisera directed against the gp120 region of the HIV-1 envelope. In keeping with previous results (Brighty & Rosenberg, 1994; Churchill et al., 1996), envelope expression from pCMV-160{Delta}SA was highly responsive to Rev; little or no envelope protein was produced in the absence of Rev and the block to envelope expression could be overcome by supplying Rev in trans (Fig. 3a). However, each of the Rev trimerization-interface mutants demonstrated substantial defects in the Rev response. Among the Rev-assembly mutants, only RevL60R had extremely weak Rev activity, and very low levels of envelope protein were detectable from cells transfected with both pCMV-160{Delta}SA and pRevL60R (Fig. 3a). In contrast, RevI52D and the mutants with multiple mutations, Rev{Delta}H and RevL12E/V16D, like the control mutant RevM10, lacked functional Rev activity, as cells transfected with these Rev variants did not express detectable levels of envelope protein (Fig. 3a). Most importantly, all of the Rev variants were expressed in transfected cells (Fig. 3b). Only the control class three mutant RevI52D was detected at low levels, due in part to a reduced stability of this particular Rev variant, but also due to reduced recognition by the mAb, which recognizes an epitope within this region of Rev (data not shown). Surprisingly, the trimerization mutants displayed anomalous migration patterns by Western blotting (Fig. 3b). It is suspected that mutations within the helical regions of Rev permit anomalous covalent modification of Rev in cells or alternatively that, compared with viral Rev, these mutants adopt somewhat extended structures under SDS-PAGE conditions.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 3. Expression and activity of Rev mutants in HeLa cells. (a) Cells were co-transfected with 0·5 µg envelope-derived reporter pCMV-160{Delta}SA and 0·5 µg RevFlag or mutant RevFlag expression constructs as indicated above the lanes. Irrelevant plasmid (pKS) was transfected to keep the total amount of DNA transfected constant at 1 µg. The envelope open reading frame in pCMV-160{Delta}SA carries a deletion removing the transmembrane region of gp41; consequently, the gp160-derived primary-translation product and mature gp120 are secreted into the culture medium. Cell-culture supernatants were harvested 72 h post-transfection and assayed for envelope expression by Western blotting using polyclonal anti-gp120 antibody; gp120 and gp160 precursors are indicated. (b) Cells transfected with control vector (pKS) or RevFlag or mutant RevFlag expression vectors (as indicated above the lanes) were examined by Western blotting using the anti-Rev mAb287. To demonstrate equal loading, the samples were additionally probed with an anti-actin mAb (Sigma). (c) HeLa cells were transfected with 0·5 µg envelope-expression construct pCMV-160{Delta}SA, 0·25 µg RevFlag and 0·25 µg each of the mutant RevFlag-expression constructs as indicated above the lanes. Each transfection mix was adjusted to a total of 1 µg with carrier DNA (pKS). Cell-culture supernatants were analysed by Western blotting using anti-gp120 antibody. At high levels of wild-type Rev vector (pRevFlag; lane WT), a slight but reproducible drop in envelope expression was observed. It is suspected that this is due to titration or ‘squelching’ of factors required for Rev-dependent nuclear export; this highlights the sensitivity of the assay. Marker sizes are in kDa.

 
An intriguing aspect of the model of Jain & Belasco (2001) is that it appears to suggest that multimerization mutants, which affect the key amino acids required for trimerization, but leave the dimerization interface unaffected, should exert a trans-dominant-negative effect on Rev-dependent gene expression (Fig. 1a). In particular, heterodimerization of functional Rev with an inactive, trimerization-defective Rev molecule would be expected to block higher-order Rev assembly and thereby inhibit or ‘squelch’ the activity of ‘wild-type’ Rev. To investigate this view, HeLa cells were co-transfected with each of the Rev-assembly mutants in combination with pRevFlag and the envelope reporter construct pCMV-160{Delta}SA; subsequently, the production of viral envelope was monitored by Western blotting. As a control for this assay, the prototypic trans-dominant-negative Rev mutant, RevM10, was also assessed for inhibition of Rev-dependent gene expression. As expected, in the presence of control Rev, abundant levels of HIV envelope were produced by cells transfected with pCMV-160{Delta}SA (Fig. 3c) and, most importantly, co-expression of RevM10 abolished envelope production completely (Fig. 3c). In stark contrast, it was found that none of the Rev-assembly mutants were capable of inhibiting the activity of wild-type Rev significantly. In each case, cells expressing both an assembly-mutant and a functional Rev were also capable of expressing substantial amounts of envelope protein in a Rev-responsive manner (Fig. 3c). Thus, contrary to expectation, trimerization-defective Rev mutants did not display significant trans-dominant-negative activity.

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 protein–RNA 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.


   ACKNOWLEDGEMENTS
 
We thank AVERT for supporting this work through a prize studentship awarded to R. T.


   REFERENCES
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
Brighty, D. W. & Rosenberg, M. (1994). A cis-acting repressive sequence that overlaps the Rev-responsive element of human immunodeficiency virus type 1 regulates nuclear retention of env mRNAs independently of known splice signals. Proc Natl Acad Sci U S A 91, 8314–8318.[Abstract/Free Full Text]

Churchill, M. J., Moore, J. L., Rosenberg, M. & Brighty, D. W. (1996). The Rev-responsive element negatively regulates human immunodeficiency virus type 1 env mRNA expression in primate cells. J Virol 70, 5786–5790.[Abstract]

Daly, T. J., Cook, K. S., Gray, G. S., Maione, T. E. & Rusche, J. R. (1989). Specific binding of HIV-1 recombinant Rev protein to the Rev-responsive element in vitro. Nature 342, 816–819.[CrossRef][Medline]

Fasken, M. B., Saunders, R., Rosenberg, M. & Brighty, D. W. (2000). A leptomycin B-sensitive homologue of human CRM1 promotes nuclear export of nuclear export sequence-containing proteins in Drosophila cells. J Biol Chem 275, 1878–1886.[Abstract/Free Full Text]

Hope, T. J., McDonald, D., Huang, X., Low, J. & Parslow, T. G. (1990). Mutational analysis of the human immunodeficiency virus type 1 Rev transactivator: essential residues near the amino terminus. J Virol 64, 5360–5366.[Medline]

Jain, C. & Belasco, J. G. (2001). Structural model for the cooperative assembly of HIV-1 Rev multimers on the RRE as deduced from analysis of assembly-defective mutants. Mol Cell 7, 603–614.[CrossRef][Medline]

Madore, S. J., Tiley, L. S., Malim, M. H. & Cullen, B. R. (1994). Sequence requirements for Rev multimerization in vivo. Virology 202, 186–194.[CrossRef][Medline]

Malim, M. H. & Cullen, B. R. (1991). HIV-1 structural gene expression requires the binding of multiple Rev monomers to the viral RRE: implications for HIV-1 latency. Cell 65, 241–248.[CrossRef][Medline]

Malim, M. H., Böhnlein, S., Hauber, J. & Cullen, B. R. (1989). Functional dissection of the HIV-1 Rev trans-activator – derivation of a trans-dominant repressor of Rev function. Cell 58, 205–214.[CrossRef][Medline]

Malim, M. H., Tiley, L. S., McCarn, D. F., Rusche, J. R., Hauber, J. & Cullen, B. R. (1990). HIV-1 structural gene expression requires binding of the Rev trans-activator to its RNA target sequence. Cell 60, 675–683.[CrossRef][Medline]

Olsen, H. S., Cochrane, A. W., Dillon, P. J., Nalin, C. M. & Rosen, C. A. (1990). Interaction of the human immunodeficiency virus type 1 Rev protein with a structured region in env mRNA is dependent on multimer formation mediated through a basic stretch of amino acids. Genes Dev 4, 1357–1364.[Abstract]

Pollard, V. W. & Malim, M. H. (1998). The HIV-1 Rev protein. Annu Rev Microbiol 52, 491–532.[CrossRef][Medline]

Thomas, S. L., Oft, M., Jaksche, H., Casari, G., Heger, P., Dobrovnik, M., Bevec, D. & Hauber, J. (1998). Functional analysis of the human immunodeficiency virus type 1 Rev protein oligomerization interface. J Virol 72, 2935–2944.[Abstract/Free Full Text]

Truant, R. & Cullen, B. R. (1999). The arginine-rich domains present in human immunodeficiency type 1 Tat and Rev function as direct importin {beta}-dependent nuclear localization signals. Mol Cell Biol 19, 1210–1217.[Abstract/Free Full Text]

Zapp, M. L., Hope, T. J., Parslow, T. G. & Green, M. R. (1991). Oligomerization and RNA binding domains of the type 1 human immunodeficiency virus Rev protein: a dual function for an arginine-rich binding motif. Proc Natl Acad Sci U S A 88, 7734–7738.[Abstract/Free Full Text]

Received 1 September 2004; accepted 5 February 2005.



This Article
Abstract
Full Text (PDF)
Supplementary figure
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Trikha, R.
Articles by Brighty, D. W.
Articles citing this Article
PubMed
PubMed Citation
Articles by Trikha, R.
Articles by Brighty, D. W.
Agricola
Articles by Trikha, R.
Articles by Brighty, D. W.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS