Oligomerization of HIV-1 Rev Mutants in the Cytoplasm and during Nuclear Import

Szilvay, Anne Marie; Brokstad, Karl Albert; Bøe, Stig-Ove; Haukenes, Gunnar; Kalland, Karl‐Henning

doi:10.1006/viro.1997.8671

Cited by 33 publications

(44 citation statements)

References 32 publications

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“…As reported above and in earlier studies (32,42,43), Rev is localized in the nucleolus of cells; however, Rev mutants of the NLS localize mainly in the cytoplasm. These observations prompted us to determine whether Rev is able to oligomerize in the cytoplasm of cells.…”

Section: Visualization Of Rev Multimerization In the Nucleoli Of Livisupporting

confidence: 82%

In Vivo HIV-1 Rev Multimerization in the Nucleolus and Cytoplasm Identified by Fluorescence Resonance Energy Transfer

Daelemans¹,

Costes

Cho

et al. 2004

Journal of Biological Chemistry

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Nuclear export of intron-containing human immunodeficiency virus type 1 RNA is mediated by the viral Rev protein. Rev is a nucleocytoplasmic transport protein that directly binds to its cis-acting Rev-responsive element RNA. Rev function depends on its ability to multimerize. The in vivo dynamics and the subcellular dependence of this process are still largely unexplored. To visualize and quantitatively analyze the mechanism of Rev multimeric assembly in live cells, we used high resolution in vivo fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching. By using two different dynamic FRET approaches (acceptor photobleaching and donor bleaching time measurements), we observed a strong Rev-Rev interaction in the nucleoli of living cells. Most interestingly, we could also detect Rev multimerization in the cytoplasm; however, FRET efficiency in the cytoplasm was significantly lower than in the nucleolus. By using fluorescence recovery after photobleaching, we investigated the mobility of Rev within the nucleolus. Mathematical modeling of the fluorescence recovery after photobleaching recoveries enabled us to extract relative association and dissociation constants and the diffusion coefficient of Rev in the nucleolus. Our results show that Rev multimerizes in the nucleolus of living cells, suggesting an important role of the nucleolus in nucleocytoplasmic transport.The replication of the human immunodeficiency virus, type 1 (HIV-1), 1 is regulated in a temporal manner by its viral mRNA expression. Control of HIV RNA expression is complex and involves the interplay of cis-acting viral transactivators and several cellular proteins. Rev is a viral trans-activator protein that controls the differential expression of viral proteins at the post-transcriptional level by allowing the accumulation in the cytoplasm of unspliced and singly spliced viral RNA containing the Rev-responsive element (RRE) (1-7). Rev interacts directly with a purine-rich stem-loop within RRE (6,8), and through multimerization additional Rev molecules bind throughout the RRE. In the absence of Rev, the only viral RNA species that accumulate in the cytoplasm are multiply spliced and encode for the viral regulatory proteins. In the presence of Rev, the incompletely spliced mRNAs accumulate in the cytoplasm to serve as templates for viral structural protein synthesis or as viral genomes. Rev activity is therefore essential for virus replication.Rev is a small protein of 116 amino acids or 18 kDa. Its cellular localization is nucleolar, but it has been shown to shuttle continuously between the nucleus and the cytoplasm (1, 9, 10). Nuclear localization is mediated by a short stretch of basic amino acids characterized by eight arginine residues located near the amino terminus of Rev that serves both as nuclear localization signal (NLS) and as an RNA binding domain (5,11,12). It is flanked on both sides by sequences that contribute to Rev oligomerization on the RRE but have no detectable role in RRE binding. The NLS of Re...

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Section: Visualization Of Rev Multimerization In the Nucleoli Of Livisupporting

confidence: 82%

In Vivo HIV-1 Rev Multimerization in the Nucleolus and Cytoplasm Identified by Fluorescence Resonance Energy Transfer

Daelemans¹,

Costes

Cho

et al. 2004

Journal of Biological Chemistry

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“…The presence of NES(Ϫ)RanBP1 in such a complex may interfere with its transport, resulting in the observed inhibition of Rev activity. A similar mechanism has been described for the inhibition of Rev function by TD Rev (28,35). Our model would predict that the inhibitory activity of wild type and NES(Ϫ)RanBP1 will specifically affect this NESspecific pathway, but not other regulated nuclear export.…”

Section: Resultssupporting

confidence: 53%

Mutations in the Nuclear Export Signal of Human Ran-binding Protein RanBP1 Block the Rev-mediated Posttranscriptional Regulation of Human Immunodeficiency Virus Type 1

Zolotukhin¹,

Felber²

1997

Journal of Biological Chemistry

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We identified a region in the human Ran GTPasebinding protein RanBP1 that shares similarities to the nuclear export signal of the inhibitor of the cAMP-dependent protein kinase. Mutational analysis confirmed that this region is responsible for the cytoplasmic accumulation of RanBP1 and can functionally replace the nuclear export signal of Rev of human immunodeficiency virus type 1. We showed that RanBP1 interferes with Rev-mediated expression of human immunodeficiency virus type 1, whereas the RanBP1 with inactivated nuclear export signal abrogates Rev function. Expression of a Rev-independent molecular clone, which is regulated via the constitutive transport element (CTE) of the simian retrovirus type 1, is not affected. These findings indicate that Rev and RanBP1 compete for the same nuclear export pathway, whereas Rev-and the CTE-mediated pathways are distinct. The inhibition of Rev function is independent of the ability of RanBP1 to associate with Ran and therefore, it is not likely a result of interference with Ran function. These data suggest that RanBP1 interacts with Rev at the putative nuclear receptor and, hence, shares a step in posttranscriptional pathway with Rev.One of the best studied examples of regulation at the posttranscriptional level is that of the Rev-responsive element (RRE) 1 -containing mRNAs of HIV-1 (for reviews, see Refs. 1-5). The Rev protein binds to the RRE and promotes the export of the RRE-containing mRNA to the cytoplasm and their expression. Rev contains an RNA-binding domain and a nuclear export signal (NES). Mutations in the NES are transdominant (TD) and lead to inhibition of Rev's nucleocytoplasmic export resulting in inhibition of HIV expression. Nucleoporin-like proteins hRip/Rab have been shown to interact with the NES of Rev and are believed to be involved in the nuclear export of Rev (6, 7). Similar NES elements have been identified in the inhibitor of cAMP-dependent protein kinase (PKI) (8) and the 5 S RNA-binding protein TFIIIA (9) and have been shown to functionally replace the NES of HIV-1 Rev. We identified a related NES region in the Ran GTPase-binding protein 1 of human (hRanBP1) (10, 11) and mouse RanBP1/ HTF9-A (mRanBP1) (12) origin. They belong to a family of proteins that bind to the GTP-bound Ran that includes known and putative nucleoporins as well as proteins of unknown function (13-19). Ran GTPase is required for active transport of macromolecules through the nuclear pore (for recent reviews, see Refs. 20 and 21). Here, we show that the NES signals of RanBP1 and Rev functionally cross-interfere, indicating a competition for a common export pathway. NES(Ϫ)RanBP1 mutants completely abrogate Rev-mediated regulation of HIV-1 without affecting the regulation mediated by the posttranscriptional control element (CTE) of simian retrovirus type 1. Taken together, our data suggest that RanBP1 and Rev share a nuclear export pathway, which is distinct from the CTE-mediated pathway. EXPERIMENTAL PROCEDURESRecombinant DNA and Transfections-HIV-1 molecular clone pNL4...

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“…Although the two-hybrid system is an important tool to probe for protein interactions, the protein interactions required to justapose the GAL4 DNA binding domain with the VP16 trans-activator may not necessarily re¯ect the strength of multimerization required to form stable Rex oligomers in vivo, in particular on the RxRE target sequence. For example Rev mutants which failed to oligomerize with WT Rev in the two-hybrid system (Bogerd and Greene, 1993;Thomas et al, 1998) were clearly capable of forming mixed heteromultimers by coexpression and to retain WT Rev in the nucleus by protein-protein interactions (Stauber et al, 1995(Stauber et al, , 1998aSzilvay et al, 1995). Thus, data obtained by the di erent assay system have to be judged critically and should not simply be extrapolated to the in vivo situation.…”

Section: Discussionmentioning

confidence: 99%

“…Coexpression of Rev together with Rexp21-GFP or the highly TD mutant RevM10 inhibited Rev mediated trans-activation (Figure 4b). Since RevM10 was shown to inhibit Rev activity directly by the formation of inactive heteromultimers (Hope et al, 1992;Stauber et al, 1995;Szilvay et al, 1995), RevM10 was more trans-dominant compared to Rexp21-GFP. These results indicate that Rexp21-GFP is titrating factor(s) essential for Rex and Rev protein tra cking.…”

Section: Rexp21-gfp Is Actively Shuttling Between the Nucleus And Thementioning

confidence: 99%

Titration of cellular export factors, but not heteromultimerization, is the molecular mechanism of trans-dominant HTLV-1 Rex mutants

et al. 1999

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The HTLV-1 Rex protein is an essential shuttle protein required for nuclear export of unspliced and incompletely-spliced viral RNAs. Several trans-dominant (TD) mutant Rex proteins have been reported, however, the mechanism of trans-dominance is not known. We compared TD Rex mutants and found that a natural occurring Rex mutant, Rexp21, lacking the RNA binding domain, was highly TD and inhibited also HIV-1 Rev function. Using fusions to the green uorescent protein (GFP) we observed that Rexp21-GFP displayed a cytoplasmic localization but was actively shuttling between the nucleus and the cytoplasm in live human cells.

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Oligomerization of HIV-1 Rev Mutants in the Cytoplasm and during Nuclear Import

Cited by 33 publications

References 32 publications

In Vivo HIV-1 Rev Multimerization in the Nucleolus and Cytoplasm Identified by Fluorescence Resonance Energy Transfer

In Vivo HIV-1 Rev Multimerization in the Nucleolus and Cytoplasm Identified by Fluorescence Resonance Energy Transfer

Mutations in the Nuclear Export Signal of Human Ran-binding Protein RanBP1 Block the Rev-mediated Posttranscriptional Regulation of Human Immunodeficiency Virus Type 1

Titration of cellular export factors, but not heteromultimerization, is the molecular mechanism of trans-dominant HTLV-1 Rex mutants

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