HIV-1 Rev Is Capable of Shuttling between the Nucleus and Cytoplasm

doi:10.1006/viro.1994.1516

Virology

Volume 204, Issue 1, October 1994, Pages 123-131

https://doi.org/10.1006/viro.1994.1516 Get rights and content

Abstract

The mechanism by which Rev facilitates the export, and consequently, the translation of the structural protein mRNAs of the human immunodeficiency virus type 1 remains undefined. Previous immunolocalization has determined that Rev is predominately in the nucleus with significant accumulation in the nucleolus, a localization consistent with the assumed site of Rev action. To determine whether the subcellular distribution is more dynamic than what was indicated by the original studies, the capacity of Rev to shuttle between the nucleus and cytoplasm was examined. It was observed that treatment of cells with DRB or actinomycin D resulted in a dramatic alteration in Rev distribution, the majority of the protein being found in the cytoplasm. Removal of the drug resulted in a rapid accumulation of Rev in the nucleus indicating that the block to nuclear import was reversible. Subsequent studies indicated that the movement of Rev into the cytoplasm was a passive process while its accumulation in the nucleus was an active one, given that only the latter displayed sensitivity to temperature. Finally, it was demonstrated that, while extensive redistribution of Rev could be attained by inhibition of RNA polymerase I alone, Rev was still capable of inducing expression of HIV structural gene expression under these conditions. Consequently, Rev activity does not appear to be dependent on either an intact nucleolus or the accumulation of the protein in the nucleus.

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Cited by (85)

Effects of positively charged arginine residues on membrane pore forming activity of Rev-NIS peptide in bacterial cells
2011, Biochimica et Biophysica Acta - Biomembranes
Citation Excerpt :
This series of processes are carried out by the direct interaction between the Rev protein and its structural RNA target, on such HIV-1 mRNAs, the specific region entitled the Rev responsive element (RRE) [35–37]. Furthermore, many studies regarding HIV-1 have suggested that Rev is shuttling between the cytoplasm and the nucleus of the host cells [12–15], mediated by the NLS (nuclear localization signal), the NOS (nucleolar targeting signal) [38] and the NES (nuclear export signal) of Rev itself [39–41]. As mentioned above, NIS region is also crucial for the cycle survival of HIV-1.
Here, we investigated antibacterial effects of Rev–NIS and suggested the role of positively charged amino acids on membrane pore forming activity of the peptide in bacterial cells, by synthesizing two analogs, Anal R and Anal S. Based on the amphipathic property of Rev–NIS, Anal R and Anal S were designed by substituting E¹ and L³ to R and L³ to S, respectively. The circular dichroism (CD) spectroscopy showed that Anal R and Anal S have the same conformation of Rev–NIS, with a significant fraction of helical structure. In succession, the antibacterial susceptibility testing showed that Rev–NIS and its analogs possessed significant activities (Anal R > Rev–NIS > Anal S), without hemolytic effects, against bacterial pathogens including antibiotics-resistant strains. Moreover, the membrane studies, 3,3′-dipropylthiadicarbocyanine iodide (diSC₃5) staining and FITC-dextran (FD) leakage assay demonstrated that the analogs as well as Rev–NIS acted on the bacterial membranes and potently made pores, with the hydrodynamic radius between 1.4 nm and 2.3 nm. Especially, Anal R made larger pores than other peptides, with the radius between 2.3 nm and 3.3 nm. These results also corresponded to the result of antibacterial susceptibility testing. In summary, this study indicates that the two arginine residues are more influential than the hydrophobicity or the helicity, regarding the molecular activity of the peptide, and finally suggests that Anal R peptide may be applied to novel antibacterial agents.
Antifungal properties of a peptide derived from the signal peptide of the HIV-1 regulatory protein, Rev
2009, FEBS Letters
Antifungal effects of nuclear entry inhibitory signal peptide of HIV-1 Rev protein (Rev-NIS) were investigated. Rev-NIS contained potent antifungal activities without hemolytic effects. To understand the antifungal mechanism(s), in vivo and in vitro fluorescent studies were conducted. Flow cytometric analysis with bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC₄(3)] and calcein-leakage measurement from large unilamellar vesicles (LUVs) indicated that Rev-NIS depolarized and disrupted the fungal membranes. These results were further confirmed by using giant unilamellar vesicles (GUVs). The current study suggests that Rev-NIS exerts its antifungal activity with membrane-active mechanism(s).
Posttranscriptional Control of HIV-1 and Other Retroviruses and Its Practical Applications
2007, Advances in Pharmacology
Citation Excerpt :
Rev is a small 116‐aa nuclear/nucleolar protein (Cochrane et al., 1990b; Cullen et al., 1988; Felber et al., 1989b; Perkins et al., 1989; Venkatesh et al., 1990), and its nucleolar localization is critical for Rev function (Michienzi et al., 2006; Stauber et al., 1995, 1998). Rev was found to shuttle rapidly between the nucleus and cytoplasm (D'Agostino et al., 1995; Kalland et al., 1994; Love et al., 1998; Meyer and Malim, 1994; Neumann et al., 2001; Richard et al., 1994; Stauber et al., 1995; Szilvay et al., 1995; Wolff et al., 2006). Rev contains four functional determinants: nuclear localization signal (NLS), RNA‐binding domain (RBD), oligomerization domain flanking the NLS/RBD, and nuclear export signal (NES) (for reviews see Cochrane, 2004; Hope, 1999; Pavlakis and Felber, 1990; Pollard and Malim, 1998).
This chapter describes the post-transcriptional control of human immunodeficiency virus type 1 (HIV‐1) and other retroviruses and its practical applications. Post-transcriptional control is a key step essential for the expression of cellular and viral mRNAs. After synthesis, processing, and assembly into ribonucleoprotein complexes (messenger ribonucleoprotein, mRNP), the mRNA is exported into the cytoplasm, which involves complex interactions of the mRNPs with transport receptors and with the components of the nuclear pore complex (NPC). Splicing mark mRNA as “export‐ready” and plays a critical role in promoting mRNA transport. Whereas, HIV and all the complex retroviruses produce several alternatively spliced mRNAs; only two types of mRNAs are produced from simple retroviruses. The unspliced primary transcript of all retroviruses has to exit the nucleus because it serves as genomic RNA, as well as gag/pol mRNA. The transport of unspliced RNA to the cytoplasm requires a special export mechanism. The chapter focuses on the cis‐acting viral RNA export elements and the viral and cellular factors promoting the export of retroviral mRNAs.
Human T-cell leukemia virus type I p30 nuclear/nucleolar retention is mediated through interactions with RNA and a constituent of the 60 S ribosomal subunit
2006, Journal of Biological Chemistry
Citation Excerpt :
However, both proteins appeared to localize more in the nucleoplasm at the higher concentration of AMD (Fig. 9A), suggesting that their nucleolar retention is partly dependent on RNA polymerase II and III transcription. In the same experiment, human immunodeficiency virus Rev-GFP was used as a positive control and was efficiently relocated to the cytoplasm following AMD treatment at either concentration, as reported previously (18, 19). We next used live cell imaging to investigate GFP-p30 recovery kinetics in response to AMD treatment.
Human T-cell leukemia virus type I is the etiological agent of adult T-cell leukemia/lymphoma, an aggressive and fatal lymphoproliferative malignancy. The virus has evolved strategies to escape immune clearance by remaining latent in most infected cells in vivo. We demonstrated previously that virally encoded p30 protein is a potent post-transcriptional inhibitor of virus replication (Nicot, C., Dundr, M., Johnson, J. M., Fullen, J. R., Alonzo, N., Fukumoto, R., Princler, G. L., Derse, D., Misteli, T., and Franchini, G. (2004) Nat. Med. 10, 197-201). p30 is unable to shuttle out of the nucleus in heterokaryon assays, suggesting the existence of specific retention signals. Because suppression of virus replication relies on nuclear retention of the tax/rex mRNA by p30, determining the retention features of p30 will offer hints to break latency in infected cells and insights into new therapeutic approaches. In this study, we used live cell imaging technologies to study the kinetics of p30 and to delineate its retention signals and their function in virus replication. Notably, this is the first study to identify p30 nucleolar retention domains. Using mutants of p30 that localized in different cellular compartments, we show that post-transcriptional control of virus replication by p30 occurs in the nucleoplasm. We further demonstrate that p30 nuclear/nucleolar retention is dependent upon de novo RNA transcripts and interactions with components of the ribosomal machinery.
Laminopathy-inducing lamin A mutants can induce redistribution of lamin binding proteins into nuclear aggregates
2006, Experimental Cell Research
Lamins, members of the family of intermediate filaments, form a supportive nucleoskeletal structure underlying the nuclear envelope and can also form intranuclear structures. Mutations within the A-type lamin gene cause a variety of degenerative diseases which are collectively referred to as laminopathies. At the molecular level, laminopathies have been shown to be linked to a discontinuous localization pattern of A-type lamins, with some laminopathies containing nuclear lamin A aggregates. Since nuclear aggregate formation could lead to the mislocalization of proteins interacting with A-type lamins, we set out to examine the effects of FLAG-lamin A N195K and R386K protein aggregate formation on the subnuclear distribution of the retinoblastoma protein (pRb) and the sterol responsive element binding protein 1a (SREBP1a) after coexpression as GFP-fusion proteins in HeLa cells. We observed strong recruitment of both proteins into nuclear aggregates. Nuclear aggregate recruitment of the NPC component nucleoporin NUP153 was also observed and found to be dependent on the N-terminus. That these effects were specific was implied by the fact that a number of other coexpressed karyophilic GFP-fusion proteins, such as the nucleoporin NUP98 and kanadaptin, did not coaggregate with FLAG-lamin A N195K or R386K. Immunofluorescence analysis further indicated that the precursor form of lamin A, pre-lamin A, could be found in intranuclear aggregates. Our results imply that redistribution into lamin A-/pre-lamin A-containing aggregates of proteins such as pRb and SREBP1a could represent a key aspect underlying the molecular pathogenesis of certain laminopathies.
Characterization of nuclear localization and export signals of the major tegument protein VP8 of bovine herpesvirus-1
2004, Virology
Bovine herpesvirus-1 (BHV-1) VP8 is found in the nucleus immediately after infection. Transient expression of VP8 fused to yellow fluorescent protein (YFP) in COS-7 cells confirmed the nuclear localization of VP8 in the absence of other viral proteins. VP8 has four putative nuclear localization signals (NLS). Deletion of pat4 (⁵¹RRPR⁵⁴) or pat7 (⁴⁸PRVRRPR⁵⁴) NLS2 abrogated nuclear accumulation, whereas deletion of ⁴⁸PRV⁵⁰ did not, so pat4 NLS2 is critical for nuclear localization of VP8. Furthermore, NLS1 (¹¹RRPRR¹⁵), pat4 NLS2, and pat7 NLS2 were all capable of transporting the majority of YFP to the nucleus. Finally, a 12-amino-acid peptide with the sequence RRPRRPRVRRPR directed all of YFP into the nucleus, suggesting that reiteration of the RRPR motif makes the nuclear localization more efficient. Heterokaryon assays demonstrated that VP8 is also capable of shuttling between the nucleus and cytoplasm of the cell. Deletion mutant analysis revealed that this property is attributed to a leucine-rich nuclear export sequence (NES) consisting of amino acids ⁴⁸⁵LSAYLTLFVAL⁴⁹⁵. This leucine-rich NES caused transport of YFP to the cytoplasm. These results demonstrate that VP8 shuttles between the nucleus and cytoplasm.

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Regular ArticleHIV-1 Rev Is Capable of Shuttling between the Nucleus and Cytoplasm

Abstract

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HIV-1 Rev Is Capable of Shuttling between the Nucleus and Cytoplasm