A cis-acting peptide signal in human immunodeficiency virus type I Rev which inhibits nuclear entry of small proteins

Kubota, Satoshi; Pomerantz, Roger J.

doi:10.1038/sj.onc.1201738

Cited by 19 publications

(15 citation statements)

References 44 publications

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“…However, we cannot completely exclude the possibility that, in addition to its anti-multimerization effect, in cells the nanobody exerts an extra anti-Rev activity that involves the same or overlapping Lys-20 and Tyr-23 residues. The N terminus of Rev is essential for Rev function and plays a role in its multimerization (17,24,25,47). The DEAD box helicase DDX1 is a co-factor that was also found to interact with the N terminus of Rev (48).…”

Section: Discussionmentioning

confidence: 99%

An Intrabody Based on a Llama Single-domain Antibody Targeting the N-terminal α-Helical Multimerization Domain of HIV-1 Rev Prevents Viral Production

Vercruysse

Pardon

Vanstreels

et al. 2010

Journal of Biological Chemistry

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The human immunodeficiency virus, type 1 (HIV-1)-encoded Rev protein is essential for the expression of late viral mRNAs. Rev forms a large organized multimeric protein-protein complex on the Rev response element of these viral mRNA species and transports them from the nucleus to the cytoplasm, exploiting the CRM1-mediated cellular machinery. Here we report the selection of a nanobody, derived from a llama heavy-chain only antibody, that efficiently blocks the assembly of Rev multimers. The nanobody inhibits HIV-1 replication in cells and specifically suppresses the Rev-dependent expression of partially spliced and unspliced HIV-1 RNA. In HIV-susceptible cells, this nanobody thus has potential as an effective anti-HIV agent using genetic immunization strategies. Its binding site was mapped to Rev residues Lys-20 and Tyr-23 located in the N-terminal ␣-helical multimerization domain. In the presence of this nanobody, we observed an accumulation of dimeric Rev species, supporting a head-to-head/tail-to-tail molecular model for Rev assembly. The results indicate that the oligomeric assembly of Rev follows an ordered stepwise process and identify a new epitope within Rev that could guide strategies for the development of novel HIV inhibitors.Nuclear export of viral mRNA is critical for the life cycle of the human immunodeficiency virus (HIV).2 Fully spliced viral mRNA is exported to the cytoplasm of the infected cell through cellular mechanisms. However, in contrast to cellular systems, HIV uses a sophisticated molecular machinery to transport unspliced and partially spliced forms of its viral mRNA. The nuclear export of these late viral messengers is required for both the expression of late viral genes and packaging of genomic RNA and is mediated by the HIV-encoded regulatory protein Rev (1). The viral Rev protein forms a multimeric complex on a secondary structured RNA element (the Rev response element (RRE)) present in all unspliced and partially spliced mRNAs (2-4) and exploits the CRM1-mediated cellular machinery to transport these RNAs from the nucleus to the cytoplasm (5-7).Rev is a small protein of 116 amino acid residues. Under steady state conditions, Rev localizes mainly to the nucleoli of cells (8). However, different functional elements cause Rev to continuously shuttle between the nucleus and the cytoplasm (9 -11). A short stretch of basic amino acids characterized by 10 arginine residues serves both as a nuclear localization signal for the nuclear import of Rev and as an RNA-binding domain during the export of RNA-Rev complexes (4). This arginine-rich region is flanked on both sides by sequences that contribute to the oligomerization of Rev on the RRE. Its strong tendency to oligomerize has hampered the structure determination of Rev by x-ray crystallography or NMR spectroscopy. However, circular dichroism, nuclear magnetic resonance, and Raman spectroscopy studies strongly suggest that the oligomerization domains and the nuclear localization signal are components of a helix-turn-helix motif (12-14). A...

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Section: Discussionmentioning

confidence: 99%

An Intrabody Based on a Llama Single-domain Antibody Targeting the N-terminal α-Helical Multimerization Domain of HIV-1 Rev Prevents Viral Production

Vercruysse

Pardon

Vanstreels

et al. 2010

Journal of Biological Chemistry

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“…Domains in Rev that mediate splicing inhibition or translational activation either have not been defined or have been suggested to coincide with domains important for Crm1-dependent nuclear RNA export (9,22). As a result, it has not been possible to clearly address the functional relevance of these hypothetical Rev activities.…”

Section: Discussionmentioning

confidence: 99%

“…These groups have therefore suggested that Rev also activates the translation of RRE-containing HIV-1 transcripts. A proposed functional domain that allows the association of Rev with currently undefined cytoplasmic components and that coincides with sequences required for Rev multimerization could play a role in this hypothetical Rev activity (22). Finally, in addition to these two novel activities for Rev, it has also been suggested that other proteins, in addition to Crm1, can interact directly with Rev to promote nuclear mRNA export.…”

Section: Discussionmentioning

confidence: 99%

Recruitment of the Crm1 Nuclear Export Factor Is Sufficient To Induce Cytoplasmic Expression of Incompletely Spliced Human Immunodeficiency Virus mRNAs

Yi¹,

Bogerd²,

Cullen

2002

J Virol

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Cytoplasmic expression of the incompletely spliced RNA transcripts that encode the late, structural proteins of human immunodeficiency virus type 1 (HIV-1) is dependent on the viral Rev regulatory protein. General agreement exists that Rev acts, at least in part, by recruiting the cellular Crm1 nuclear export factor to HIV-1 transcripts bearing the Rev response element RNA target, and thereby inducing their nuclear egress. However, several groups have argued that Crm1 recruitment may not be sufficient for Rev function. Thus, several additional candidate cofactors for Rev have been proposed, and Rev has also been suggested to also inhibit the nuclear splicing of HIV-1 transcripts and/or to directly enhance their cytoplasmic translation. To examine whether Crm1 recruitment is, instead, sufficient to activate the nuclear export of viral mRNAs, we targeted a leucine-rich Crm1 binding domain, derived from a heterologous protein that normally plays no role in RNA metabolism, to HIV-1 RNAs and showed that this tethered Crm1 binding domain is sufficient to induce the nuclear export and cytoplasmic translation of late HIV-1 mRNA species. More importantly, we show that direct tethering of the Crm1 nuclear export factor to target mRNAs, by fusion to a heterologous RNA binding domain, is in and of itself sufficient to induce the nuclear export and cytoplasmic expression of the unspliced HIV-1 mRNAs that encode the viral Gag proteins.Retroviral replication requires the cytoplasmic expression of both fully spliced and incompletely spliced forms of the initial, genome-length viral RNA transcript (reviewed in references 7 and 37). However, cells have evolved mechanisms to prevent the nuclear export of incompletely spliced cellular mRNAs, i.e., pre-mRNAs (4, 23). Retroviruses have therefore had to develop mechanisms that allow intron-containing viral transcripts to exit the nucleus in the face of this cellular proofreading mechanism. In several simple retroviruses, nuclear export of the genome-length transcript is mediated by cellular factors that are recruited to a cis-acting RNA target termed a constitutive transport element (CTE) (3,36,49). The cellular protein specific for the CTE found in simian type D viruses has been identified as Tap, a nuclear export factor that also plays a key role in mediating cellular mRNA export (7,15,19,43).In contrast to simple retroviruses, complex retroviruses encode a regulatory protein that is critical for the cytoplasmic expression of their incompletely spliced transcripts. In human immunodeficiency virus type 1 (HIV-1), this protein is termed Rev. Rev interacts with a cis-acting viral RNA target site, the Rev response element (RRE), and with Crm1, a host cell protein that is a member of the karyopherin or importin/exportin family of nucleocytoplasmic transport factors (12,14,29,31,35,42,50). Crm1 binds specifically to a short leucinerich motif found in the Rev protein that also functions as a nuclear export signal (NES) (2,11,30,48). NES binding by Crm1 requires a cellular cofactor, the GTP...

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“…The NIS, a 15 amino acid peptide motif in the N-terminal of HIV-1 Rev, modulates nucleocytoplasmic protein trafficking and intracellular stability of the HIV-1 Rev [3][4][5]. When HIV-1 Rev enters the nucleus, the NIS stays around the nuclear membrane by behaving like an anchor and interferes with the nuclear entry of passive diffusion small proteins [3][4][5]. The NIS consists of an amphipathic helix, thought to allow binding to the lipid-bilayer of biomembranes, which include the nuclear membrane [3].…”

Section: Introductionmentioning

confidence: 99%

“…In addition, the existence of a nuclear diffusion inhibitory signal (NIS) has been recently identified. The NIS, a 15 amino acid peptide motif in the N-terminal of HIV-1 Rev, modulates nucleocytoplasmic protein trafficking and intracellular stability of the HIV-1 Rev [3][4][5]. When HIV-1 Rev enters the nucleus, the NIS stays around the nuclear membrane by behaving like an anchor and interferes with the nuclear entry of passive diffusion small proteins [3][4][5].…”

Section: Introductionmentioning

confidence: 99%

Antimicrobial Properties of Nuclear Diffusion Inhibitory Signal of HIV-1 Rev

Kobayashi

Yoshida²

2006

PPL

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Nuclear Diffusion Inhibitory Signal (NIS) has been identified in human immunodeficiency virus type 1 (HIV-1) Rev as a nuclear signal peptide which modulates nucleocytoplasmic protein trafficking and intracellular stability of the HIV-1 Rev. In this study, it was discovered that antimicrobial properties are inherent in the NIS. This is a significant finding that the NIS, which does not exist solely for self defense, in fact possesses antimicrobial properties.

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A cis-acting peptide signal in human immunodeficiency virus type I Rev which inhibits nuclear entry of small proteins

Cited by 19 publications

References 44 publications

An Intrabody Based on a Llama Single-domain Antibody Targeting the N-terminal α-Helical Multimerization Domain of HIV-1 Rev Prevents Viral Production

An Intrabody Based on a Llama Single-domain Antibody Targeting the N-terminal α-Helical Multimerization Domain of HIV-1 Rev Prevents Viral Production

Recruitment of the Crm1 Nuclear Export Factor Is Sufficient To Induce Cytoplasmic Expression of Incompletely Spliced Human Immunodeficiency Virus mRNAs

Antimicrobial Properties of Nuclear Diffusion Inhibitory Signal of HIV-1 Rev

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