The roles of nucleolar structure and function in the subcellular location of the HIV-1 Rev protein

The human immunodeficiency virus 1 (HIV-1) Rev transactivator protein plays a critical role in the regulation of expression of structural proteins by controlling the pathway of mRNA transport. The Rev protein is located predominantly in the nucleoli of HIV-1 infected or Rev-expressing cells. Previous studies demonstrated that the Rev protein forms a specific complex in vitro with protein B23 which is suggested to be a nucleolar receptor and/or carrier for the Rev protein. To study the role of the nucleolus and nucleolar proteins in Rev function, transfected COS-7 or transformed CMT3 cells expressing the Rev protein were examined for subcellular locations of Rev and other proteins using indirect immunofluorescence and immunoelectron microscopy. One day after transfection the Rev protein was found in most cells only in the nucleolar dense fibrillar and granular components where it colocalized with protein B23. These were designated class 1 cells. In a second class of cells Rev and B23 accumulated in the nucleoplasm as well as in nucleoli. Treatment of class 1 cells with actinomycin D (AMD) under conditions that blocked only RNA polymerase I transcription caused Rev to completely redistribute from nucleoli to the cytoplasm. Simultaneously, protein B23 was partially released from nucleoli, mostly into the nucleoplasm, with detectable amounts in the cytoplasm. In cells recovering from AMD treatment in the presence of cycloheximide Rev and B23 showed coincident relocation to nucleoli. Class 2 cells were resistant to AMD-induced Rev redistribution. Selective inhibition of RNA polymerase II transcription by alpha-amanitin or by DRB did not cause Rev to be released into the cytoplasm suggesting that active preribosomal RNA transcription is required for the nucleolar location of Rev. However, treatment with either of the latter two drugs at higher doses and for longer times caused partial disruption of nucleoli accompanied by translocation of the Rev protein to the cytoplasm. These results suggest that the nucleolar location of Rev depends on continuous preribosomal RNA transcription and a substantially intact nucleolar structure.

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Competent transcription initiation by RNA polymerase II in cell-free extracts from xeroderma pigmentosum groups B and D in an optimized RNA transcription assay

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression ◽

10.1016/s0167-4781(97)00102-4 ◽

1997 ◽

Vol 1354 (3) ◽

pp. 241-251 ◽

Cited By ~ 13

Author(s):

Masahiko S Satoh ◽

Philip C Hanawalt

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Xeroderma Pigmentosum ◽

Rna Transcription ◽

Transcription Assay

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Origin of Hepatitis Delta Virus mRNA

Journal of Virology ◽

10.1128/jvi.74.16.7204-7210.2000 ◽

2000 ◽

Vol 74 (16) ◽

pp. 7204-7210 ◽

Cited By ~ 45

Author(s):

Severin Gudima ◽

Shwu-Yuan Wu ◽

Cheng-Ming Chiang ◽

Gloria Moraleda ◽

John Taylor

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Hepatitis Delta Virus ◽

Rna Synthesis ◽

Genome Replication ◽

Rna Transcription ◽

Hepatitis Delta ◽

New Findings ◽

Delta Virus

ABSTRACT Hepatitis delta virus (HDV) is unique relative to all known animal viruses, especially in terms of its ability to redirect host RNA polymerase(s) to transcribe its 1,679-nucleotide (nt) circular RNA genome. During replication there accumulates not only more molecules of the genome but also its exact complement, the antigenome. In addition, there are relatively smaller amounts of an 800-nt RNA of antigenomic polarity that is polyadenylated and considered to act as mRNA for translation of the single and essential HDV protein, the delta antigen. Characterization of this mRNA could provide insights into the in vivo mechanism of HDV RNA-directed RNA transcription and processing. Previously, we showed that the 5′ end of this RNA was located in the majority of species, at nt 1630. The present studies show that (i) at least some of this RNA, as extracted from the liver of an HDV-infected woodchuck, behaved as if it contained a 5′-cap structure; (ii) in the infected liver there were additional polyadenylated antigenomic HDV RNA species with 5′ ends located at least 202 nt and even 335 nt beyond the nt 1630 site, (iii) the 5′ end at nt 1630 was not detected in transfected cells, following DNA-directed HDV RNA transcription, in the absence of genome replication, and (iv) nevertheless, using in vitro transcription with purified human RNA polymerase II holoenzyme and genomic RNA template, we did not detect initiation of template-dependent RNA synthesis; we observed only low levels of 3′-end addition to the template. These new findings support the interpretation that the 5′ end detected at nt 1630 during HDV replication represents a specific site for the initiation of an RNA-directed RNA synthesis, which is then modified by capping.

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Novel critical role of a human Mediator complex for basal RNA polymerase II transcription

EMBO Reports ◽

10.1093/embo-reports/kve186 ◽

2001 ◽

Vol 2 (9) ◽

pp. 808-813 ◽

Cited By ~ 93

Author(s):

Gerhard Mittler ◽

Elisabeth Kremmer ◽

H Th. Marc Timmers ◽

Michael Meisterernst

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Critical Role ◽

Mediator Complex ◽

Rna Polymerase Ii Transcription

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An N-Terminal Region of Lassa Virus L Protein Plays a Critical Role in Transcription but Not Replication of the Virus Genome

Journal of Virology ◽

10.1128/jvi.01657-09 ◽

2009 ◽

Vol 84 (4) ◽

pp. 1934-1944 ◽

Cited By ~ 41

Author(s):

Michaela Lelke ◽

Linda Brunotte ◽

Carola Busch ◽

Stephan Günther

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Large Scale ◽

Critical Role ◽

Terminal Region ◽

Lassa Virus ◽

Mrna Synthesis ◽

L Protein ◽

Mrna Pool ◽

Capping Enzymes

ABSTRACT The central domain of the 200-kDa Lassa virus L protein is a putative RNA-dependent RNA polymerase. N- and C-terminal domains may harbor enzymatic functions important for viral mRNA synthesis, including capping enzymes or cap-snatching endoribonucleases. In the present study, we have employed a large-scale mutagenesis approach to map functionally relevant residues in these regions. The main targets were acidic (Asp and Glu) and basic residues (Lys and Arg) known to form catalytic and binding sites of capping enzymes and endoribonucleases. A total of 149 different mutants were generated and tested in the Lassa virus replicon system. Nearly 25% of evolutionarily highly conserved acidic and basic side chains were dispensable for function of L protein in the replicon context. The vast majority of the remaining mutants had defects in both transcription and replication. Seven residues (Asp-89, Glu-102, Asp-119, Lys-122, Asp-129, Glu-180, and Arg-185) were selectively important for mRNA synthesis. The phenotype was particularly pronounced for Asp-89, Glu-102, and Asp-129, which were indispensable for transcription but could be replaced by a variety of amino acid residues without affecting genome replication. Bioinformatics disclosed the remote similarity of this region to type IIs endonucleases. The mutagenesis was complemented by experiments with the RNA polymerase II inhibitor α-amanitin, demonstrating dependence of viral transcription from the cellular mRNA pool. In conclusion, this paper describes an N-terminal region in L protein being important for mRNA, but not genome synthesis. Bioinformatics and cell biological experiments lend support to the hypothesis that this region could be part of a cap-snatching enzyme.

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Assembly and Functional Organization of the Nucleolus: Ultrastructural Analysis ofSaccharomyces cerevisiaeMutants

Molecular Biology of the Cell ◽

10.1091/mbc.11.6.2175 ◽

2000 ◽

Vol 11 (6) ◽

pp. 2175-2189 ◽

Cited By ~ 38

Author(s):

Stéphanie Trumtel ◽

Isabelle Léger-Silvestre ◽

Pierre-Emmanuel Gleizes ◽

Frédéric Teulières ◽

Nicole Gas

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Functional Organization ◽

Ultrastructural Localization ◽

Rna Polymerase I ◽

Wild Type ◽

Rdna Transcription ◽

Nucleolar Structure ◽

Fibrillar Component ◽

Polymerase I

Using Saccharomyces cerevisiae strains with genetically modified nucleoli, we show here that changing parameters as critical as the tandem organization of the ribosomal genes and the polymerase transcribing rDNA, although profoundly modifying the position and the shape of the nucleolus, only partially alter its functional subcompartmentation. High-resolution morphology achieved by cryofixation, together with ultrastructural localization of nucleolar proteins and rRNA, reveals that the nucleolar structure, arising upon transcription of rDNA from plasmids by RNA polymerase I, is still divided in functional subcompartments like the wild-type nucleolus. rRNA maturation is restricted to a fibrillar component, reminiscent of the dense fibrillar component in wild-type cells; a granular component is also present, whereas no fibrillar center can be distinguished, which directly links this latter substructure to rDNA chromosomal organization. Although morphologically different, the mininucleoli observed in cells transcribing rDNA with RNA polymerase II also contain a fibrillar subregion of analogous function, in addition to a dense core of unknown nature. Upon repression of rDNA transcription in this strain or in an RNA polymerase I thermosensitive mutant, the nucleolar structure falls apart (in a reversible manner), and nucleolar constituents partially relocate to the nucleoplasm, indicating that rRNA is a primary determinant for the assembly of the nucleolus.

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Iron Chelator ICL670 Inhibits HIV-1 Transcription.

Blood ◽

10.1182/blood.v110.11.3863.3863 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3863-3863

Author(s):

Zufan Debebe ◽

Tatyana Ammosova ◽

Hanspeter Nick ◽

Xiaomei Niu ◽

Marina Jerebtsova ◽

...

Keyword(s):

Cell Cycle ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Protein Level ◽

Iron Chelator ◽

Iron Chelators ◽

Cyclin T1 ◽

Terminal Domain ◽

Cycle Dependent ◽

Hiv 1

Abstract HIV-1 replication is induced by the excess of iron and iron chelation by desferrioxamine (DFO) inhibits viral replication in HIV-1 infected CEM T cells [1]. Treatment of cells with DFO or 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone inhibits expression of proteins that regulate cell-cycle progression, including cycle-dependent kinase 2 (CDK2) [2]. HIV-1 transcription is activated by Tat protein, which recruits transcriptional co-activators to the HIV-1 promoter. Elongation of HIV-1 transcription is mediated by the interaction of HIV Tat with host cell cycle-dependent kinase 9 (CDK9)/cyclin T1, which phosphorylates the C-terminal domain of RNA polymerase II. Our recent studies showed that CDK2 participates in HIV-1 transcription by phosphorylating Tat [3]. Thus inhibition of CDK2 by iron chelators might present a new approach to inhibit HIV-1 transcription. We evaluated the effect of a clinically approved orally effective iron chelator, 4-[3,5-bis-(hydroxyphenyl) -1,2,4-triazol-1-yl]-benzoic acid (ICL670 or deferasirox) on HIV-1 transcription. ICL670 inhibited Tat-induced HIV-1 transcription in CEM, 293T and HeLa cells at concentrations that did not induce cytotoxicity. The chelator decreased cellular activity of CDK2 but not its protein level and reduced HIV-1 Tat phosphorylation by CDK2. ICL670 did not decrease CDK9 protein level but significantly reduced association of CDK9 with cyclin T1 and reduced phosphorylation of Ser-2 residues of RNA polymerase II C-terminal domain. In conclusion, our findings add to the evidence that iron chelators may inhibit HIV-1 transcription by deregulating CDK2 and Cdk9. Further consideration should be given to the evaluation of ICL670 for future anti-retroviral therapeutics and to the development of iron chelators specifically as anti-retroviral agents.

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Transcription elongation regulator 1 (TCERG1) regulates competent RNA polymerase II-mediated elongation of HIV-1 transcription and facilitates efficient viral replication

Retrovirology ◽

10.1186/1742-4690-10-124 ◽

2013 ◽

Vol 10 (1) ◽

pp. 124 ◽

Cited By ~ 9

Author(s):

Mayte Coiras ◽

Marta Montes ◽

Immaculada Montanuy ◽

María López-Huertas ◽

Elena Mateos ◽

...

Keyword(s):

Rna Polymerase ◽

Viral Replication ◽

Rna Polymerase Ii ◽

Transcription Elongation ◽

Hiv 1 ◽

Efficient Viral Replication

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The Role of RNA Polymerase II Elongation Control in HIV-1 Gene Expression, Replication, and Latency

Genetics Research International ◽

10.4061/2011/726901 ◽

2011 ◽

Vol 2011 ◽

pp. 1-9 ◽

Cited By ~ 2

Author(s):

Kyle A. Nilson ◽

David H. Price

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Elongation Factor ◽

Health Concern ◽

Pol Ii ◽

Positive Transcription Elongation Factor ◽

Elongation Control ◽

Hiv 1 ◽

Latent Phase

HIV-1 usurps the RNA polymerase II elongation control machinery to regulate the expression of its genome during lytic and latent viral stages. After integration into the host genome, the HIV promoter within the long terminal repeat (LTR) is subject to potent downregulation in a postinitiation step of transcription. Once produced, the viral protein Tat commandeers the positive transcription elongation factor, P-TEFb, and brings it to the engaged RNA polymerase II (Pol II), leading to the production of viral proteins and genomic RNA. HIV can also enter a latent phase during which factors that regulate Pol II elongation may play a role in keeping the virus silent. HIV, the causative agent of AIDS, is a worldwide health concern. It is hoped that knowledge of the mechanisms regulating the expression of the HIV genome will lead to treatments and ultimately a cure.

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Domains in the SPT5 Protein That Modulate Its Transcriptional Regulatory Properties

Molecular and Cellular Biology ◽

10.1128/mcb.20.9.2970-2983.2000 ◽

2000 ◽

Vol 20 (9) ◽

pp. 2970-2983 ◽

Cited By ~ 141

Author(s):

Dmitri Ivanov ◽

Youn Tae Kwak ◽

Jun Guo ◽

Richard B. Gaynor

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Repression ◽

Transcriptional Elongation ◽

Tat Protein ◽

C Terminus ◽

Heptad Repeats ◽

And Function ◽

Hiv 1

ABSTRACT SPT5 and its binding partner SPT4 regulate transcriptional elongation by RNA polymerase II. SPT4 and SPT5 are involved in both 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB)-mediated transcriptional inhibition and the activation of transcriptional elongation by the human immunodeficiency virus type 1 (HIV-1) Tat protein. Recent data suggest that P-TEFb, which is composed of CDK9 and cyclin T1, is also critical in regulating transcriptional elongation by SPT4 and SPT5. In this study, we analyze the domains of SPT5 that regulate transcriptional elongation in the presence of either DRB or the HIV-1 Tat protein. We demonstrate that SPT5 domains that bind SPT4 and RNA polymerase II, in addition to a region in the C terminus of SPT5 that contains multiple heptad repeats and is designated CTR1, are critical for in vitro transcriptional repression by DRB and activation by the Tat protein. Furthermore, the SPT5 CTR1 domain is a substrate for P-TEFb phosphorylation. These results suggest that C-terminal repeats in SPT5, like those in the RNA polymerase II C-terminal domain, are sites for P-TEFb phosphorylation and function in modulating its transcriptional elongation properties.

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