Cooperative dimerization of a stably folded protein directed by a flexible RNA in the assembly of the HIV Rev dimer–RRE stem II complex

The binding of the HIV‐1 Rev protein as an oligomer to a viral RNA element, the Rev‐response element (RRE), mediates nuclear export of genomic RNA. Assembly of the Rev–RRE ribonucleoprotein (RNP) complex is nucleated by the binding of the first Rev molecule to stem IIB of the RRE. This is followed by stepwise addition of a total of ~six Rev molecules along the RRE through a combination of RNA–protein and protein–protein interactions. RRE stem II, which forms a three‐way junction consisting of stems IIA, IIB and IIC, has been shown to bind to two Rev molecules in a cooperative manner, with the second Rev molecule binding to the junction region of stem II. The results of base substitutions at the stem II junction, and characterization of stem II junction variants selected from a randomized library showed that an “open” flexible structure is preferred for binding of the second Rev molecule, and that binding of the second Rev molecule to the junction region is not sequence‐specific. Alanine substitutions of a number of Rev amino acid residues implicated to be important for Rev folding in previous structural studies were found to result in a dramatic decrease in the binding of the second Rev molecule. These results support the model that proper folding of Rev is critical in ensuring that the flexible RRE is able to correctly position Rev molecules for specific RNP assembly, and suggests that targeting Rev folding may be effective in the inhibition of Rev function. Copyright © 2015 John Wiley & Sons, Ltd.     

Studies on the subcellular localization of the membrane-bound fraction of intestinal calcium-binding protein

Sorbitol density gradient centrifugation applied to intestinal mucosa homogenates resulted in a complete separation of soluble calcium-binding protein from the bound fraction of calcium-binding protein, providing further documentation of the bound pool of calcium-binding protein. The peak of the bound calcium-binding protein was not associated with the major peaks of any of the markers used, but was associated with minor peaks of alkaline phosphatase, RNA, and glucose-6-phosphatase. Lack of association of bound calcium-binding protein with (Na⁺ + K⁺)-ATPase indicated that the bound calcium-binding protein is not on the basolateral membrane. Differential centrifugation fractionation indicated that the bound calcium-binding protein is not associated with nuclei or mitochondria. The bound calcium-binding protein also could not be detected in partially pyrified brush borders. Exclusion of the brush border and basolateral membranes as the location of the bound calcium-binding protein suggests an intracellular locate.     

Modulation of HIV-1 Rev protein abundance and activity by polyubiquitination with unconventional Lys-33 branching

The HIV-1 Rev protein plays a key role in virus replication by allowing export to the cytoplasm of unspliced or singly-spliced RNAs. In this report, we investigated whether Rev is modified by ubiquitination or sumoylation. Whereas no evidence of sumoylation was obtained, transient expression experiments showed that ubiquitin conjugates to Rev as high molecular weight polyubiquitin chains. Mutation of the three lysine residues of Rev showed that the site of ubiquitin conjugation is Lys-115. Experiments with ubiquitin mutants including a single lysine at every seven possible position indicated that branching of the polyubiquitin chains mainly involves Lys-33. Mutation of Rev Lys-115 to arginine reduces markedly the steady state amount of the protein, but does not impair its ability to export RNA via the Rev response element. These observations support the notion that polyubiquitination of Rev stabilizes the viral protein but hinders its activity.     

Potent inhibition of HIV-1 replication by backbone cyclic peptides bearing the Rev arginine rich motif

Summary Due to its essential role in the virus life cycle, the viral regulatory protein Rev constitutes an attractive target for the development of new antiviral molecules. In this work, a series of Backbone Cyclic Peptide (BCP) analogs that bear a conformationally constrained arginine rich motif (ARM) of Rev were tested for in vitro inhibition of HIV-1 replication. We observed a potent suppression of HIV-1 replication in chronically infected T lymphocytic cells treated with Rev-BCPs. We further investigated possible mechanisms of HIV-1 inhibition and showed that Rev-BCPs interfere slightly with the nuclear import process and are very efficient in blocking a mechanism that controls Pr55^gag and gp160^env synthesis. Interestingly, these protein precursors are known to be encoded by mRNAs that require Rev-binding for nuclear export. In situ hybridization using a Cy-3 conjugated HIV-1 gag oligonucleotide probe indicated that Rev-BCPs prevent the intracellular accumulation of unspliced viral RNA. As a model, the most promising analog, Rev-BCP 14, was studied by molecular modeling and dynamics in order to identify its binding site on the Rev Response Element (RRE). The annealing simulation suggests that upon binding on the RRE, Rev-BCP 14 widens the distorted major groove of the viral RNA. Numerous contacts between peptide and RNA were found within the complex and some were identified as key components for the interactions. Altogether, our data indicate that the use of conformationally constrained Rev-BCPs represents a promising strategy for the development of new peptide-based therapeutic agents against HIV-1.     

Prediction of protein subcellular localization by weighted gene ontology terms

We develop a new weighting approach of gene ontology (GO) terms for predicting protein subcellular localization. The weights of individual GO terms, corresponding to their contribution to the prediction algorithm, are determined by the term-weighting methods used in text categorization. We evaluate several term-weighting methods, which are based on inverse document frequency, information gain, gain ratio, odds ratio, and chi-square and its variants. Additionally, we propose a new term-weighting method based on the logarithmic transformation of chi-square. The proposed term-weighting method performs better than other term-weighting methods, and also outperforms state-of-the-art subcellular prediction methods. Our proposed method achieves 98.1%, 99.3%, 98.1%, 98.1%, and 95.9% overall accuracies for the animal BaCelLo independent dataset (IDS), fungal BaCelLo IDS, animal Höglund IDS, fungal Höglund IDS, and PLOC dataset, respectively. Furthermore, the close correlation between high-weighted GO terms and subcellular localizations suggests that our proposed method appropriately weights GO terms according to their relevance to the localizations.     

Analysis of nuclear targeting activities of transport signals in the human immunodeficiency virus Rev protein

The human immunodeficiency Rev protein shuttles between the nucleus and cytoplasm, while accumulating to high levels in the nucleus. Rev has a nuclear localization signal (NLS; AA 35-50) with an arginine-rich motif (ARM) that interacts with importin beta and a leucine-rich nuclear export signal (NES; AA 75-84) recognized by CRM1/exportin 1. Here we explore nuclear targeting activities of the transport signals of Rev. GFP tagging and quantitative fluorescence microscopy were used to study the localization behavior of Rev NLS/ARM mutants under conditions inhibiting the export of Rev. Rev mutant M5 was actively transported to the nucleus, despite its known failure to bind importin beta. Microinjection of transport substrates with Rev-NES peptides revealed that the Rev-NES has both nuclear import and export activities. Replacement of amino acid residues "PLER" (77-80) of the NES with alanines abolished bidirectional transport activity of the Rev-NES. These results indicate that both transport signals of Rev have nuclear import capabilities and that the Rev NLS has more than one nuclear targeting activity. This suggests that Rev is able to use various routes for nuclear entry rather than depending on a single pathway.     

A role for Rev in the association of HIV-1 gag mRNA with cytoskeletal β-actin and viral protein expression

Human immunodeficiency virus type-1 (HIV-1) Rev acts by inducing the specific nucleocytoplasmic transport of a class of incompletely spliced RNAs that encodes the viral structural proteins. The transfection of HeLA cells with a rev-defective HIV-1 expression plasmid, however, resulted in the export of overexpressed, intron-containing species of viral RNAs, possibly through a default process of nuclear retention. Thus, this system enabled us to directly compare Rev⁺ and Rev⁻ cells as to the usage of RRE-containing mRNAs by the cellular translational machinery. Biochemical examination of the transfected cells revealed that although significant levels of gag and env mRNAs were detected in both the presence and absence of Rev, efficient production of viral proteins was strictly dependent on the presence of Rev. A fluoroscence in situ hybridisation assay confirmed these findings and provided further evidence that even in the presence of Rev, not all of the viral mRNA was equally translated. At the early phase of RNA export in Rev⁺ cells, gag mRNA was observed throughout both the cytoplasm and nucleoplasm as uniform fine stippling. In addition, the mRNA formed clusters mainly in the perinuclear region, which were not observed in Rev⁻ cells. In the presence of Rev, expression of the gag protein was limited to these perinuclear sites where the mRNA accumulated. Subsequent staining of the cytoskeletal proteins demonstrated that in Rev⁺ cells gag mRNA is colocalized with β-actin in the sites where the RNA formed clusters. In the absence of Rev, in contrast, the gag mRNA failed to associate with the cytoskeletal proteins. These results suggest that in addition to promoting the emergence of intron-containing RNA from the nucleus, Rev plays an important role in the compartmentation of translation by directing RRE-containing mRNAs to the β-actin to form the perinuclear clusters at which the synthesis of viral structural proteins begins.     

Distinct roles of TIR and non-TIR regions in the subcellular localization and signaling properties of MyD88

MyD88 is a cytoplasmic adaptor protein that is critical for Toll-like receptor (TLR) signaling. The subcellular localization of MyD88 is characterized as large condensed forms in the cytoplasm. The mechanism and significance of this localization with respect to the signaling function, however, are currently unknown. Here, we demonstrate that MyD88 localization depends on the entire non-TIR region and that the correct cellular targeting of MyD88 is indispensable for its signaling function. The Toll-interleukin I receptor-resistance (TIR) domain does not determine the subcellular localization, but it mediates interaction with specific TLRs. These findings reveal distinct roles for the TIR and non-TIR regions in the subcellular localization and signaling properties of MyD88.     

Human immunodeficiency virus-1 Rev protein activates hepatitis C virus gene expression by directly targeting the HCV 5'-untranslated region

Coinfection with human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV) accelerates hepatitis C disease progression; however, the mechanism underlying this effect is unknown. Here, we investigated the role of HIV-1 in HCV gene expression and the mechanism involved in this regulation. We discovered that HIV-1 Rev protein activates HCV gene expression. We further revealed that Rev binds to the internal loop of the HCV 5′-untranslated region (5′-UTR) to stimulate HCV IRES-mediated translation.     

Molecular mechanisms of gene expression regulation by the apoptosis-promoting protein TIA-1

TIA-1 and the related protein TIAR promote DNA fragmentation in digitonin-permeabilized thymocytes. These proteins contain RNA Recognition Motifs (RRMs) and bind uridine-rich sequences. These observations suggested that TIA-1/TIAR are pro-apoptotic factors that influence some aspect of RNA metabolism. Here we review recent data implicating TIA-1 as a regulator of translation of Tumor Necrosis Factor- mRNA and as regulator of alternative splicing of a variety of pre-mRNAs, including those of the Fibroblast Growth Factor Receptor 2 and the Fas receptor. We also discuss how some of these activities could be integrated in the control of programmed of cell death.     

Functional expression and subcellular localization of the Nectria haematococca Mak1 phytoalexin detoxification enzyme in transgenic tobacco

Medicarpin and maackiain are antifungal pterocarpan phytoalexins produced by many legumes, and are thought to be important components of the defense response of these legumes to certain fungal pathogens. The Mak1 gene from the fungal pathogen Nectria haematococca encodes an FAD-dependent mono-oxygenase, known to specifically hydroxylate the phytoalexins medicarpin and maackiain, converting them to less fungitoxic derivatives. Two binary vector constructs were made containing the coding regions from two fungal clones, a Mak1 cDNA (intronless) and a genomic (including three fungal introns) clone, regulated by an enhanced cauliflower mosaic virus 35S promoter. The constructs were introduced into tobacco to check for expression of active fungal enzyme in plant cells and for splicing of fungal introns. Leaves of tobacco plants transformed with the Mak1 cDNA construct readily metabolized infiltrated medicarpin to 1a-hydroxymedicarpin, indicating high levels of active enzyme. RT-PCR analysis of tobacco plants transformed with the Mak1 genomic construct indicated no processing of Mak1 introns, and no Mak1 activity was detected in these plants. When using plants containing the Mak1 cDNA construct, immunolocalization with a Mak1-specific antibody together with cellular fractionation indicated that Mak1 protein accumulated in the plant cytoplasm, associated with endoplasmic reticulum membranes; medicarpin biosynthetic enzymes have been localized to the same subcellular region. The Mak1 cDNA construct is therefore suitable for use in studies to selectively eliminate medicarpin accumulation to assess the relative importance of medicarpin in the antifungal defense mechanisms of alfalfa and other legumes.     

The extreme N-terminal domain of a hordeivirus TGB1 movement protein mediates its localization to the nucleolus and interaction with fibrillarin

The hordeiviral movement protein encoded by the first gene of the triple gene block (TGBp1) of Poa semilatent virus (PSLV), interacts with viral genomic RNAs to form RNP particles which are considered to be a form of viral genome capable of cell-to-cell and long-distance transport in infected plants. The PSLV TGBp1 contains a C-terminal NTPase/helicase domain (HELD) and an N-terminal extension region consisting of two structurally and functionally distinct domains: an extreme N-terminal domain (NTD) and an internal domain (ID). This study demonstrates that transient expression of TGBp1 fused to GFP in Nicotiana benthamiana leaves results in faint but obvious fluorescence in the nucleolus in addition to cytosolic distribution. Mutagenesis of the basic amino acids inside the NTD clusters A ¹¹⁶KSKRKKKNKK¹²⁵ and B ¹⁷⁵KKATKKESKKQTK¹⁸⁷ reveals that these clusters are indispensable for nuclear and nucleolar targeting of PSLV TGBp1 and may contain nuclear and nucleolar localization signals or their elements. The PSLV TGBp1 is able to bind to fibrillarin, the major nucleolar protein (AtFib2 from Arabidopsis thaliana) in vitro. This protein–protein interaction occurs between the glycine-arginine-rich (GAR) domain of fibrillarin and the first 82 amino acid residues of TGBp1. The interaction of TGBp1 with fibrillarin is also visualized in vivo by bimolecular fluorescence complementation (BiFC) during co-expression of TGBp1 or its deletion mutants, and fibrillarin as fusions to different halves of YFP in N. benthamiana plants. The sites responsible for nuclear/nucleolar localization and fibrillarin binding, have been located within the intrinsically disordered TGBp1 NTD. These data could suggest that specific functions of hordeivirus TGBp1 may depend on its interaction with nucleolar components.     

Regulation of subcellular localization of the antiproliferative protein Tob by its nuclear export signal and bipartite nuclear localization signal sequences

Tob, a member of the Tob and BTG antiproliferative protein family, plays an important role in many cellular processes including cell proliferation. In this study, we have addressed molecular mechanisms regulating subcellular localization of Tob. Treatment with leptomycin B, an inhibitor of nuclear export signal (NES) receptor, resulted in a change in subcellular distribution of Tob from its pan-cellular distribution to nuclear accumulation, indicating the existence of NES in Tob. Our results have then identified an N-terminal region (residues 2-14) of Tob as a functional NES. They have also shown that Tob has a functional, bipartite nuclear localization signal (NLS) in residues 18-40. Thus, Tob is shuttling between the nucleus and the cytoplasm by its NES and NLS. To examine a possible relationship between subcellular distribution of Tob and its function, we exogenously added a strong NLS sequence or a strong NES sequence or both to Tob. The obtained results have demonstrated that the strong NLS-added Tob has a much weaker activity to inhibit cell cycle progression from G0/G1 to S phase. These results suggest that cytoplasmic localization or nucleocytoplasmic shuttling is important for the antiproliferative function of Tob.     

In vitro expression of the HIV-2 genomic RNA is controlled by three distinct internal ribosome entry segments that are regulated by the HIV protease and the Gag polyprotein

The HIV-2 genomic RNA serves both as a messenger for protein synthesis and as a genome for viral assembly and particle production. Our previous work has shown that the HIV-2 genomic RNA encodes two additional Gag proteins that are N-terminal truncated isoforms of the p57 Gag polyprotein. In this study, by the use of mono- and bicistronic RNAs we show that translation at the three AUGs is driven by three distinct and independent internal ribosome entry segments both in vitro and ex vivo. Furthermore we used the recombinant Gag and HIV-2 protease to show that, in vitro, translation is tightly regulated by these two viral proteins. This regulation is exerted both at the level of protein production and also on the selection of the AUG initiation site which changes the ratio at which the three different Gag isoforms are produced.