scholarly journals Nucleolin stabilizes G-quadruplex structures folded by the LTR promoter and silences HIV-1 viral transcription

2015 ◽  
Vol 43 (18) ◽  
pp. 8884-8897 ◽  
Author(s):  
Elena Tosoni ◽  
Ilaria Frasson ◽  
Matteo Scalabrin ◽  
Rosalba Perrone ◽  
Elena Butovskaya ◽  
...  
Keyword(s):  
Transcriptional Activity ◽  
Cellular Protein ◽  
Spectrometric Analysis ◽  
Viral Transcription ◽  
Mobility Shift ◽  
Binding Preference ◽  
G Quadruplex ◽  
Mobility Shift Assay ◽  
Ltr Promoter ◽  
Hiv 1

Abstract Folding of the LTR promoter into dynamic G-quadruplex conformations has been shown to suppress its transcriptional activity in HIV-1. Here we sought to identify the proteins that control the folding of this region of proviral genome by inducing/stabilizing G-quadruplex structures. The implementation of electrophorethic mobility shift assay and pull-down experiments coupled with mass spectrometric analysis revealed that the cellular protein nucleolin is able to specifically recognize G-quadruplex structures present in the LTR promoter. Nucleolin recognized with high affinity and specificity the majority, but not all the possible G-quadruplexes folded by this sequence. In addition, it displayed greater binding preference towards DNA than RNA G-quadruplexes, thus indicating two levels of selectivity based on the sequence and nature of the target. The interaction translated into stabilization of the LTR G-quadruplexes and increased promoter silencing activity; in contrast, disruption of nucleolin binding in cells by both siRNAs and a nucleolin binding aptamer greatly increased LTR promoter activity. These data indicate that nucleolin possesses a specific and regulated activity toward the HIV-1 LTR promoter, which is mediated by G-quadruplexes. These observations provide new essential insights into viral transcription and a possible low mutagenic target for antiretroviral therapy.

scholarly journals A dynamic i-motif with a duplex stem-loop in the long terminal repeat promoter of the HIV-1 proviral genome modulates viral transcription

2019 ◽  
Vol 47 (21) ◽  
pp. 11057-11068 ◽  
Author(s):  
Emanuela Ruggiero ◽  
Sara Lago ◽  
Primož Šket ◽  
Matteo Nadai ◽  
Ilaria Frasson ◽  
...  
Keyword(s):  
Long Terminal Repeat ◽  
Antiviral Drug ◽  
Cellular Protein ◽  
Terminal Repeat ◽  
Proviral Genome ◽  
Stem Loop ◽  
Hnrnp K ◽  
Induced Folding ◽  
Ltr Promoter ◽  
Hiv 1

Abstract I-motifs are non-canonical nucleic acids structures characterized by intercalated H-bonds between hemi-protonated cytosines. Evidence on the involvement of i-motif structures in the regulation of cellular processes in human cells has been consistently growing in the recent years. However, i-motifs within non-human genomes have never been investigated. Here, we report the characterization of i-motifs within the long terminal repeat (LTR) promoter of the HIV-1 proviral genome. Biophysical and biochemical analysis revealed formation of a predominant i-motif with an unprecedented loop composition. One-dimensional nuclear magnetic resonance investigation demonstrated formation of three G-C H-bonds in the long loop, which likely improve the structure overall stability. Pull-down experiments combined with mass spectrometry and protein crosslinking analysis showed that the LTR i-motif is recognized by the cellular protein hnRNP K, which induced folding at physiological conditions. In addition, hnRNP K silencing resulted in an increased LTR promoter activity, confirming the ability of the protein to stabilize the i-motif-forming sequence, which in turn regulates the LTR-mediated HIV-1 transcription. These findings provide new insights into the complexity of the HIV-1 virus and lay the basis for innovative antiviral drug design, based on the possibility to selectively recognize and target the HIV-1 LTR i-motif.

scholarly journals NUCKS1, a novel Tat coactivator, plays a crucial role in HIV-1 replication by increasing Tat-mediated viral transcription on the HIV-1 LTR promoter

Retrovirology ◽  
2014 ◽  
Vol 11 (1) ◽  
Author(s):  
Hye-Young Kim ◽  
Byeong-Sun Choi ◽  
Sung Soon Kim ◽  
Tae-Young Roh ◽  
Jihwan Park ◽  
...  
Keyword(s):  
Crucial Role ◽  
Viral Transcription ◽  
Ltr Promoter ◽  
Hiv 1

scholarly journals The cellular protein hnRNP A2/B1 enhances HIV-1 transcription by unfolding LTR promoter G-quadruplexes

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Matteo Scalabrin ◽  
Ilaria Frasson ◽  
Emanuela Ruggiero ◽  
Rosalba Perrone ◽  
Elena Tosoni ◽  
...  
Keyword(s):  
Cellular Protein ◽  
Ltr Promoter ◽  
Hiv 1

scholarly journals Regulation of lactase–phlorizin hydrolase gene expression by the caudal-related homoeodomain protein Cdx-2

1997 ◽  
Vol 322 (3) ◽  
pp. 833-838 ◽  
Author(s):  
Jesper T. TROELSEN ◽  
Cathy MITCHELMORE ◽  
Nikolaj SPODSBERG ◽  
Anette M. JENSEN ◽  
Ove NORÉN ◽  
...  
Keyword(s):  
Gene Expression ◽  
Small Intestine ◽  
Binding Site ◽  
Electrophoretic Mobility Shift Assay ◽  
Gene Transcription ◽  
Transcriptional Activity ◽  
Nuclear Factor ◽  
Mobility Shift ◽  
Cis Element ◽  
Mobility Shift Assay

Lactase–phlorizin hydrolase is exclusively expressed in the small intestine and is often used as a marker for the differentiation of enterocytes. The cis-element CE-LPH1 found in the lactase–phlorizin hydrolase promoter has previously been shown to bind an intestinal-specific nuclear factor. By electrophoretic mobility-shift assay it was shown that the factor Cdx-2 (a homoeodomain-protein related to caudal) binds to a TTTAC sequence in the CE-LPH1. Furthermore it was demonstrated that Cdx-2 is able to activate reporter gene transcription by binding to CE-LPH1. A mutation in CE-LPH1, which does not affect Cdx-2 binding, results in a higher transcriptional activity, indicating that the CE-LPH1 site contains other binding site(s) in addition to the Cdx-2-binding site.

Cloning, Characterization, and Overexpression in Arabidopsis to Determine the Function of SPL Genes from Woodland Strawberry (Fragaria Vesca)

2021 ◽  
Author(s):  
Yibo Bai ◽  
Morong Liang ◽  
Chuangju Ma ◽  
Zongming Cheng ◽  
Jinsong Xiong
Keyword(s):  
Untranslated Region ◽  
Transcriptional Activity ◽  
Biochemical Analysis ◽  
Activity Analysis ◽  
Fragaria Vesca ◽  
Mobility Shift ◽  
Woodland Strawberry ◽  
Squamosa Promoter Binding Protein ◽  
Spl Genes ◽  
Mobility Shift Assay

Abstract SQUAMOSA promoter binding protein-like (SPL) proteins is a class of plant specific transcription factors that play important roles during plant development. However, the majority of SPL genes in strawberry are functionally uncharacterized. In this study, three SPL genes, i.e. FvSPL1, FvSPL2, and FvSPL11 (FvSPL1/2/11), from woodland strawberry were cloned and characterized. Phylogenetic analysis with SPL genes from Arabidopsis, tomato and chrysanthemum indicated that FvSPL1/2/11 were clustered into the same group with those of miR156 target site located at the 3’-untranslated region (UTR). Further biochemical analysis indicated that FvSPL1 was exclusively localized in the nucleus. Electrophoretic mobility shift assay demonstrated FvSPL1 could specifically recognized the GTAC motif. Transcriptional activity analysis showed FvSPL1 was a transcriptional activator that could activate the expression of FvAP1 gene. Finally, all of the transgenic Arabidopsis that overexpression the three FvSPL genes were exhibited significantly early flowering phenotype. Taken together, our study indicated that FvSPL1/2/11 similar to their orthologs in Arabidopsis mainly functions in regulating plant flowering. These results enriched our understanding to the functions of SPL genes in strawberry and might be utilized for strawberry flowering time manipulation in the future.

scholarly journals Structure and possible function of a G-quadruplex in the long terminal repeat of the proviral HIV-1 genome

2016 ◽  
Vol 44 (13) ◽  
pp. 6442-6451 ◽  
Author(s):  
Beatrice De Nicola ◽  
Christopher J. Lech ◽  
Brahim Heddi ◽  
Sagar Regmi ◽  
Ilaria Frasson ◽  
...  
Keyword(s):  
Long Terminal Repeat ◽  
Terminal Repeat ◽  
Competent Cell ◽  
Cell Infection ◽  
Transcription Analysis ◽  
Single Nucleotide ◽  
Biologically Relevant ◽  
G Quadruplex ◽  
Ltr Promoter ◽  
Hiv 1

Abstract The long terminal repeat (LTR) of the proviral human immunodeficiency virus (HIV)-1 genome is integral to virus transcription and host cell infection. The guanine-rich U3 region within the LTR promoter, previously shown to form G-quadruplex structures, represents an attractive target to inhibit HIV transcription and replication. In this work, we report the structure of a biologically relevant G-quadruplex within the LTR promoter region of HIV-1. The guanine-rich sequence designated LTR-IV forms a well-defined structure in physiological cationic solution. The nuclear magnetic resonance (NMR) structure of this sequence reveals a parallel-stranded G-quadruplex containing a single-nucleotide thymine bulge, which participates in a conserved stacking interaction with a neighboring single-nucleotide adenine loop. Transcription analysis in a HIV-1 replication competent cell indicates that the LTR-IV region may act as a modulator of G-quadruplex formation in the LTR promoter. Consequently, the LTR-IV G-quadruplex structure presented within this work could represent a valuable target for the design of HIV therapeutics.

scholarly journals Using capillary electrophoresis to study methylation effect on RNA-peptide interaction.

2003 ◽  
Vol 50 (3) ◽  
pp. 857-864 ◽  
Author(s):  
Piotr Mucha ◽  
Agnieszka Szyk ◽  
Piotr Rekowski ◽  
Paul F Agris
Keyword(s):  
Capillary Electrophoresis ◽  
Amino Acid ◽  
Gene Expression Regulation ◽  
Phage Display Library ◽  
Significant Shift ◽  
Mobility Shift ◽  
Amino Acid Peptide ◽  
Peptide Concentration ◽  
Mobility Shift Assay ◽  
Hiv 1

Methylation of RNA and proteins is one of a broad spectrum of post-transcriptional/translational mechanisms of gene expression regulation. Its functional signification is only beginning to be understood. A sensitive capillary electrophoresis mobility shift assay (CEMSA) for qualitative study of the methylation effect on biomolecules interaction is presented. Two RNA-peptide systems were chosen for the study. The first one consists of a 17-nucleotide analogue (+27-+43) of the yeast tRNA(Phe) anticodon stem and loop domain (ASL(Phe)) containing three of the five naturally occurring modifications (2'-O-methylcytidine (Cm(32)), 2'-O-methylguanine (Gm(34)) and 5-methylcytidine (m(5)C(40))) (ASL(Phe)-Cm(32),Gm(34),m(5)C(40)) and a 15-amino-acid peptide (named t(F)2: Ser(1)-Ile-Ser-Pro-Trp(5)-Gly-Phe-Ser-Gly-Leu(10)-Leu- Arg-Trp-Ser-Tyr(15)) selected from a random phage display library (RPL). A peptide-concentration-dependent formation of an RNA-peptide complex was clearly observable by CEMSA. In the presence of the peptide the capillary electrophoresis (CE) peak for triply methylated ASL(Phe) shifted from 18.16 to 20.90 min. Formation of the complex was not observed when an unmethylated version of ASL(Phe) was used. The second system studied consisted of the (+18)-(+44) fragment of the trans-activation response element of human immunodeficiency virus type 1 (TAR RNA HIV-1) and a 9-amino-acid peptide of the trans-activator of transcription protein (Tat HIV-1) Tat(49-57)-NH(2) (named Tat1: Arg(49)-Lys-Lys-Arg(52)-Arg-Gln-Arg-Arg- Arg(57)-NH(2)). In the presence of Tat(49-57)-NH(2) a significant shift of migration time of TAR from 18.66 min to 20.12 min was observed. Methylation of a residue Arg(52)-->Arg(Me)(2), crucial for TAR binding, strongly disrupted formation of the complex. Only at a high micromolar peptide concentration a poorly shaped, broad peak of the complex was observed. CE was found to be an efficient and sensitive method for the analysis of methylation effects on interaction of biomolecules.

Interaction of long telomeric DNAs with macrocyclic hexaoxazole as a G-quadruplex ligand

MedChemComm ◽  
2013 ◽  
Vol 4 (1) ◽  
pp. 260-264 ◽  
Author(s):  
Keisuke Iida ◽  
Gen Tsubouchi ◽  
Takahiro Nakamura ◽  
Satoki Majima ◽  
Hiroyuki Seimiya ◽  
...  
Keyword(s):  
Circular Dichroism ◽  
Living Cells ◽  
Dna Melting ◽  
Mobility Shift ◽  
Electrophoresis Mobility Shift Assay ◽  
G Quadruplex ◽  
Mobility Shift Assay ◽  
Circular Dichroïsm

The interactions of long telomeric DNAs, which mimic telomeres in living cells, with a macrocyclic hexaoxazole ligand L2H2-6OTD (2) were investigated by means of electrophoresis mobility shift assay, circular dichroism (CD) titration analysis, and DNA melting measurements.

scholarly journals Naf1 Regulates HIV-1 Latency by Suppressing Viral Promoter-Driven Gene Expression in Primary CD4+ T Cells

2016 ◽  
Vol 91 (1) ◽  
Author(s):  
Chuan Li ◽  
Hai-Bo Wang ◽  
Wen-Dong Kuang ◽  
Xiao-Xin Ren ◽  
Shu-Ting Song ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell Activation ◽  
Nuclear Export ◽  
Therapeutic Strategy ◽  
Host Protein ◽  
Viral Reactivation ◽  
Viral Transcription ◽  
Dependent Manner ◽  
Ltr Promoter ◽  
Hiv 1

ABSTRACT HIV-1 latency is characterized by reversible silencing of viral transcription driven by the long terminal repeat (LTR) promoter of HIV-1. Cellular and viral factors regulating LTR activity contribute to HIV-1 latency, and certain repressive cellular factors modulate viral transcription silencing. Nef-associated factor 1 (Naf1) is a host nucleocytoplasmic shuttling protein that regulates multiple cellular signaling pathways and HIV-1 production. We recently reported that nuclear Naf1 promoted nuclear export of unspliced HIV-1 gag mRNA, leading to increased Gag production. Here we demonstrate new functions of Naf1 in regulating HIV-1 persistence. We found that Naf1 contributes to the maintenance of HIV-1 latency by inhibiting LTR-driven HIV-1 gene transcription in a nuclear factor kappa B-dependent manner. Interestingly, Naf1 knockdown significantly enhanced viral reactivation in both latently HIV-1-infected Jurkat T cells and primary central memory CD4+ T cells. Furthermore, Naf1 knockdown in resting CD4+ T cells from HIV-1-infected individuals treated with antiretroviral therapy significantly increased viral reactivation upon T-cell activation, suggesting an important role of Naf1 in modulating HIV-1 latency in vivo. Our findings provide new insights for a better understanding of HIV-1 latency and suggest that inhibition of Naf1 activity to activate latently HIV-1-infected cells may be a potential therapeutic strategy. IMPORTANCE HIV-1 latency is characterized mainly by a reversible silencing of LTR promoter-driven transcription of an integrated provirus. Cellular and viral proteins regulating LTR activity contribute to the modulation of HIV-1 latency. In this study, we found that the host protein Naf1 inhibited HIV-1 LTR-driven transcription of HIV genes and contributed to the maintenance of HIV-1 latency. Our findings provide new insights into the effects of host modulation on HIV-1 latency, which may lead to a potential therapeutic strategy for HIV persistence by targeting the Naf1 protein.

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