26 research outputs found
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Nucleolar Localization of HIV-1 Rev Is Required, Yet Insufficient for Production of Infectious Viral Particles.
Combination antiretroviral therapy fails in complete suppression of HIV-1 due to drug resistance and persistent latency. Novel therapeutic intervention requires knowledge of intracellular pathways responsible for viral replication, specifically those untargeted by antiretroviral drugs. An understudied phenomenon is the nucleolar localization of Rev phosphoprotein, which completes nucleocytoplasmic transport of unspliced/partially spliced HIV mRNA through multimerization with intronic cis-acting targets-the Rev-response element (RRE). Rev contains a nucleolar localization signal (NoLS) comprising the COOH terminus of the arginine-rich motif for accumulation within nucleoli-speculated as the interaction ground for Rev with cellular proteins mediating mRNA-independent nuclear export and splicing. Functionality of Rev nucleolar access during HIV-1 production and infection was investigated in the context of deletion and single-point mutations within Rev-NoLS. Mutations induced upon Rev-NoLS are hypothesized to inactivate the HIV-1 infectious cycle. HIV-1HXB2 replication ceased with Rev mutations lacking nucleolar access due to loss or replacement of multiple arginine residues. Rev mutations missing single arginine residues remained strictly nucleolar in pattern and participated in proviral production, however, with reduced efficiency. Viral RNA packaging also decreased in efficiency after expression of nucleolar-localizing mutations. These results were observed during propagation of variant HIV-1NL4-3 containing nucleolar-localizing mutations within the viral backbone (M4, M5, and M6). Lentiviral particles produced with Rev single-point mutations were transducible at extremely low frequency. Similarly, HIV-1NL4-3 Rev-NoLS variants lost infectivity, unlike virulent WT (wild type) HIV-1NL4-3. HIV-1NL4-3 variants were capable of CD4+ host entry and reverse transcription as WT HIV-1NL4-3, but lacked ability to complete a full infectious cycle. We currently reveal that viral integration is deregulated in the presence of Rev-NoLS mutations
Identification of functional microRNAs released through asymmetrical processing of HIV-1 TAR elementā
The interaction between human immunodeficiency virus type 1 (HIV-1) and RNA silencing pathways is complex and multifaceted. Essential for efficient viral transcription and supporting Tat-mediated transactivation of viral gene expression, the trans-activation responsive (TAR) element is a structured RNA located at the 5ā² end of all transcripts derived from HIV-1. Here, we report that this element is a source of microRNAs (miRNAs) in cultured HIV-1-infected cell lines and in HIV-1-infected human CD4+ T lymphocytes. Using primer extension and ribonuclease (RNase) protection assays, we delineated both strands of the TAR miRNA duplex deriving from a model HIV-1 transcript, namely miR-TAR-5p and miR-TAR-3p. In vitro RNase assays indicate that the lack of a free 3ā² extremity at the base of TAR may contribute to its low processing reactivity in vivo. Both miR-TAR-5p and miR-TAR-3p down-regulated TAR miRNA sensor activity in a process that required an integral miRNA-guided RNA silencing machinery. miR-TAR-3p exerted superior gene downregulatory effects, probably due to its preferential release from HIV-1 TAR RNA by the RNase III Dicer. Our study suggests that the TAR element of HIV-1 transcripts releases functionally competent miRNAs upon asymmetrical processing by Dicer, thereby providing novel insights into viral miRNA biogenesis
321. Deletion of Mutated GAA Repeats from the Intron 1 of the Frataxin Gene Using the CRISPR System Restores the Protein Expression in a Friedreich Ataxia Model
The CRISPR system is now widely used as a molecular tool to edit the genome. We used this technique in Friedreich Ataxia (FRDA), an inherited autosomal disease known to cause a decrease of the mitochondrial frataxin protein. Genetic analysis revealed a GAA repeat expansion within the intron 1 of the frataxin (FXN) gene. We used cells derived from the YG8sR mouse model where the mouse frataxin gene is knockout but contain a human FXN mutated transgene on one allele. We then deleted the GAA trinucleotide repeat using 2 specific guide RNAs (gRNAs) co-expressed with either S. pyogenes (Sp) or S. aureus (Sa) Cas9. We were able to monitored an increase up to 2-fold of frataxin mRNA and protein levels in clone cells. We also confirmed these results in vivo using DNA electroporation in the Tibialis anterior muscle of the YG8R mice. Ongoing in vivo investigation of a systemically injected AAV-DJ vector expressing the SaCas9 and 2 successful selected gRNAs in the mouse model YG8sR will hopefully provide more details answers on the efficacy of the approach and give us preliminary data to go forward for clinical trial. The deletion of the GAA repeats expansion then might be a highly valuable gene therapy approach for FRDA patients
Refractoriness of hepatitis C virus internal ribosome entry site to processing by Dicer in vivo
<p>Abstract</p> <p>Background</p> <p>Hepatitis C virus (HCV) is a positive-strand RNA virus harboring a highly structured internal ribosome entry site (IRES) in the 5' nontranslated region of its genome. Important for initiating translation of viral RNAs into proteins, the HCV IRES is composed of RNA structures reminiscent of microRNA precursors that may be targeted by the host RNA silencing machinery.</p> <p>Results</p> <p>We report that HCV IRES can be recognized and processed into small RNAs by the human ribonuclease Dicer in vitro. Furthermore, we identify domains II, III and VI of HCV IRES as potential substrates for Dicer in vitro. However, maintenance of the functional integrity of the HCV IRES in response to Dicer overexpression suggests that the structure of the HCV IRES abrogates its processing by Dicer in vivo.</p> <p>Conclusion</p> <p>Our results suggest that the HCV IRES may have evolved to adopt a structure or a cellular context that is refractory to Dicer processing, which may contribute to viral escape of the host RNA silencing machinery.</p
Characterization of the Trans Watson-Crick GU Base Pair Located in the Catalytic Core of the Antigenomic HDV Ribozyme
The HDV ribozymeās folding pathway is, by far, the most complex folding
pathway elucidated to date for a small ribozyme. It includes 6 different steps
that have been shown to occur before the chemical cleavage. It is likely that
other steps remain to be discovered. One of the most critical of these unknown
steps is the formation of the trans Watson-Crick GU base
pair within loop III. The U23 and G28 nucleotides that
form this base pair are perfectly conserved in all natural variants of the
HDV ribozyme, and therefore are considered as being part of the signature
of HDV-like ribozymes. Both the formation and the transformation of this base
pair have been studied mainly by crystal structure and by molecular dynamic
simulations. In order to obtain physical support for the formation of this
base pair in solution, a set of experiments, including direct mutagenesis,
the site-specific substitution of chemical groups, kinetic studies, chemical
probing and magnesium-induced cleavage, were performed with the specific goal
of characterizing this trans Watson-Crick GU base pair in
an antigenomic HDV ribozyme. Both U23 and G28 can be
substituted for nucleotides that likely preserve some of the H-bond interactions
present before and after the cleavage step. The formation of the more stable trans
Watson-Crick base pair is shown to be a post-cleavage event, while a possibly
weaker trans Watson-Crick/Hoogsteen interaction seems to
form before the cleavage step. The formation of this unusually stable post-cleavage
base pair may act as a driving force on the chemical cleavage by favouring
the formation of a more stable ground state of the product-ribozyme complex.
To our knowledge, this represents the first demonstration of a potential stabilising
role of a post-cleavage conformational switch event in a ribozyme-catalyzed
reaction
MicroRNAs in Gene Regulation: When the Smallest Governs It All
Encoded by the genome of most eukaryotes examined so far,
microRNAs (miRNAs) are small ~21-nucleotide (nt) noncoding
RNAs (ncRNAs) derived from a biosynthetic cascade involving
sequential processing steps executed by the ribonucleases
(RNases) III Drosha and Dicer. Following their recent
identification, miRNAs have rapidly taken the center stage as key
regulators of gene expression. In this review, we will summarize
our current knowledge of the miRNA biosynthetic pathway and its
protein components, as well as the processes it regulates via
miRNAs, which are known to exert a variety of biological functions
in eukaryotes. Although the relative importance of miRNAs remains
to be fully appreciated, deregulated protein expression resulting
from either dysfunctional miRNA biogenesis or abnormal miRNA-based
gene regulation may represent a key etiologic factor in several,
as yet unidentified, diseases. Hence is our need to better understand
the complexity of the basic mechanisms underlying miRNA biogenesis
and function
Increased Frataxin Expression Induced in Friedreich Ataxia Cells by Platinum TALE-VP64s or Platinum TALE-SunTag
Frataxin gene (FXN) expression is reduced in Friedreichās ataxia patients due to an increase in the number of GAA trinucleotides in intron 1. The frataxin protein, encoded by that gene, plays an important role in mitochondriaās iron metabolism. Platinum TALE (plTALE) proteins targeting the regulatory region of the FXN gene, fused with a transcriptional activator (TA) such as VP64 or P300, were used to increase theĀ expression of that gene. Many effectors, plTALEVP64, plTALEp300, and plTALESunTag, targeting 14 sequences of the FXN gene promoter or intron 1 were produced. This permitted selection of 3 plTALEVP64s and 2 plTALESunTag that increased FXN gene expression by up to 19-fold in different Friedreich ataxia (FRDA) primary fibroblasts. Adeno-associated viruses were used to deliver the best effectors to the YG8R mouse model to validate their efficiencies inĀ vivo. Our results showed that these selected plTALEVP64s or plTALESunTag induced transcriptional activity of the endogenous FXN gene as well as expression of the frataxin protein in YG8R mouse heart by 10-fold and in skeletal muscles by up to 35-fold. The aconitase activity was positively modulated by the frataxin level in mitochondria, and it was, thus, increased inĀ vitro and inĀ vivo by the increased frataxin expression. Keywords: TAL effector, VP64, p300, SunTag, TALE-SunTag, AAV9, frataxin, transcription regulation, initiation of transcription, epigenetics, gene regulatio
Recommended from our members
Nucleolar Localization of HIV-1 Rev Is Required, Yet Insufficient for Production of Infectious Viral Particles.
Combination antiretroviral therapy fails in complete suppression of HIV-1 due to drug resistance and persistent latency. Novel therapeutic intervention requires knowledge of intracellular pathways responsible for viral replication, specifically those untargeted by antiretroviral drugs. An understudied phenomenon is the nucleolar localization of Rev phosphoprotein, which completes nucleocytoplasmic transport of unspliced/partially spliced HIV mRNA through multimerization with intronic cis-acting targets-the Rev-response element (RRE). Rev contains a nucleolar localization signal (NoLS) comprising the COOH terminus of the arginine-rich motif for accumulation within nucleoli-speculated as the interaction ground for Rev with cellular proteins mediating mRNA-independent nuclear export and splicing. Functionality of Rev nucleolar access during HIV-1 production and infection was investigated in the context of deletion and single-point mutations within Rev-NoLS. Mutations induced upon Rev-NoLS are hypothesized to inactivate the HIV-1 infectious cycle. HIV-1HXB2 replication ceased with Rev mutations lacking nucleolar access due to loss or replacement of multiple arginine residues. Rev mutations missing single arginine residues remained strictly nucleolar in pattern and participated in proviral production, however, with reduced efficiency. Viral RNA packaging also decreased in efficiency after expression of nucleolar-localizing mutations. These results were observed during propagation of variant HIV-1NL4-3 containing nucleolar-localizing mutations within the viral backbone (M4, M5, and M6). Lentiviral particles produced with Rev single-point mutations were transducible at extremely low frequency. Similarly, HIV-1NL4-3 Rev-NoLS variants lost infectivity, unlike virulent WT (wild type) HIV-1NL4-3. HIV-1NL4-3 variants were capable of CD4+ host entry and reverse transcription as WT HIV-1NL4-3, but lacked ability to complete a full infectious cycle. We currently reveal that viral integration is deregulated in the presence of Rev-NoLS mutations
Dicer-Derived MicroRNAs Are Utilized by the Fragile X Mental Retardation Protein for Assembly on Target RNAs
In mammalian cells, fragile X mental retardation protein (FMRP)
has been reported to be part of a microRNA (miRNA)-containing
effector ribonucleoprotien (RNP) complex believed to mediate
translational control of specific mRNAs. Here, using recombinant
proteins, we demonstrate that human FMRP can act as a miRNA
acceptor protein for the ribonuclease Dicer and facilitate the
assembly of miRNAs on specific target RNA sequences. The miRNA
assembler property of FMRP was abrogated upon deletion of its
single-stranded (ss) RNA binding K-homology domains. The
requirement of FMRP for efficient RNA interference (RNAi) in vivo
was unveiled by reporter gene silencing assays using various small
RNA inducers, which also supports its involvement in an ss small
interfering RNA (siRNA)-containing RNP (siRNP) effector complex in
mammalian cells. Our results define a possible role for FMRP in
RNA silencing and may provide further insight into the molecular
defects in patients with the fragile X syndrome