ABSTRACT
BACKGROUND: The murine leukemia virus (MLV) has been a powerful model of pathogenesis for the discovery of genes involved in cancer. Its splice donor (SD')-associated retroelement (SDARE) is important for infectivity and tumorigenesis, but the mechanism remains poorly characterized. Here, we show for the first time that P50 protein, which is produced from SDARE, acts as an accessory protein that transregulates transcription and induces cell transformation. RESULTS: By infecting cells with MLV particles containing SDARE transcript alone (lacking genomic RNA), we show that SDARE can spread to neighbouring cells as shown by the presence of P50 in infected cells. Furthermore, a role for P50 in cell transformation was demonstrated by CCK8, TUNEL and anchorage-independent growth assays. We identified the integrase domain of P50 as being responsible for transregulation of the MLV promoter using luciferase assay and RTqPCR with P50 deleted mutants. Transcriptomic analysis furthermore revealed that the expression of hundreds of cellular RNAs involved in cancerogenesis were deregulated in the presence of P50, suggesting that P50 induces carcinogenic processes via its transcriptional regulatory function. CONCLUSION: We propose a novel SDARE-mediated mode of propagation of the P50 accessory protein in surrounding cells. Moreover, due to its transforming properties, P50 expression could lead to a cellular and tissue microenvironment that is conducive to cancer development.
Subject(s)
Gene Expression Profiling , Gene Expression Regulation , Mice , Animals , Genomics , Leukemia Virus, Murine/genetics , Promoter Regions, Genetic , RNAABSTRACT
In cells, positive strand RNA viruses, such as Retroviridae, must selectively recognize their full-length RNA genome among abundant cellular RNAs to assemble and release particles. How viruses coordinate the intracellular trafficking of both RNA and protein components to the assembly sites of infectious particles at the cell surface remains a long-standing question. The mechanisms ensuring packaging of genomic RNA are essential for viral infectivity. Since RNA packaging impacts on several essential functions of retroviral replication such as RNA dimerization, translation and recombination events, there are many studies that require the determination of RNA packaging efficiency and/or RNA packaging ability. Studies of RNA encapsidation rely upon techniques for the identification and quantification of RNA species packaged by the virus. This review focuses on the different approaches available to monitor RNA packaging: Northern blot analysis, ribonuclease protection assay and quantitative reverse transcriptase-coupled polymerase chain reaction as well as the most recent RNA imaging and sequencing technologies. Advantages, disadvantages and limitations of these approaches will be discussed in order to help the investigator to choose the most appropriate technique. Although the review was written with the prototypic simple murine leukemia virus (MLV) and complex human immunodeficiency virus type 1 (HIV-1) in mind, the techniques were described in order to benefit to a larger community.
Subject(s)
Molecular Biology/methods , RNA, Viral/metabolism , Retroviridae/physiology , Virion/metabolism , Virology/methods , Virus Assembly , Animals , HumansABSTRACT
Dimerization is a unique and vital characteristic of retroviral genomes. It is commonly accepted that genomic RNA (gRNA) must be dimeric at the plasma membrane of the infected cells to be packaged during virus assembly. However, where, when and how HIV-1 gRNA find each other and dimerize in the cell are long-standing questions that cannot be answered using conventional approaches. Here, we combine two state-of-the-art, multicolor RNA labeling strategies with two single-molecule microscopy technologies to address these questions. We used 3D-super-resolution structured illumination microscopy to analyze and quantify the spatial gRNA association throughout the cell and monitored the dynamics of RNA-RNA complexes in living-cells by cross-correlation fluctuation analysis. These sensitive and complementary approaches, combined with trans-complementation experiments, reveal for the first time the presence of interacting gRNA in the cytosol, a challenging observation due to the low frequency of these events and their dilution among the bulk of other RNAs, and allow the determination of the subcellular orchestration of the HIV-1 dimerization process.
Subject(s)
Dimerization , HIV-1/genetics , Imaging, Three-Dimensional , Microscopy/methods , Nucleic Acid Conformation , RNA, Viral/chemistry , Cell Membrane/metabolism , Cell Survival , Color , Cytosol/metabolism , Genome, Viral , HeLa Cells , Humans , In Situ Hybridization, Fluorescence , RNA, Viral/genetics , Staining and Labeling , gag Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
The retroviral genome consists of an intron-containing transcript that has essential cytoplasmic functions in the infected cell. This viral transcript can escape splicing, circumvent the nuclear checkpoint mechanisms and be transported to the cytoplasm by hijacking the host machinery. Once in the cytoplasm, viral unspliced RNA acts as mRNA to be translated and as genomic RNA to be packaged into nascent viruses. The murine leukemia virus (MLV) is among the first retroviruses discovered and is classified as simple Retroviridae due to its minimal encoding capacity. The oncogenic and transduction abilities of MLV are extensively studied, whereas surprisingly the crucial step of its nuclear export has remained unsolved until 2014. Recent work has revealed the recruitment by MLV of the cellular NXF1/Tap-dependent pathway for export. Unconventionally, MLV uses of Tap to export both spliced and unspliced viral RNAs. Unlike other retroviruses, MLV does not harbor a unique RNA signal for export. Indeed, multiple sequences throughout the MLV genome appear to promote export of the unspliced MLV RNA. We review here the current understanding of the export mechanism and highlight the determinants that influence MLV export. As the molecular mechanism of MLV export is elucidated, we will gain insight into the contribution of the export pathway to the cytoplasmic fate of the viral RNA.
Subject(s)
Active Transport, Cell Nucleus , Introns , Leukemia Virus, Murine/physiology , RNA, Viral/genetics , RNA, Viral/metabolism , Alternative Splicing , Animals , Genome, Viral , Humans , Inverted Repeat Sequences , Mice , Nucleic Acid Conformation , Nucleocytoplasmic Transport Proteins/metabolism , Nucleotide Motifs , Open Reading Frames , Protein Binding , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/chemistry , Regulatory Sequences, Ribonucleic Acid , Signal Transduction , Terminal Repeat SequencesABSTRACT
HIV-1, the agent of the AIDS pandemic, is an RNA virus that reverse transcribes its RNA genome (gRNA) into DNA, shortly after its entry into cells. Within cells, retroviral assembly requires thousands of structural Gag proteins and two copies of gRNA as well as cellular factors, which converge to the plasma membrane in a finely regulated timeline. In this process, the nucleocapsid domain of Gag (GagNC) ensures gRNA selection and packaging into virions. Subsequent budding and virus release require the recruitment of the cellular ESCRT machinery. Interestingly, mutating GagNC results into the release of DNA-containing viruses, by promo-ting reverse transcription (RTion) prior to virus release, through an unknown mechanism. Therefore, we explored the biogenesis of these DNA-containing particles, combining live-cell total internal-reflection fluorescent microscopy, electron microscopy, trans-complementation assays and biochemical characterization of viral particles. Our results reveal that DNA virus production is the consequence of budding defects associated with Gag aggregation at the plasma membrane and deficiency in the recruitment of Tsg101, a key ESCRT-I component. Indeed, targeting Tsg101 to virus assembly sites restores budding, restricts RTion and favors RNA packaging into viruses. Altogether, our results highlight the role of GagNC in the spatiotemporal control of RTion, via an ESCRT-I-dependent mechanism.
Subject(s)
DNA-Binding Proteins/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , HIV-1/physiology , Transcription Factors/metabolism , Virus Assembly , gag Gene Products, Human Immunodeficiency Virus/metabolism , Cell Line , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Cell Membrane/virology , DNA, Viral/biosynthesis , HIV-1/genetics , HIV-1/metabolism , HeLa Cells , Humans , Reverse Transcription , Sequence Deletion , Virion/metabolism , Zinc Fingers , gag Gene Products, Human Immunodeficiency Virus/chemistryABSTRACT
Biopsies from patients show that hepadnaviral core proteins and capsids - collectively called core - are found in the nucleus and cytoplasm of infected hepatocytes. In the majority of studies, cytoplasmic core localization is related to low viraemia while nuclear core localization is associated with high viral loads. In order to better understand the molecular interactions leading to core localization, we analysed transfected hepatoma cells using immune fluorescence microscopy. We observed that expression of core protein in the absence of other viral proteins led to nuclear localization of core protein and capsids, while expression of core in the context of the other viral proteins resulted in a predominantly cytoplasmic localization. Analysis of which viral partner was responsible for cytoplasmic retention indicated that the HBx, surface proteins and HBeAg had no impact but that the viral polymerase was the major determinant. Further analysis revealed that ϵ, an RNA structure to which the viral polymerase binds, was essential for cytoplasmic retention. Furthermore, we showed that core protein phosphorylation at Ser 164 was essential for the cytoplasmic core localization phenotype, which is likely to explain differences observed between individual cells.
Subject(s)
Capsid Proteins/metabolism , DNA-Directed DNA Polymerase/genetics , Hepatitis B virus/genetics , Hepatitis B virus/metabolism , Phosphorylation/genetics , Viral Core Proteins/metabolism , Capsid Proteins/genetics , Cell Line, Tumor , Cell Nucleus/genetics , Cell Nucleus/metabolism , Cytoplasm/genetics , Cytoplasm/metabolism , Hep G2 Cells , Hepatitis B e Antigens/genetics , Hepatitis B e Antigens/metabolism , Humans , Transfection/methods , Viral Core Proteins/geneticsABSTRACT
BACKGROUND: Eukaryotic cells have evolved stringent proofreading mechanisms to ensure that intron-containing mRNAs do not leave the nucleus. However, all retroviruses must bypass this checkpoint for replication. Indeed, their primary polycistronic transcript (Full-Length) must reach the cytoplasm to be either translated or packaged as genomic RNA in progeny viruses.Murine leukemia virus (MLV) is a prototype of simple retroviruses with only two well-regulated splicing events that directly influence viral leukemogenic properties in mice. Several cis-elements have been identified in the FL RNA that regulate its cytoplasmic accumulation. However, their connection with an export mechanism is yet unknown. Our goal was to identify the cellular pathway used by MLV to export its RNAs into the cytoplasm of the host cells. RESULTS: Since other retroviruses use the CRM1 and/or the Tap/NXF1 pathways to export their unspliced RNA from the nucleus, we investigated the role of these two pathways in MLV replication by using specific inhibitors. The effects of export inhibition on MLV protein synthesis, RNA levels and RNA localization were studied by Western blotting, RT-qPCR, fluorescence microscopy and ribonucleoprotein immunoprecipitation assays. Taken together, our results show for the first time that MLV requires the Tap/NXF1-mediated export pathway, and not the CRM1 pathway, for the expression of its spliced and unspliced RNAs and for FL RNA nuclear export. CONCLUSIONS: By contrast to HIV-1, MLV recruits the same pathway for the cytoplasmic expression of its spliced and unspliced RNAs. Thus, MLV RNA expression depends upon coordinated splicing/export processes. In addition, FL RNA translation relies on Tap/NXF1-dependent export, raising the critical question of whether the pool of FL RNA to be packaged is also exported by Tap/NXF1.
Subject(s)
Antigens, Ly/metabolism , Gene Expression , Leukemia Virus, Murine/physiology , Membrane Proteins/metabolism , Nucleocytoplasmic Transport Proteins/metabolism , RNA, Messenger/metabolism , RNA, Viral/metabolism , Active Transport, Cell Nucleus , Animals , Blotting, Western , Cell Line , Cytoplasm/metabolism , Leukemia Virus, Murine/genetics , Mice , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Viral Proteins/biosynthesisABSTRACT
Generation of infectious retroviral particles rely on the targeting of all structural components to the correct cellular sites at the correct time. Gag, the main structural protein, orchestrates the assembly process and the mechanisms that trigger its targeting to assembly sites are well described. Gag is also responsible for the packaging of the viral genome and the molecular details of the Gag/RNA interaction are well characterized. Until recently, much less was understood about the cell biology of retrovirus RNA packaging. However, novel biochemical and live-cell microscopic approaches have identified where in the cell the initial events of genome recognition by Gag occur. These recent developments have shed light on the role played by the viral genome during virion assembly. Other central issues of the cell biology of RNA packaging, such as how the Gag-RNA complex traffics through the cytoplasm towards assembly sites, await characterization.
Subject(s)
Gene Products, gag/metabolism , Genome, Viral , RNA, Viral/genetics , RNA, Viral/metabolism , Retroviridae/physiology , Animals , Cell Membrane/virology , Cytoplasm/metabolism , Gene Products, gag/genetics , Humans , Retroviridae/genetics , Virus Assembly/genetics , Virus Assembly/physiologyABSTRACT
RNA-based compounds are promising agents to inactivate viruses. New specific hepatitis delta virus (HDV)-derived ribozymes are natural molecules that can be engineered to specifically target a viral RNA. We have designed specific on-off adaptor (SOFA)-HDV ribozymes targeting the tat and rev sequences of the human immunodeficiency virus type 1 (HIV-1) RNA. We show that the SOFA-HDV ribozymes cleave their RNA target in vitro. They inhibit the Tat-mediated transactivation of HIV-1 from 62% to 86% in different assays. In vivo, the amount of HIV RNA was decreased by 60 and 86% with two distinct ribozymes, which indicates that the inhibition of HIV production is directly correlated to the decline in spliced and unspliced viral RNAs. These SOFAHDV- ribozymes inhibited the expression and the viral production of four HIV-1 strains, indicating an extended potential to act on multiple HIV variants. In HEK 293T and HeLa cells transfected with pNL4-3 and the SOFA-HDV-ribozymes, the reduced RNA levels consequently decreased the Gag protein expression in the cell and virus production in the supernatant. When transfected before HIV-1 infection, the ribozymes prevented the incoming virus from being expressed. The ribozymes inhibited HIV production up to 90% when transfected in combination with the HIV protease inhibitor Atazanavir. Our results strongly suggest that SOFA-HDV ribozymes have a great potential to target HIV-1 and to be used as therapeutic agents in combination therapy.
Subject(s)
HIV-1/enzymology , RNA, Catalytic/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Virus Replication , Atazanavir Sulfate , Base Sequence , Gene Products, gag/biosynthesis , Gene Products, gag/genetics , Gene Products, rev/genetics , Gene Products, rev/metabolism , HEK293 Cells , HIV Infections/genetics , HIV-1/genetics , HeLa Cells , Hepatitis Delta Virus/enzymology , Hepatitis Delta Virus/genetics , Humans , Oligopeptides/pharmacology , Pyridines/pharmacology , RNA Splicing , RNA, Catalytic/genetics , tat Gene Products, Human Immunodeficiency Virus/genetics , tat Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
BACKGROUND: Dicer, Ago2 and TRBP are the minimum components of the human RNA-induced silencing complex (RISC). While Dicer and Ago2 are RNases, TRBP is the double-stranded RNA binding protein (dsRBP) that loads small interfering RNA into the RISC. TRBP binds directly to Dicer through its C-terminal domain. RESULTS: We show that the TRBP binding site in Dicer is a 165 amino acid (aa) region located between the ATPase and the helicase domains. The binding site in TRBP is a 69 aa domain, called C4, located at the C-terminal end of TRBP. The TRBP1 and TRBP2 isoforms, but not TRBPs lacking the C4 site (TRBPsDeltaC4), co-immunoprecipitated with Dicer. The C4 domain is therefore necessary to bind Dicer, irrespective of the presence of RNA. Immunofluorescence shows that while full-length TRBPs colocalize with Dicer, TRBPsDeltaC4 do not. tarbp2-/- cells, which do not express TRBP, do not support RNA interference (RNAi) mediated by short hairpin or micro RNAs against EGFP. Both TRBPs, but not TRBPsDeltaC4, were able to rescue RNAi function. In human cells with low RNAi activity, addition of TRBP1 or 2, but not TRBPsDeltaC4, rescued RNAi function. CONCLUSION: The mapping of the interaction sites between TRBP and Dicer show unique domains that are required for their binding. Since TRBPsDeltaC4 do not interact or colocalize with Dicer, we suggest that TRBP and Dicer, both dsRBPs, do not interact through bound dsRNA. TRBPs, but not TRBPsDeltaC4, rescue RNAi activity in RNAi-compromised cells, indicating that the binding of Dicer to TRBP is critical for RNAi function.
Subject(s)
RNA Interference , RNA-Binding Proteins/chemistry , Ribonuclease III/metabolism , Animals , Binding Sites , Cells, Cultured , HeLa Cells , Humans , Protein Binding , Protein Structure, Tertiary , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Ribonuclease III/chemistry , Ribonuclease III/geneticsABSTRACT
In Escherichia coli the rare codons AGG, AGA and CGA are reported to have a detrimental effect on protein synthesis, especially during the expression of heterologous proteins. In the present work, we have studied the impact of successive clusters of these rare codons on the accuracy of mRNA translation in E. coli. For this purpose, we have analyzed the expression of an mRNA which contains in its 3' region a triplet and a tandem of AGA codons. This mRNA is derived from the human hepatitis B virus (HBV) preC gene. Both in eukaryotic cells and in eukaryotic cell-free translation system, this mRNA, directs the synthesis of a single 25 kDa protein. However, in a conventional E. coli strain BL 21 (DE3), transformed with a plasmid expressing this protein the synthesis of four polypeptides ranging from 30 to 21.5 kDa can be observed. Using different approaches, notably expression of i) precore mutated proteins or ii) chimeric proteins containing HA- and Myc-tags downstream of the AGA clusters (respectively in the -1 or +1 frame), we have found that when the ribosome encounters the AGA clusters, it can then resume the translation in both +1 and -1 frames. This result is in agreement with the model proposed recently by Baranov et al. (Baranov, P.V., Gesteland, R.F., Atkins, J.F., 2004. P-site tRNA is a crucial initiator of ribosomal frameshifting. RNA 10, 221-230), thus confirming that AGA/AGG codons can serve as sites for -1 frameshifting events. Only +1 frameshifting was suggested previously to occur at the AGA/AGG clusters.
Subject(s)
Escherichia coli/genetics , Base Sequence , Codon/genetics , DNA Primers/genetics , DNA, Bacterial/genetics , Escherichia coli/metabolism , Frameshifting, Ribosomal , Molecular Sequence Data , Open Reading Frames , Plasmids/genetics , Protein Biosynthesis , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Proteins/genetics , Ribosomes/metabolismABSTRACT
RNA interference (RNAi) is now widely used for gene silencing in mammalian cells. The mechanism uses the RNA-induced silencing complex, in which Dicer, Ago2, and the human immunodeficiency virus type 1 (HIV-1) TAR RNA binding protein (TRBP) are the main components. TRBP is a protein that increases HIV-1 expression and replication by inhibition of the interferon-induced protein kinase PKR and by increasing translation of viral mRNA. After HIV infection, TRBP could restrict the viral RNA through its activity in RNAi or could contribute more to the enhancement of viral replication. To determine which function will be predominant in the virological context, we analyzed whether the inhibition of its expression could enhance or decrease HIV replication. We have generated small interfering RNAs (siRNAs) against TRBP and found that they decrease HIV-1 long terminal repeat (LTR) basal expression 2-fold, and the LTR Tat transactivated level up to 10-fold. In the context of HIV replication, siRNAs against TRBP decrease the expression of viral genes and inhibit viral production up to fivefold. The moderate increase in PKR expression and activation indicates that it contributes partially to viral gene inhibition. The moderate decrease in micro-RNA (miRNA) biogenesis by TRBP siRNAs suggests that in the context of HIV replication, TRBP functions other than RNAi are predominant. In addition, siRNAs against Dicer decrease viral production twofold and impede miRNA biogenesis. These results suggest that, in the context of HIV replication, TRBP contributes mainly to the enhancement of virus production and that Dicer does not mediate HIV restriction by RNAi.
Subject(s)
HIV Long Terminal Repeat/physiology , HIV-1/physiology , RNA Interference , RNA, Small Interfering/genetics , RNA-Binding Proteins/metabolism , Virus Replication , Gene Expression , Genes, Reporter , Green Fluorescent Proteins , HIV Reverse Transcriptase/antagonists & inhibitors , HeLa Cells , Humans , Luciferases , MicroRNAs/biosynthesis , RNA-Binding Proteins/antagonists & inhibitors , RNA-Binding Proteins/genetics , Ribonuclease III/antagonists & inhibitors , Ribonuclease III/biosynthesis , Ribonuclease III/geneticsABSTRACT
HIV-1 viral production is restricted intracellularly in astrocytes compared with lymphocytes due to the limited expression of viral structural proteins. The poor translation of HIV-1 mRNA and consequent limited virion production can be restored by overexpression of TRBP proteins in the astrocytoma U251MG cells. TRBP1 and TRBP2 are double-stranded RNA binding proteins that increase HIV-1 gene expression. Both proteins are produced from a single gene that possesses two independent promoters and an alternative first exon. Endogenous expression is restricted in astrocytes due to limited TRBP promoter expression compared to lymphocytes. We examined the transcriptional regulation of TRBP1 and TRBP2 by in vivo genomic footprinting in the lymphocytic Jurkat and in the astrocytic U251MG cells. We identified one AP4 and one AP2-binding site that regulate the TRBP2 promoter in both cell types, and one Sp1 and two CCAAT-binding sites that control TRBP1 expression. Mutations in the TRBP1 promoter modulate its expression specifically in Jurkat and in U251MG. The analysis of the CCAAT-390 site by EMSA and by ChIP demonstrates that NF-Y/CBF transcription factor binds specifically to the promoter in vitro and in vivo. Furthermore, each NF-Y subunit was more highly expressed in the lymphocytic cells, compared to astrocytic cells. An NF-YA trans-dominant mutant decreased TRBP1 promoter expression fourfold in Jurkat cells, thus demonstrating the functional importance of NF-Y factors in lymphocytes. These studies suggest that the cell specifity of HIV-1 expression and replication may be regulated, in part, through the control of TRBP1 expression.
Subject(s)
Astrocytes/physiology , CCAAT-Binding Factor/metabolism , Intracellular Signaling Peptides and Proteins , Lymphocytes/physiology , Promoter Regions, Genetic , Protein Isoforms , Astrocytes/cytology , Astrocytoma , Base Sequence , Binding Sites , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Genes, Reporter , HIV-1/genetics , HIV-1/metabolism , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Jurkat Cells , Lymphocytes/cytology , Molecular Sequence Data , Nuclear Receptor Coactivators , Protein Isoforms/genetics , Protein Isoforms/metabolism , Tissue Distribution , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
Increasing evidence indicates that RNA interference (RNAi) may be used to provide antiviral immunity in mammalian cells. Human micro (mi)RNAs can inhibit the replication of a primate virus, whereas a virally-encoded miRNA from HIV inhibits its own replication. Indirect proof comes from RNAi suppressors encoded by mammalian viruses. Influenza NS1 and Vaccinia E3L proteins can inhibit RNAi in plants, insects and worms. HIV-1 Tat protein and Adenovirus VA RNAs act as RNAi suppressors in mammalian cells. Surprisingly, many RNAi suppressors are also inhibitors of the interferon (IFN)-induced protein kinase R (PKR) but the potential overlap between the RNAi and the IFN pathways remains to be determined. The link between RNAi as an immune response and the IFN pathway may be formed by a cellular protein, TRBP, which has a dual role in HIV replication and RNAi. TRBP has been isolated as an HIV-1 TAR RNA binding protein that increases HIV expression and replication by inhibiting PKR and by increasing translation of structured RNAs. A recent report published in the Journal of Virology shows that the poor replication of HIV in astrocytes is mainly due to a heightened PKR response that can be overcome by supplying TRBP exogenously. In two recent papers published in Nature and EMBO Reports, TRBP is now shown to interact with Dicer and to be required for RNAi mediated by small interfering (si) and micro (mi)RNAs. The apparent discrepancy between TRBP requirement in RNAi and in HIV replication opens the hypotheses that RNAi may be beneficial for HIV-1 replication or that HIV-1 may evade the RNAi restriction by diverting TRBP from Dicer and use it for its own benefit.
Subject(s)
HIV/physiology , RNA Interference , RNA-Binding Proteins/physiology , Virus Replication , HIV/immunology , Humans , Immunity, Innate , Ribonuclease III/metabolismABSTRACT
Dicer is a key enzyme involved in RNA interference (RNAi) and microRNA (miRNA) pathways. It is required for biogenesis of miRNAs and small interfering RNAs (siRNAs), and also has a role in the effector steps of RNA silencing. Apart from Argonautes, no proteins are known to associate with Dicer in mammalian cells. In this work, we describe the identification of TRBP (human immunodeficiency virus (HIV-1) transactivating response (TAR) RNA-binding protein) as a protein partner of human Dicer. We show that TRBP is required for optimal RNA silencing mediated by siRNAs and endogenous miRNAs, and that it facilitates cleavage of pre-miRNA in vitro. TRBP had previously been assigned several functions, including inhibition of the interferon-induced double-stranded RNA-regulated protein kinase PKR and modulation of HIV-1 gene expression by association with TAR. The TRBP-Dicer interaction shown raises interesting questions about the potential interplay between RNAi and interferon-PKR pathways.
Subject(s)
HIV-1/genetics , MicroRNAs/metabolism , RNA Interference/physiology , RNA, Small Interfering/metabolism , Ribonuclease III/metabolism , eIF-2 Kinase/genetics , Cell Line , Gene Expression Regulation, Viral , Genes, Regulator , HIV Long Terminal Repeat , Humans , Immunoprecipitation , Interferons , MicroRNAs/biosynthesis , RNA, Small Interfering/biosynthesis , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , RNA-Induced Silencing Complex , Ribonuclease III/genetics , Trans-ActivatorsABSTRACT
Circumstantial evidence suggests that the secreted hepatitis B virus (HBV) e antigen (HBeAg) and/or its 22 kDa precursor (P22) have an essential role in the establishment of persistent infection. In order to identify cellular proteins that could interact with P22, large amounts of this protein are required to perform pull-down assays. A plasmid was constructed encoding a recombinant P22 with a Histidine-tag at its N-terminal extremity (P22r). The initial attempts to overexpress P22r in a conventional Escherichia coli strain failed, most likely due to the presence of rare AGA/AGG codon clusters in the 3' part of the gene. To overcome this difficulty, P22r was overexpressed in the Epicurian coli BL21-codonplus (DE3)-RIL strain, which possesses extra copies of the ArgU gene that encodes the tRNA(AGA/AGG). In this strain, P22r was overexpressed successfully and then purified in milligram quantities by metal affinity chromatography on Ni2+-chelated His-Bind resin. The purified recombinant protein P22r was able to interact with a cellular protein (P32), which had previously been shown to co-immunoprecipitate with native P22, indicating that at least some of the P22r molecules were folded correctly.