A fast and tuneable auxin-inducible degron for depletion of target proteins in budding yeast.

The auxin-inducible degron (AID) is a useful technique to rapidly deplete proteins of interest in nonplant eukaryotes. Depletion is achieved by addition of the plant hormone auxin to the cell culture, which allows the auxin-binding receptor, TIR1, to target the AID-tagged protein for degradation by the proteasome. Fast depletion of the target protein requires good expression of TIR1 protein, but as we show here, high levels of TIR1 may cause uncontrolled depletion of the target protein in the absence of auxin. To enable conditional expression of TIR1 to a high level when required, we regulated the expression of TIR1 using the ß-estradiol expression system. This is a fast-acting gene induction system that does not cause secondary effects on yeast cell metabolism. We demonstrate that combining the AID and ß-estradiol systems results in a tightly controlled and fast auxin-induced depletion of nuclear target proteins. Moreover, we show that depletion rate can be tuned by modulating the duration of ß-estradiol preincubation. We conclude that TIR1 protein is a rate-limiting factor for target protein depletion in yeast, and we provide new tools that allow tightly controlled, tuneable, and efficient depletion of essential proteins whereas minimising secondary effects.

Functional signature for the recognition of specific target mRNAs by human Staufen1 protein.

Cellular messenger RNAs (mRNAs) are associated to proteins in the form of ribonucleoprotein particles. The double-stranded RNA-binding (DRB) proteins play important roles in mRNA synthesis, modification, activity and decay. Staufen is a DRB protein involved in the localized translation of specific mRNAs during Drosophila early development. The human Staufen1 (hStau1) forms RNA granules that contain translation regulation proteins as well as cytoskeleton and motor proteins to allow the movement of the granule on microtubules, but the mechanisms of hStau1-RNA recognition are still unclear. Here we used a combination of affinity chromatography, RNAse-protection, deep-sequencing and bioinformatic analyses to identify mRNAs differentially associated to hStau1 or a mutant protein unable to bind RNA and, in this way, defined a collection of mRNAs specifically associated to wt hStau1. A common sequence signature consisting of two opposite-polarity Alu motifs was present in the hStau1-associated mRNAs and was shown to be sufficient for binding to hStau1 and hStau1-dependent stimulation of protein expression. Our results unravel how hStau1 identifies a wide spectrum of cellular target mRNAs to control their localization, expression and fate.

Human Staufen1 protein interacts with influenza virus ribonucleoproteins and is required for efficient virus multiplication.

The influenza A virus genome consists of 8 negative-stranded RNA segments. NS1 is a nonstructural protein that participates in different steps of the virus infectious cycle, including transcription, replication, and morphogenesis, and acts as a virulence factor. Human Staufen1 (hStau1), a protein involved in the transport and regulated translation of cellular mRNAs, was previously identified as a NS1-interacting factor. To investigate the possible role of hStau1 in the influenza virus infection, we characterized the composition of hStau1-containing granules isolated from virus-infected cells. Viral NS1 protein and ribonucleoproteins (RNPs) were identified in these complexes by Western blotting, and viral mRNAs and viral RNAs (vRNAs) were detected by reverse transcription (RT)-PCR. Also, colocalization of hStau1 with NS1, nucleoprotein (NP), and PA in the cytosol of virus-infected cells was shown by immunofluorescence. To analyze the role of hStau1 in the infection, we downregulated its expression by gene silencing. Human HEK293T cells or A549 cells were silenced using either short hairpin RNAs (shRNAs) or small interfering RNAs (siRNAs) targeting four independent sites in the hStau1 mRNA. The yield of influenza virus was reduced 5 to 10 times in the various hStau1-silenced cells compared to that in control silenced cells. The expression levels of viral proteins and their nucleocytoplasmic localization were not affected upon hStau1 silencing, but virus particle production, as determined by purification of virions from supernatants, was reduced. These results indicate a role for hStau1 in late events of the influenza virus infection, possibly during virus morphogenesis.

hCLE/C14orf166, a cellular protein required for viral replication, is incorporated into influenza virus particles.

The influenza A virus polymerase associates with a number of cellular transcription-related factors, including the RNA polymerase II (RNAP II). We previously described that the cellular protein hCLE/C14orf166 interacts with and stimulates influenza virus polymerase as well as RNAP II activities. Here we show that, despite the considerable cellular shut-off observed in infected cells, which includes RNAP II degradation, hCLE protein levels increase throughout infection in a virus replication-dependent manner. Human and avian influenza viruses of various subtypes increase hCLE levels, but other RNA or DNA viruses do not. hCLE colocalises and interacts with viral ribonucleoproteins (vRNP) in the nucleus, as well as in the cytoplasm late in infection. Furthermore, biochemical analysis of purified virus particles and immunoelectron microscopy of infected cells show hCLE in virions, in close association with viral vRNP. These findings indicate that hCLE, a cellular protein important for viral replication, is one of the very few examples of transcription factors that are incorporated into particles of an RNA-containing virus.

Hepatitis C virus core protein transactivates the inducible nitric oxide synthase promoter via NF-kappaB activation.

Intrahepatic levels of the inducible nitric oxide synthase (iNOS) are increased in chronic hepatitis C patients. As iNOS gene promoter contains Nuclear Factor (NF)-kappaB binding sites and hepatitis C virus (HCV) core protein activates NF-kappaB, the aim of this work was to study if HCV core protein transactivates iNOS promoter through NF-kappaB activation. iNOS mRNA and protein were determined by RT-PCR and western blot in HepG2 cells. The effect of HCV core protein on iNOS promoter was assayed by cotransfecting HepG2 cells with the core protein expression plasmid pHCV-Co and p1iNOS-CAT or p2iNOS-CAT plasmids. Formation of NF-kappaB-DNA complexes was determined by electrophoretic mobility shift assay. Transfection of HepG2 cells with pHCV-Co plasmid results in an increase in iNOS mRNA and protein levels. Cotransfection with pHCV-Co and p1iNOS-CAT or p2iNOS-CAT plasmids results in a transactivation of iNOS promoter, the presence of the proximal NF-kappaB binding site in the promoter being sufficient for the transactivation. Furthermore, the HCV core protein increases the formation of complexes between NF-kappaB and its binding sequence in the iNOS promoter. The expression of the NF-kappaB inhibitor IKB reverts the effect of the HCV core protein on the iNOS promoter. In conclusion, HCV core protein transactivates iNOS gene promoter through NF-kappaB activation.

Human Staufen1 associates to miRNAs involved in neuronal cell differentiation and is required for correct dendritic formation.

Double-stranded RNA-binding proteins are key elements in the intracellular localization of mRNA and its local translation. Staufen is a double-stranded RNA binding protein involved in the localised translation of specific mRNAs during Drosophila early development and neuronal cell fate. The human homologue Staufen1 forms RNA-containing complexes that include proteins involved in translation and motor proteins to allow their movement within the cell, but the mechanism underlying translation repression in these complexes is poorly understood. Here we show that human Staufen1-containing complexes contain essential elements of the gene silencing apparatus, like Ago1-3 proteins, and we describe a set of miRNAs specifically associated to complexes containing human Staufen1. Among these, miR-124 stands out as particularly relevant because it appears enriched in human Staufen1 complexes and is over-expressed upon differentiation of human neuroblastoma cells in vitro. In agreement with these findings, we show that expression of human Staufen1 is essential for proper dendritic arborisation during neuroblastoma cell differentiation, yet it is not necessary for maintenance of the differentiated state, and suggest potential human Staufen1 mRNA targets involved in this process.

Hepatitis C virus core protein down-regulates transcription of interferon-induced antiviral genes.

BACKGROUND: Hepatitis C virus (HCV) proteins interfere with the interferon (IFN)-alpha-induced Jak/signal transducer and activator of transcription (STAT) pathway. Which protein is responsible for this effect and whether this interference results in down-regulation of IFN-induced genes remain controversial. We analyzed the effect of HCV core (HCV-Co) protein on expression of IFN-induced antiviral genes. METHODS: HepG2 cells were transfected with the plasmid pHCV-Co, and, after treatment with IFN-alpha , levels of MxA, protein kinase R (PKR), and 2'-5' oligoadenylate synthetase (2'-5'OAS) mRNA were determined. Chloramphenycol acethyl transferase (CAT) analysis was performed on cells cotransfected with pHCV-Co and pMx4CAT (containing the MxA gene promoter) and treated with IFN. Electrophoretic mobility shift assays were used, and Western-blot analysis of STAT 1 and 2 was performed. RESULTS: Levels of MxA mRNA in pHCV-Co-transfected cells decreased in a dose-dependent manner, by down-regulation of the MxA gene promoter. HCV-Co protein inhibits binding of IFN-stimulated gene factor 3 (ISGF3) to the IFN-stimulated response element (ISRE). Intracellular distribution of STAT 1 and 2 was not modified after treatment with IFN. Expression of HCV-Co protein also results in down-regulation of expression of PKR and 2'-5'OAS genes. CONCLUSION: HCV-Co protein inhibits IFN-alpha-induced transcription of antiviral genes by decreasing binding of ISGF3 to the ISRE.

Nitric oxide and TGF-beta1 inhibit HNF-4alpha function in HEPG2 cells.

This study analyzes if the profibrogenic factors nitric oxide and transforming growth factor-beta1 (TGF-beta1) affect hepatocyte nuclear factor-4alpha (HNF-4alpha) function. For this purpose, HepG2 cells were treated with TGF-beta1 or with a nitric oxide donor to determine mRNA levels of coagulation factor VII and HNF-4alpha. Treatment effect on factor VII gene promoter was assessed by chloramphenicol acetyl-transferase assays in cells transfected with the pFVII-CAT plasmid. HNF-4alpha binding and protein levels were determined by gel shift assays and Western blot. TGF-beta1 and nitric oxide downregulated factor VII mRNA levels by inhibiting its gene promoter activity. This inhibition is caused by a decrease in the DNA binding of HNF-4alpha. TGF-beta1 induces degradation of HNF-4alpha in the proteasome while nitric oxide provokes nitrosylation of cysteine residues in this factor. TGF-beta1 and nitric oxide inhibit HNF-4alpha activity. These findings may explain the loss of liver functions that occurs during fibrosis progression.

TT virus replicates in stimulated but not in nonstimulated peripheral blood mononuclear cells.

TT virus (TTV) is an unenveloped, single-stranded, circular-DNA virus which resembles members of the Circoviridae, that is commonly found in humans and which lacks pathological consequences for the infected host. TTV replication has been demonstrated in bone marrow cells but not in peripheral blood mononuclear cells (PBMC), suggesting that hematopoietic cells must be activated to support TTV replication. To test this hypothesis, PBMC from two naturally TTV-infected individuals and from two healthy TTV-DNA negative donors infected in vitro with a TTV-DNA-positive serum were cultured in the presence (stimulated) or absence (unstimulated) of phytohemagglutinin, lipopolysaccharide, and interleukin-2. TTV-DNA was detected in both stimulated and unstimulated PBMC. However, TTV-DNA replicative intermediates and mRNA were detected only in stimulated PBMC. Furthermore, TTV-DNA and mRNA were detected in PBMC from two TTV negative donors reinfected with supernatants from TTV-infected stimulated cells but not when using culture supernatants from unstimulated cells. These results demonstrate that TTV replicates in PBMC only when stimulated.

Occult hepatitis C virus infection in patients in whom the etiology of persistently abnormal results of liver-function tests is unknown.

BACKGROUND: There are patients in whom the etiology of long-standing abnormal results of liver-function tests is unknown (ALF-EU) after exclusion of all known causes of liver diseases. We analyzed the presence of hepatitis C virus (HCV) RNA in liver-biopsy specimens from 100 patients who were negative for anti-HCV antibodies and for serum HCV RNA and who had ALF-EU. METHODS: HCV RNA status was tested by reverse-transcription polymerase chain reaction (RT-PCR) and by in situ hybridization, in liver and peripheral-blood mononuclear cells (PBMCs). RESULTS: HCV RNA was detected in liver-biopsy specimens from 57 of 100 patients negative for anti-HCV antibodies and for serum HCV RNA (i.e., who had occult HCV infection). HCV RNA of negative polarity was found in the liver of 48 (84.2%) of these 57 patients with occult HCV infection. Nucleotide-sequence analysis confirmed the specificity of detection of HCV RNA and that patients were infected with the HCV 1b genotype. Of these 57 patients with intrahepatic HCV RNA, 40 (70%) had viral RNA in their PBMCs. With regard to liver histology, patients with occult HCV infection were more likely to have necroinflammatory activity (P=.017) and fibrosis (P=.022) than were patients without intrahepatic HCV RNA. CONCLUSIONS: Patients with ALF-EU may have intrahepatic HCV RNA in the absence of anti-HCV antibodies and of serum HCV RNA.