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Hepatitis D virus (HDV) super-infection of Hepatitis B virus (HBV)-infected patients is the most aggressive form of viral hepatitis. HDV infection is not susceptible to direct anti-HBV drugs, and only suboptimal antiviral responses are obtained with interferon (IFN)-alpha-based therapy. To get insights on HDV replication and interplay with HBV in physiologically relevant hepatocytes, differentiated HepaRG (dHepaRG) cells, previously infected or not with HBV, were infected with HDV, and viral markers were extensively analyzed. Innate and IFN responses to HDV were monitored by measuring pro-inflammatory and interferon-stimulated gene (ISG) expression. Both mono- and super-infected dHepaRG cells supported a strong HDV intracellular replication, which was accompanied by a strong secretion of infectious HDV virions only in the super-infection setting and despite the low number of co-infected cells. Upon HDV super-infection, HBV replication markers including HBeAg, total HBV-DNA and pregenomic RNA were significantly decreased, confirming the interference of HDV on HBV. Yet, no decrease of circular covalently closed HBV DNA (cccDNA) and HBsAg levels was evidenced. At the peak of HDV-RNA accumulation and onset of interference on HBV replication, a strong type-I IFN response was observed, with interferon stimulated genes, RSAD2 (Viperin) and IFI78 (MxA) being highly induced. We established a cellular model to characterize in more detail the direct interference of HBV and HDV, and the indirect interplay between the two viruses via innate immune responses. This model will be instrumental to assess molecular and immunological mechanisms of this viral interference.

Varying length cDNAs encoding the N-terminal nucleotide-binding domain (NBD1) from mouse mdr1 P-glycoprotein were prepared on the basis of structure predictions. Corresponding recombinant proteins were overexpressed in Escherichia coli, and the shortest one containing amino acids 395-581 exhibited the highest solubility. Insertion of an N-terminal hexahistidine tag allowed domain purification by nickel-chelate affinity chromatography.NBD1 efficiently interacted with nucleotides. Fluorescence methods showed that ATP bound at millimolar concentrations and its 2, Multidrug resistance of tumor cells is often associated with overexpression of P-glycoprotein, a membrane transporter that extrudes chemotherapeutic drugs using ATP hydrolysis as energy source (1, 2). The protein is encoded by the mdr gene family comprising two members in man, mdr1 and mdr2, or three in mouse, mdr1 (or mdr1b), mdr2, and mdr3 (or mdr1a). Only mdr1, and to a lower extent mdr3, was found to convey cellular multidrug resistance; mdr3 appears to be involved in detoxification/protection processes and mdr2 in phospholipid translocation (3). The function of mdr1 P-glycoprotein in normal tissues is still questioned although its relative abundance in mouse pregnant uterus and adrenal glands (4) favors a role in steroid hormone secretion (5).Structural analysis of the P-glycoprotein sequence, composed of 1276 amino acids in mouse (6), predicts two homologous halves, each containing up to six putative membrane-spanning ␣-helices and one cytoplasmically sided nucleotide-binding domain with characteristic Walker motifs A and B (7). P-glycoprotein structural organization is typical of the ATP-binding cassette (ABC) 1 superfamily including yeast (8) and protozoan parasite (9) drug transporters, and a series of different members from eukaryotic proteins, like the cystic fibrosis gene product CFTR, to bacterial transporters (10). The ATPase activity and related drug transport of P-glycoprotein require both functional nucleotide-binding sites (11,12) and are sensitive to the cysteine-specific modifier N-ethylmaleimide (NEM) (13)(14)(15)(16)(17). The lack of structural data about P-glycoprotein is due to its low abundance, difficult purification and membrane character, and to the lack of a highly overexpressing system (18). A recent approach to circumvent such problems was to overexpress in bacteria recombinant domains predicted to be soluble, in fusion with either the glutathione S-transferase or the maltose-binding protein to allow their purification by affinity chromatography: this was achieved with the C-terminal nucleotidebinding domain (NBD2) from 20), or with HlyB or CFTR domains (21,22). However, the presence of a relatively high-size fusion protein might be undesirable when studying protein/protein interactions, and its release by specific proteolytic cleavage was only partial or led to unstable nucleotide-binding domains. An alternative was to use a hexahistidine tag for fusion, in order to increase protein solubility and allow its purification by ...

Growth of Escherichia coli on acetate requires operation of the anaplerotic sequence known as the glyoxylate bypass. In this pathway three different enzymes are activated: malate synthase, isocitrate lyase and isocitrate dehydrogenase kinase/phosphatase which are encoded by genes aceB, aceA and aceK, respectively. These three genes are clustered, in that order, in the same acetate (ace) operon whose expression is under the transcriptional control of the iclR gene located downstream from aceK. We have cloned the iclR gene in the pKK233–2 vector which allows optimization of both transcription and translation initiation. The IclR repressor has been overproduced, then purified to homogeneity in a one‐step procedure by cation exchange chromatography after ammonium sulfate fractionation. Its specific interaction with the operator/promoter region of the ace operon has been analyzed by gel retardation and DNase I footprinting experiments. The IclR repressor has been shown to recognize a 35 bp palindromic sequence which largely overlaps the ‐35 recognition site of RNA polymerase. Moreover, the formation of the complex between IclR and the operator/promoter region has been found to be impaired by phosphoenol pyruvate but insensitive to acetate, acetyl‐CoA, pyruvate, and oxaloacetate. These results are discussed in terms of primary regulation of the expression of the ace operon.

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Using a C-terminal domain (PCT) of the measles virus (MV) phosphoprotein (P protein) as bait in a yeast two-hybrid screen, a cDNA identical to the recently described human p53-induced-RING-H2 (hPIRH2) cDNA was isolated. A glutathione S-transferase-hPIRH2 fusion protein expressed in bacteria was able to pull down P protein when mixed with an extract from P-expressing HeLa cells in vitro, and myc-tagged hPIRH2 could be reciprocally coimmunoprecipitated with MV P protein from human cells. Additionally, immunoprecipitation experiments demonstrated that hPIRH2-myc, MV P, and nucleocapsid (N) proteins form a ternary complex. The hPIRH2 binding site was mapped to the C-terminal X domain region of the P protein by using a yeast two-hybrid assay. The PCT binding site was mapped on hPIRH2 by using a novel yeast two-hybrid tagged PCR approach and by coimmunoprecipitation of hPIRH2 cysteine mutants and mouse/human PIRH2 chimeras. The hPIRH2 C terminus could mediate the interaction with MV P which was favored by the RING-H2 motif. When coexpressed with an enhanced green fluorescent protein-tagged hPIRH2 protein, MV P alone or in a complex with MV N was able to redistribute hPIRH2 to outside the nucleus, within intracellular aggregates. Finally, MV P efficiently stabilized hPIRH2-myc expression and prevented its ubiquitination in vivo but had no effect on the stability or ubiquitination of an alternative ubiquitin E3 ligase, Mdm2. Thus, MV P protein is the first protein from a pathogen that is able to specifically interact with and stabilize the ubiquitin E3 ligase hPIRH2 by preventing its ubiquitination.Measles virus (MV) is responsible for an acute childhood disease that, in 2002, infected almost 40 million infants and caused 0.8 million deaths (42). It is an enveloped virus belonging to the Mononegavirales order, the Paramyxoviridae family, and the Morbillivirus genus. The genome is a nonsegmented, negative-strand RNA. It comprises six genes, the genes for nucleoprotein (N) and phosphoprotein (P protein) and matrix (M), fusion (F), hemagglutinin (H), and large (L; polymerase) proteins, in that order, flanked by two short sequences, the leader (Le) and the trailer (Tr), which contain the genome and antigenome promoters, respectively. The MV RNA-dependent RNA polymerase comprising the L and P proteins uses a ribonucleoprotein template composed of the genomic RNA in complex with the N protein. The P protein from Paramyxoviridae acts as a bridge between L and ribonucleoprotein and has partially overlapping binding sites for both L (40) and N proteins. P has multiple functions: (i) it binds to the N-terminal domain of L (7), (ii) it acts as a molecular chaperone and stabilizes the L protein (20), (iii) it places the polymerase complex on the nucleocapsid, (iv) it allows viral transcription and replication, and (v) it prevents the illegitimate encapsidation of cellular RNA by keeping N in a monomeric N°state (22

The phosphorylated proteins of Escherichia coli, radioactively labeled with [32P]orthophosphate, have been analyzed by the O'Farrell gel technique and autoradiography. The effects of various culture conditions on the pattern of protein phosphorylation have been studied, including growth on different carbon sources in either exponential or stationary phase, treatment of cells with ethanol, heat shock and amino acid starvation. A total number of 128 different phosphoproteins, labeled to a varying extent, have been detected and each of them has been characterized by both its molecular mass and isoelectric point. These proteins are located mainly in the cytosoic fraction of cells, none of them being present within either ribosomes or nucleoids, and only three being associated with membranes. Analysis of their phosphoamino acid content has shown that they are phosphorylated mostly at serine residues and, less frequently, at threonine and tyrosine residues.