Frédéric Picard-Jean

Characterization of the DNA- and dNTP-binding activities of the human cytomegalovirus DNA polymerase catalytic subunit UL54

The catalytic subunit of the human cytomegalovirus DNA polymerase is critical for the replication of the virus. In the present study, we report the expression and purification of a recombinant catalytic subunit of the human cytomegalovirus DNA polymerase expressed in bacteria which retains polymerase activity. As a first step towards elucidating the nature of the interaction between the enzyme, DNA and dNTPs, we have utilized endogenous tryptophan fluorescence to evaluate the binding of ligands to the enzyme. Using this technique, we demonstrate that the minimal DNA-binding site of the enzyme is 6 nt. We also report the first detailed study of the binding kinetics and thermodynamic parameters involved in the interaction between the enzyme, DNA and dNTPs. Our thermodynamic analyses indicate that the initial formation of the enzyme-DNA binary complex is driven by a favourable entropy change, but is also clearly associated with an unfavourable enthalpic contribution. In contrast, the interaction of dNTPs to the binary complex was shown to depend on a completely different mode of binding that is dominated by a favourable enthalpy change and associated with an unfavourable entropy change. In order to provide additional insights into the structural modifications that occur during catalysis, we correlated the effect of DNA and dNTP binding on protein structure using CD. Our results indicate that the enzyme undergoes a first conformational change upon the formation of the protein-DNA binary complex, which is followed by a second structural modification upon dNTP binding. The present study provides a better understanding of the molecular basis of DNA and dNTP recognition by the catalytic subunit of the human cytomegalovirus DNA polymerase.

The RNA capping machinery as an anti‐infective target

A number of different human pathogens code for their own enzymes involved in the synthesis of the RNA cap structure. Although the RNA cap structures originating from human and microbial enzymes are often identical, the subunit composition, structure and catalytic mechanisms of the microbial‐encoded enzymes involved in the synthesis of the RNA cap structure are often significantly different from those of host cells. As a consequence, these pathogenic cap‐forming enzymes are potential targets for antimicrobial drugs. During the past few years, experimental studies have started to demonstrate that inhibition of the RNA capping activity is a reasonable approach for the development of antimicrobial agents. The combination of structural, biochemical, and molecular modeling studies are starting to reveal novel molecules that can serve as starting blocks for the design of more potent and specific antimicrobial agents. Here, we examine various strategies that have been developed to inhibit microbial enzymes involved in the synthesis of the RNA cap structure, emphasizing the challenges remaining to design potent and selective drugs. WIREs RNA 2011 2 184–192 DOI: 10.1002/wrna.43

Energetics of RNA binding by the West Nile virus RNA triphosphatase

The West Nile virus (WNV) RNA genome harbors the characteristic methylated cap structure present at the 5′ end of eukaryotic mRNAs. In the present study, we report a detailed study of the binding energetics and thermodynamic parameters involved in the interaction between RNA and the WNV RNA triphosphatase, an enzyme involved in the synthesis of the RNA cap structure. Fluorescence spectroscopy assays revealed that the initial interaction between RNA and the enzyme is characterized by a high enthalpy of association and that the minimal RNA binding site of NS3 is 13 nucleotides. In order to provide insight into the relationship between the enzyme structure and RNA binding, we also correlated the effect of RNA binding on protein structure using both circular dichroism and denaturation studies as structural indicators. Our data indicate that the protein undergoes structural modifications upon RNA binding, although the interaction does not significantly modify the stability of the protein.

Transcriptional pausing at the translation start site operates as a critical checkpoint for riboswitch regulation

On the basis of nascent transcript sequencing, it has been postulated but never demonstrated that transcriptional pausing at translation start sites is important for gene regulation. Here we show that the Escherichia coli thiamin pyrophosphate (TPP) thiC riboswitch contains a regulatory pause site in the translation initiation region that acts as a checkpoint for thiC expression. By biochemically probing nascent transcription complexes halted at defined positions, we find a narrow transcriptional window for metabolite binding, in which the downstream boundary is delimited by the checkpoint. We show that transcription complexes at the regulatory pause site favour the formation of a riboswitch intramolecular lock that strongly prevents TPP binding. In contrast, cotranscriptional metabolite binding increases RNA polymerase pausing and induces Rho-dependent transcription termination at the checkpoint. Early transcriptional pausing may provide a general mechanism, whereby transient transcriptional windows directly coordinate the sensing of environmental cues and bacterial mRNA regulation.

The Immunosuppressive Agent Mizoribine Monophosphate Is an Inhibitor of the Human RNA Capping Enzyme

Mizoribine monophosphate (MZP) is a specific inhibitor of the cellular inosine-5′-monophosphate dehydrogenase (IMPDH), the enzyme catalyzing the rate-limiting step of de novo guanine nucleotide biosynthesis. MZP is a highly potent antagonistic inhibitor of IMPDH that blocks the proliferation of T and B lymphocytes that use the de novo pathway of guanine nucleotide synthesis almost exclusively. In the present study, we investigated the ability of MZP to directly inhibit the human RNA capping enzyme (HCE), a protein harboring both RNA 5′-triphosphatase and RNA guanylyltransferase activities. HCE is involved in the synthesis of the cap structure found at the 5′ end of eukaryotic mRNAs, which is critical for the splicing of the cap-proximal intron, the transport of mRNAs from the nucleus to the cytoplasm, and for both the stability and translation of mRNAs. Our biochemical studies provide the first insight that MZP can inhibit the formation of the RNA cap structure catalyzed by HCE. In the presence of MZP, the RNA 5′-triphosphatase activity appears to be relatively unaffected while the RNA guanylyltransferase activity is inhibited, indicating that the RNA guanylyltransferase activity is the main target of MZP inhibition. Kinetic studies reveal that MZP is a non-competitive inhibitor that likely targets an allosteric site on HCE. Mizoribine also impairs mRNA capping in living cells, which could account for the global mechanism of action of this therapeutic agent. Together, our study clearly demonstrates that mizoribine monophosphate inhibits the human RNA guanylyltransferase in vitro and impair mRNA capping in cellulo.

The intracellular inhibition of HCV replication represents a novel mechanism of action by the innate immune Lactoferrin protein.

Hepatitis C virus (HCV) is a major public-health problem with 130-170 million individuals chronically infected worldwide. In order to halt the epidemic, therapy against HCV will need to be both effective and widely available. Studies focusing on safe and affordable natural product active against HCV have revealed the antiviral activity of the human Lactoferrin (hLF) protein which binds and neutralizes the circulating virion. In the current study, investigation of hLF activity on the HCV subgenomic replicon system, which is independent from viral entry and shedding, revealed a distinct antireplicative activity of hLF against HCV. Hepatocellular uptake of hLF was confirmed and correlated with qualitative HCV staining reduction. Quantitative dose-response inhibition assays confirmed an hLF-mediated and dose-dependent HCV replication reduction reaching up to 60%. The in cellulo anti-HCV activity of hLF was additive to both Ribavirin and Interferon-α-2b. Further investigation of hLF activity against the essential viral proteins involved in HCV genome replication revealed an inhibitory activity against the HCV ATPase/Helicase NS3 protein but not against the HCV RNA-dependent RNA polymerase (NS5B protein). NS3 inhibition was mediated by a direct and specific interaction between hLF and an allosteric binding site on NS3. Taken together, our findings reveal a new antiviral mechanism of action by which hLF inhibits intracellular HCV replication.

Correction: 2'-O-methylation of the mRNA cap protects RNAs from decapping and degradation by DXO

[This corrects the article DOI: 10.1371/journal.pone.0193804.].

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or localization of a target molecule in cells and tissues. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell. Both direct and indirect immunofluorescence approaches can be used which rely on the use of antibodies linked with a fluorochrome. Direct immunofluorescence is less frequently used because it provides lower signal, involves higher cost and less flexibility. In contrast, indirect immunofluorescence is more commonly used because of its high sensitivity and provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In this manuscript, both epifluorescence microscopy and confocal microscopy were used to monitor the internalization of human lactoferrin, an important component of the immune system, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus using immunofluorescence. Both the advantages and disadvantages associated with these approaches are discussed.