Mutagenesis of a conserved region of the gene encoding the FLP recombinase of Saccharomyces cerevisiae. A role for arginine 191 in binding and ligation.

The FLP recombinase from the 2 microns plasmid of Saccharomyces cerevisiae contains a region from amino acid 185 to 203 that is conserved among several FLP-like proteins from different yeasts. Using site-directed mutagenesis, we have made mutations in this region of the FLP gene. Five of twelve mutations in the region yielded proteins that were unable to bind to the FLP recombination target (FRT) site. A change of arginine at position 191 to lysine resulted in a protein (FLP-R191K) that could bind to the FRT site but could not catalyze recombination. This mutant protein accumulated as a stable protein-DNA complex in which one of the two bound FLP proteins was covalently attached to the DNA. FLP-R191K was defective in strand exchange and ligation and was unable to promote protein-protein interaction with half-FRT sites. The conservation of three residues in all members of the integrase family of site-specific recombinases (His305, Arg308, Tyr343 in FLP) implies a common mechanism of recombination. The conservation of arginine 191 and the properties of the FLP-R191K mutant protein suggest that this arginine also plays an important role in the mechanism of FLP-mediated site-specific recombination.     

The DEAD-box RNA helicase DDX6 is required for efficient encapsidation of a retroviral genome

Viruses have to encapsidate their own genomes during the assembly process. For most RNA viruses, there are sequences within the viral RNA and virion proteins needed for high efficiency of genome encapsidation. However, the roles of host proteins in this process are not understood. Here we find that the cellular DEAD-box RNA helicase DDX6 is required for efficient genome packaging of foamy virus, a spumaretrovirus. After infection, a significant amount of DDX6, normally concentrated in P bodies and stress granules, re-localizes to the pericentriolar site where viral RNAs and Gag capsid proteins are concentrated and capsids are assembled. Knockdown of DDX6 by siRNA leads to a decreased level of viral nucleic acids in extracellular particles, although viral protein expression, capsid assembly and release, and accumulation of viral RNA and Gag protein at the assembly site are little affected. DDX6 does not interact stably with Gag proteins nor is it incorporated into particles. However, we find that the ATPase/helicase motif of DDX6 is essential for viral replication. This suggests that the ATP hydrolysis and/or the RNA unwinding activities of DDX6 function in moderating the viral RNA conformation and/or viral RNA-Gag ribonucleoprotein complex in a transient manner to facilitate incorporation of the viral RNA into particles. These results reveal a unique role for a highly conserved cellular protein of RNA metabolism in specifically re-locating to the site of viral assembly for its function as a catalyst in retroviral RNA packaging.     

Combining genetic and biochemical approaches to identify functional molecular contact points

Protein-protein interactions are required for many viral and cellular functions and are potential targets for novel therapies. Here we detail a series of genetic and biochemical techniques used in combination to find an essential molecular contact point on the duck hepatitis B virus polymerase. These techniques include differential immunoprecipitation, mutagenesis and peptide competition. The strength of these techniques is their ability to identify contact points on intact proteins or protein complexes employing functional assays. This approach can be used to aid identification of putative binding sites on proteins and protein complexes which are resistant to characterization by other methods.     

DNA recognition by the FLP recombinase of the yeast 2 mu plasmid. A mutational analysis of the FLP binding site

The 2 mu plasmid of the yeast Saccharomyces cerevisiae encodes a site-specific recombination system consisting of the FLP protein and two inverted recombination sites on the plasmid. The minimal fully functional substrate for in-vitro recombination in this system consists of two FLP protein binding sites separated by an eight base-pair spacer sequence. We have used site-directed mutagenesis to generate every possible mutation (36 in all) within 11 base-pairs of one FLP protein binding site and the base-pair immediately flanking it. The base-pairs within the binding site can be separated into three classes on the basis of these results. Thirty of the 36 sequence changes, including all three at seven different positions (class I) produce a negligible or modest effect on FLP protein-promoted recombination. In particular, most transition mutations are well-tolerated in this system. In only one case do all three possible mutations produce large effects (class II). At three positions, clustered near the site at which DNA is cleaved by FLP protein, one of the two possible transversions produces a large effect on recombination, while the other two changes produce modest effects (class III). For seven mutants for which FLP protein binding was measured, a direct correlation between decreases in recombination activity and in binding was observed. Positive effects on the reaction potential of mutant sites are observed when the other FLP binding site in a single recombination site is unaltered or when the second recombination site in a reaction is wild-type. This suggests a functional interaction between FLP binding sites both in cis and in trans. When two mutant recombination sites (each with 1 altered FLP binding site) are recombined, the relative orientation of the mutations (parallel or antiparallel) has no effect on the result. These results provide an extensive substrate catalog to complement future studies in this system.     

Intrinsic disorder mediates hepatitis C virus core–host cell protein interactions

Viral proteins bind to numerous cellular and viral proteins throughout the infection cycle. However, the mechanisms by which viral proteins interact with such large numbers of factors remain unknown. Cellular proteins that interact with multiple, distinct partners often do so through short sequences known as molecular recognition features (MoRFs) embedded within intrinsically disordered regions (IDRs). In this study, we report the first evidence that MoRFs in viral proteins play a similar role in targeting the host cell. Using a combination of evolutionary modeling, protein–protein interaction analyses and forward genetic screening, we systematically investigated two computationally predicted MoRFs within the N‐terminal IDR of the hepatitis C virus (HCV) Core protein. Sequence analysis of the MoRFs showed their conservation across all HCV genotypes and the canine and equine Hepaciviruses. Phylogenetic modeling indicated that the Core MoRFs are under stronger purifying selection than the surrounding sequence, suggesting that these modules have a biological function. Using the yeast two‐hybrid assay, we identified three cellular binding partners for each HCV Core MoRF, including two previously characterized cellular targets of HCV Core (DDX3X and NPM1). Random and site‐directed mutagenesis demonstrated that the predicted MoRF regions were required for binding to the cellular proteins, but that different residues within each MoRF were critical for binding to different partners. This study demonstrated that viruses may use intrinsic disorder to target multiple cellular proteins with the same amino acid sequence and provides a framework for characterizing the binding partners of other disordered regions in viral and cellular proteomes.     

Development of cell lines that provide tightly controlled temporal translation of the human cytomegalovirus IE2 proteins for complementation and functional analyses of growth-impaired and nonviable IE2 mutant viruses

The human cytomegalovirus (HCMV) IE2 86 protein is essential for viral replication. Two other proteins, IE2 60 and IE2 40, which arise from the C-terminal half of IE2 86, are important for later stages of the infection. Functional analyses of IE2 86 in the context of the infection have utilized bacterial artificial chromosomes as vectors to generate mutant viruses. One limitation is that many mutations result in debilitated or nonviable viruses. Here, we describe a novel system that allows tightly controlled temporal expression of the IE2 proteins and provides complementation of both growth-impaired and nonviable IE2 mutant viruses. The strategy involves creation of cell lines with separate lentiviruses expressing a bicistronic RNA with a selectable marker as the first open reading frame (ORF) and IE2 86, IE2 60, or IE2 40 as the second ORF. Induction of expression of the IE2 proteins occurs only following DNA recombination events mediated by Cre and FLP recombinases that delete the first ORF. HCMV encodes Cre and FLP, which are expressed at immediate-early (for IE2 86) and early-late (for IE2 40 and IE2 60) times, respectively. We show that the presence of full-length IE2 86 alone provides some complementation for virus production, but the correct temporal expression of IE2 86 and IE2 40 together has the most beneficial effect for early-late gene expression and synthesis of infectious virus. This approach for inducible protein translation can be used for complementation of other mutations as well as controlled expression of toxic cellular and microbial proteins.     

Mutations That Improve the Binding of Yeast Flp Recombinase to Its Substrate

When yeast FLP recombinase is expressed from the phage lambda PR promoter in a Salmonella host, it cannot efficiently repress an operon controlled by an operator/promoter region that includes a synthetic, target FLP site. On the basis of this phenotype, we have identified four mutant FLP proteins that function as more efficient repressors of such an operon. At least two of these mutant FLP proteins bind better to the FLP site in vivo and in vitro. One mutant changes the presumed active site tyrosine residue of FLP protein to phenylalanine, is blocked in recombination, and binds the FLP site about five-fold better than the wild-type protein. A second mutant protein that functions as a more efficient repressor retains catalytic activity. We conclude that the eukaryotic yeast FLP recombinase, when expressed in a heterologous prokaryotic host, can function as a repressor, and that mutant FLP proteins that bind DNA more tightly may be selected as more efficient repressors.     

Surface plasmon resonance analysis of the binding of high‐risk mucosal HPV E6 oncoproteins to the PDZ1 domain of the tight junction protein MAGI‐1

The E6 oncoproteins from high‐risk mucosal human papillomavirus (HPV) induce cervical cancer via two major activities, the binding and the degradation of the p53 protein and PDZ domain‐containing proteins. Human MAGI‐1 is a multi‐PDZ domain protein implicated into protein complex assembly at cell–cell contacts. High‐risk mucosal HPV E6 proteins interact with the PDZ1 domain of MAGI‐1 via a C‐terminal consensus binding motif. Here, we developed a medium throughput protocol to accurately measure by surface plasmon resonance affinity constants of protein domains binding to peptidic sequences produced as recombinant fusions to the glutathione‐S‐transferase (GST). This approach was applied to measure the binding of MAGI‐1 PDZ1 to the C‐termini of viral or cellular proteins. Both high‐risk mucosal HPV E6 C‐terminal peptides and cellular partners of MAGI‐1 PDZ1 bind to MAGI‐1 PDZ1 with comparable dissociation constants in the micromolar range. MAGI‐1 PDZ1 shows a preference for C‐termini with a valine at position 0 and a negative charge at position ?3, confirming previous studies performed with HPV18 E6. A detailed combined analysis via site‐directed mutagenesis of the HPV16 C‐terminal peptide and PDZ1 indicated that interactions mediated by charged residues upstream the PDZ‐binding motif strongly contribute to binding selectivity of this interaction. In addition, our work highlighted the K₄₉₉ residue of MAGI‐1 as a novel determinant of binding specificity. Finally, we showed that MAGI‐1 PDZ1 also binds to the C‐termini of LPP and Tax proteins, which were already known to bind to PDZ proteins but not to MAGI‐1. Copyright © 2010 John Wiley & Sons, Ltd.     

Dengue Virus Non-structural Protein 1 Modulates Infectious Particle Production via Interaction with the Structural Proteins

Non-structural protein 1 (NS1) is one of the most enigmatic proteins of the Dengue virus (DENV), playing distinct functions in immune evasion, pathogenesis and viral replication. The recently reported crystal structure of DENV NS1 revealed its peculiar three-dimensional fold; however, detailed information on NS1 function at different steps of the viral replication cycle is still missing. By using the recently reported crystal structure, as well as amino acid sequence conservation, as a guide for a comprehensive site-directed mutagenesis study, we discovered that in addition to being essential for RNA replication, DENV NS1 is also critically required for the production of infectious virus particles. Taking advantage of a trans-complementation approach based on fully functional epitope-tagged NS1 variants, we identified previously unreported interactions between NS1 and the structural proteins Envelope (E) and precursor Membrane (prM). Interestingly, coimmunoprecipitation revealed an additional association with capsid, arguing that NS1 interacts via the structural glycoproteins with DENV particles. Results obtained with mutations residing either in the NS1 Wing domain or in the β-ladder domain suggest that NS1 might have two distinct functions in the assembly of DENV particles. By using a trans-complementation approach with a C-terminally KDEL-tagged ER-resident NS1, we demonstrate that the secretion of NS1 is dispensable for both RNA replication and infectious particle production. In conclusion, our results provide an extensive genetic map of NS1 determinants essential for viral RNA replication and identify a novel role of NS1 in virion production that is mediated via interaction with the structural proteins. These studies extend the list of NS1 functions and argue for a central role in coordinating replication and assembly/release of infectious DENV particles.     

Genetic screens for the control of influenza virus replication: from meta-analysis to drug discovery

Current anti-influenza virus drugs target two viral proteins and induce a selective pressure for the generation of drug resistant variants. This stresses the need for additional therapeutic strategies including drug targeting of cellular factors that are essential for viral replication. Reverse genetics approaches can be used to identify these factors and recently six independent genomic initiatives have led to the identification of 925 host factors that are essential for the replication of influenza viruses. Here we report a meta-analysis of this dataset, first revealing that these screens are poorly overlapping at the gene level. However, a strong convergence was observed at the level of biological processes which was further supported by an interactomic analysis showing a high interconnectivity of the essential host factors in the human protein network. Plugging virus-host protein interaction data on this dataset reveals a significant targeting of these factors by viral proteins, further validating the cellular targets. Combining this information, the first drug-influenza virus target network was constructed by retrieving from DrugBank 298 molecules interacting with 100 essential host factors. Of these, 204 are FDA-approved offering interesting potential for rapid drug repositioning in the treatment of flu.     

Determination of DNA sequences essential for FLP-mediated recombination by a novel method

The yeast 2-micron circle plasmid encodes a protein, FLP, that mediates site-specific recombination across the two FLP-binding sites of the plasmid. We have used a novel technique, "exonuclease-treated substrate analysis," to determine the minimal duplex DNA sequence needed for this recombination event. A linear DNA containing two FLP sites in a direct orientation was treated with the double-strand specific 3'-exonuclease, exonuclease III, to generate molecules with a nested set of single-strand deletions that extended into one of the FLP sites. The DNA was then end-labeled at the sites of the deletions and used as a substrate for recombination in vitro. FLP-mediated recombination between two FLP sites excised a restriction endonuclease cleavage site from the DNA. Comparison of the fragments produced by restriction enzyme digestion of untreated and FLP-treated DNA showed to the nucleotide the duplex DNA sequence required for FLP-mediated recombination. To examine essential sequences in the opposite DNA strand, similar experiments were done using the 5'-exonuclease encoded by phage T7. The minimal essential duplex DNA sequence lies within the region of the FLP site that was previously shown to be protected from nuclease digestion in the presence of FLP. A modified form of this technique can be used to study the minimal sequence requirements of site-specific DNA binding proteins.     

A core of three amino acids at the carboxyl‐terminal region of glutamine synthetase defines its regulation in cyanobacteria

Glutamine synthetase (GS) type I is a key enzyme in nitrogen metabolism, and its activity is finely controlled by cellular carbon/nitrogen balance. In cyanobacteria, a reversible process that involves protein–protein interaction with two proteins, the inactivating factors IF7 and IF17, regulates GS. Previously, we showed that three arginine residues of IFs are critical for binding and inhibition of GS. In this work, taking advantage of the specificity of GS/IFs interaction in the model cyanobacteria Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120, we have constructed a different chimeric GSs from these two cyanobacteria. Analysis of these proteins, together with a site‐directed mutagenesis approach, indicates that a core of three residues (E419, N456 and R459) is essential for the inactivation process. The three residues belong to the last 56 amino acids of the C‐terminus of Synechocystis GS. A protein–protein docking modeling of Synechocystis GS in complex with IF7 supports the role of the identified core for GS/IF interaction.     

Proteolytic Processing of Human Cytomegalovirus Glycoprotein B Is Dispensable for Viral Growth in Culture

Glycoprotein B (gB) of human cytomegalovirus (HCMV), which is considered essential for the viral life cycle, is proteolytically processed during maturation. Since gB homologues of several other herpesviruses remain uncleaved, the relevance of this property of HCMV gB for viral infectivity is unclear. Here we report on the construction of a viral mutant in which the recognition site of gB for the cellular endoprotease furin was destroyed. Because mutagenesis of essential proteins may result in a lethal phenotype, a replication-deficient HCMV gB-null genome encoding enhanced green fluorescent protein was constructed, and complementation by mutant gBs was initially evaluated in transient-cotransfection assays. Cotransfection of plasmids expressing authentic gB or gB with a mutated cleavage site (gB-DeltaFur) led to the formation of green fluorescent miniplaques which were considered to result from one cycle of phenotypic complementation of the gB-null genome. To verify these results, two recombinant HCMV genomes were constructed: HCMV-BAC-DeltaMhdI, with a deletion of hydrophobic domain 1 of gB that appeared to be essential for viral growth in the cotransfection experiments, and HCMV-BACDeltaFur, in which the gB cleavage site was mutated by amino acid substitution. Consistent with the results of the cotransfection assays, only the DeltaFur mutant replicated in human fibroblasts, showing growth kinetics comparable to that of wild-type virus. gB in mutant-infected cells was uncleaved, whereas glycosylation and transport to the cell surface were not impaired. Extracellular mutant virus contained exclusively uncleaved gB, indicating that proteolytic processing of gB is dispensable for viral replication in cell culture.