The Flexible Motif V of Epstein-Barr Virus Deoxyuridine 5��-Triphosphate Pyrophosphatase Is Essential for Catalysis

Deoxyuridine 5′-triphosphate pyrophosphatases (dUTPases) are ubiquitous enzymes essential for hydrolysis of dUTP, thus preventing its incorporation into DNA. Although Epstein-Barr virus (EBV) dUTPase is monomeric, it has a high degree of similarity with the more frequent trimeric form of the enzyme. In both cases, the active site is composed of five conserved sequence motifs. Structural and functional studies of mutants based on the structure of EBV dUTPase gave new insight into the mechanism of the enzyme. A first mutant allowed us to exclude a role in enzymatic activity for the disulfide bridge involving the beginning of the disordered C terminus. Sequence alignments revealed two groups of dUTPases, based on the position in sequence of a conserved aspartic acid residue close to the active site. Single mutants of this residue in EBV dUTPase showed a highly impaired catalytic activity, which could be partially restored by a second mutation, making EBV dUTPase more similar to the second group of enzymes. Deletion of the flexible C-terminal tail carrying motif V resulted in a protein completely devoid of enzymatic activity, crystallizing with unhydrolyzed Mg²⁺-dUTP complex in the active site. Point mutations inside motif V highlighted the essential role of lid residue Phe²⁷³. Magnesium appears to play a role mainly in substrate binding, since in absence of Mg²⁺, the K_m of the enzyme is reduced, whereas the k_cat is less affected.Epstein-Barr virus, a human γ-herpesvirus, is the causative agent of infectious mononucleosis and establishes a lifelong persistent infection in over 90% of the world''s population. EBV³ is implicated in a number of cancers, such as Burkitt''s lymphoma, undifferentiated nasopharyngeal carcinoma, or Hodgkin disease. The large DNA genome of this virus codes for about 86 proteins implicated in a large number of functions related to viral latency or the lytic cycle, during which the virus replicates.One protein of the lytic cycle is deoxyuridine 5′-triphosphate pyrophosphatase, a ubiquitous enzyme catalyzing the cleavage of dUTP into dUMP and pyrophosphate (PP_i). This enzyme not only provides the precursor for the formation of dTMP by thymidylate synthase, but it also has a crucial role in maintaining a low dUTP/dTTP ratio in the cell in order to limit the incorporation of deoxyuridylate into DNA by DNA polymerases. Based on their oligomerization state, dUTPases can be divided into three families.The first family of dUTPases contains homodimeric enzymes with the prototype structure of Trypanosoma cruzi dUTPase (1).dUTPases of the second and largest family form homotrimers. Their structure is unrelated to dimeric dUTPases. Trimeric dUTPases are found in most eukaryotes, in prokaryotes, in some DNA viruses, such as poxvirus, and in a number of retroviruses. Several x-ray structures of dUTPases of this family are available: Escherichia coli (2), Homo sapiens (3), equine infectious anemia virus (4), feline immunodeficiency virus (5), Mason-Pfizer monkey virus (6), Mycobacterium tuberculosis (7), Plasmodium falciparum (8), Arabidopsis thaliana (9), and vaccinia virus (10). The active site is formed by five conserved motifs that are distributed over the entire sequence (11) (Fig. 1A). Each subunit of the trimer contributes to the formation of each of the three active sites (2). Whereas the first four motifs are well ordered, motif V is most often disordered and is only observed in some structures after inhibitor binding such as in the feline immunodeficiency virus dUTPase structure (Protein Data Bank entry 1f7r) (12) and the human dUTPase structure (Protein Data Bank entry 1q5h) (3), both in complex with dUDP. Recently, several structures showing motif V and α,β-imido-dUTP in a conformation close to the situation during catalysis became available: one of the human enzyme (Protein Data Bank entry 3ehw) (13) and one from M. tuberculosis (Protein Data Bank entry 2py4) (14). Together with kinetic information about the human enzyme (15), a model of enzymatic action became available: (i) rapid, probably diffusion-limited binding of the substrate; (ii) a substrate-induced structural change required for the formation of the catalytically competent conformation; (iii) the rate-limiting hydrolysis step; and (iv) a rapid release of the reaction products PP_i and dUMP. Hydrolysis occurs through a nucleophilic in-line attack on the α-phosphate by a water molecule activated by an aspartic acid residue (16).Open in a separate windowFIGURE 1.EBV dUTPase sequence and structure. A, sequence alignment of different dUTPases with known structures and the one from C. glutamaticum. Residue numbers are given above the sequence for the human enzyme and below the sequences for the mycobacterial and EBV enzymes. The linker region is highlighted with cyan letters. The five conserved motifs are highlighted. Key residues of motif V are printed in red. A pink background highlights residues of motif V where side chain hydroxyl and main chain amide contact γ-phosphate in the crystal structure (13, 14). Green background, main chain amide interaction with β- and γ-phosphate; residues that have been deleted in the ΔV mutant are underlined. The conserved residues interacting with the motif V arginine of human-like dUTPases are printed in blue; those interacting with the motif V arginine of mycobacterium-like dUTPases are shown in magenta. B, structure of EBV dUTPase in complex with α,β-imido-dUTP and Mg²⁺ (Protein Data Bank entry 2bt1). The catalytic residue Asp⁷⁶ is shown together with Asp¹³¹ next to it. The four visible sequence motifs of the active site are colored according to Fig. 1A, and the loop connecting the two domains (residues 114–125) is colored in cyan. The bound α,β-imido-dUTP molecule is shown as sticks and colored according to atom types. C, the ΔV structure (pink) in complex with dUTP (sticks, atom colors) and Mg²⁺. The disordered part of the connecting loop is located between the two arrows. Superposed is the dUMP-bound WT dUTPase structure (Protein Data Bank entry 2bsy; gray, connecting loop in cyan, motif I in green, disordered part dotted).The third family contains monomeric dUTPases encoded by avian and mammalian herpesviruses, which include important human pathogens, such as Epstein-Barr virus and herpes simplex virus. They have limited sequence homology to the trimeric enzymes; the five motifs forming the active site are present but reshuffled and spread out over a single polypeptide that is twice as long as the sequence of the subunit in trimeric enzymes. Working on the enzyme from EBV, we reported the first crystal structures of a monomeric dUTPase determined in complex with the product dUMP or the non-hydrolyzable substrate analogue α,β-imido-dUTP (17) (Fig. 1B). Like most dUTPase structures, those of the EBV enzyme showed four of the five conserved sequence motifs (Fig. 1A), with motif V invisible due to its flexibility, and revealed an active site that is extremely similar to those of trimeric dUTPases. EBV dUTPase furthermore exhibited similar kinetic parameters (17, 18). This therefore implies that the conclusions of studies of the enzymatic mechanism for trimeric dUTPases are also valid for the EBV enzyme and vice versa.In the previously published EBV dUTPase structures (17), the N and C termini of the ordered structure were linked by a disulfide bridge between Cys⁴ and Cys²⁴⁶. We therefore wished to check the influence of this bridge on activity and its possible regulatory role by studying mutant C4S.EBV dUTPase structures (17) revealed that residues 114–133 linking the two domains of monomeric dUTPase and containing part of motif I show different structures in the α,β-imido-dUTP-bound form (Fig. 1B) and in the dUMP-bound form (Fig. 1C). This leads indirectly to a non-productive conformation of catalytic residue Asp⁷⁶ of motif III (Fig. 1B). Motif I is always well ordered in trimeric dUTPases. We hypothesized first that the clustering of negative charges of two aspartic acid residues (catalytic residues Asp⁷⁶ and Asp¹³¹ of the linker region) and the γ-phosphate of the inhibitor leads to destabilization of this part of the structure. When dUTPase structures showing motif V in a productive conformation became available (13, 14), they showed an interaction of the residue corresponding to Asp¹³¹ with the conserved arginine residue of motif V. On the other hand, Asp¹³¹ is only partially conserved in sequence alignments. These revealed two classes of dUTPases, the first containing EBV dUTPase with a conserved aspartic acid residue in a position corresponding to Asp¹³¹ and the second group containing human and E. coli enzymes with a conserved aspartic acid residue at the position corresponding to residue 78 in EBV (Fig. 1A). We decided to study three single mutants of Asp¹³¹ (D131S, D131N, and D131E) along with a double mutant (D131S/G78D), making the EBV enzyme more similar to the second group.Finally, we report the structure of a dUTPase mutant with motif V deleted (ΔV), constructed in order to facilitate binding studies and crystallographic studies in view of potential antiviral drug design. This mutant showed the presence of intact Mg²⁺-dUTP in its active site, posing the question of why this mutant is completely inactive although most of the catalytic machinery is in place. In order to understand the precise mechanism of action of motif V, we decided to study two conserved residues of motif V, Arg²⁶⁸ and Phe²⁷³, since it has been shown that they interact with the bound substrate analogue α,β-imido-dUTP (13, 14).Since binding of an Mg²⁺-substrate complex appeared possible without catalysis, we wanted to further characterize the role of Mg²⁺. Previous studies carried out on E. coli dUTPase (16, 19, 20) indicated an important role of Mg²⁺ for catalytic activity, by promoting enzyme-substrate complex stabilization (21). Various dUTPase structures in the presence of α,β-imido-dUTP show the metal ion coordinating all three phosphate groups.     

EBV lytic-phase protein BGLF5 contributes to TLR9 downregulation during productive infection

Viruses use a wide range of strategies to modulate the host immune response. The human gammaherpesvirus EBV, causative agent of infectious mononucleosis and several malignant tumors, encodes proteins that subvert immune responses, notably those mediated by T cells. Less is known about EBV interference with innate immunity, more specifically at the level of TLR-mediated pathogen recognition. The viral dsDNA sensor TLR9 is expressed on B cells, a natural target of EBV infection. Here, we show that EBV particles trigger innate immune signaling pathways through TLR9. Furthermore, using an in vitro system for productive EBV infection, it has now been possible to compare the expression of TLRs by EBV(-) and EBV(+) human B cells during the latent and lytic phases of infection. Several TLRs were found to be differentially expressed either in latently EBV-infected cells or after induction of the lytic cycle. In particular, TLR9 expression was profoundly decreased at both the RNA and protein levels during productive EBV infection. We identified the EBV lytic-phase protein BGLF5 as a protein that contributes to downregulating TLR9 levels through RNA degradation. Reducing the levels of a pattern-recognition receptor capable of sensing the presence of EBV provides a mechanism by which the virus could obstruct host innate antiviral responses.     

The intraflagellar transport dynein complex of trypanosomes is made of a heterodimer of dynein heavy chains and of light and intermediate chains of distinct functions

Cilia and flagella are assembled by intraflagellar transport (IFT) of protein complexes that bring tubulin and other precursors to the incorporation site at their distal tip. Anterograde transport is driven by kinesin, whereas retrograde transport is ensured by a specific dynein. In the protist Trypanosoma brucei, two distinct genes encode fairly different dynein heavy chains (DHCs; ∼40% identity) termed DHC2.1 and DHC2.2, which form a heterodimer and are both essential for retrograde IFT. The stability of each heavy chain relies on the presence of a dynein light intermediate chain (DLI1; also known as XBX-1/D1bLIC). The presence of both heavy chains and of DLI1 at the base of the flagellum depends on the intermediate dynein chain DIC5 (FAP133/WDR34). In the IFT140^RNAi mutant, an IFT-A protein essential for retrograde transport, the IFT dynein components are found at high concentration at the flagellar base but fail to penetrate the flagellar compartment. We propose a model by which the IFT dynein particle is assembled in the cytoplasm, reaches the base of the flagellum, and associates with the IFT machinery in a manner dependent on the IFT-A complex.     

Application of silica aerogel encapsulated lipases in the synthesis of biodiesel by transesterification reactions

Two types of commercial lipases preparations, one from Burkholderia cepacia, the other one from Candida antartica, were encapsulated in silica aerogels reinforced with silica quartz fibre felt and dried by the CO₂ supercritical technique. These immobilized biocatalysts were applied in biodiesel synthesis by transesterification of sunflower seed oil with methyl acetate. They were found to be efficient even with mixtures of both substrates without any solvent addition. The aerogel encapsulation technique made it possible to maintain the enzymes in a dispersion state similar to the dispersion prevailing in an aqueous solution, even for further use in organic hydrophobic media. In transesterification in excess iso-octane, the two lipases encapsulated in aerogels made from 40% MTMS, were found to have activities relatively close to each other and comparable with commercial Novozyme 435. On the other in transesterification with mixture of oil and methyl acetate without any solvent, the kinetics were severely limited by substrate diffusion inside the aerogels. This was particularly true with the C. antartica, so that the corresponding aerogel encapsulated enzyme was much less active than commercial Novozyme 435, although it improved after a few tests.     

Spatial mismatch in morphological,ecological and phylogenetic diversity,in historical and contemporary European freshwater fish faunas

Biodiversity encompasses multiple facets, among which taxonomic, functional and phylogenetic aspects are the most often considered. Understanding how those diversity facets are distributed and what are their determinants has become a central concern in the current context of biodiversity crisis, but such multi‐faceted measures over large geographical areas are still pending. Here, we measured the congruence between the biogeographical patterns of freshwater fish morphological, ecological and phylogenetic diversity across Europe and identified the natural and anthropogenic drivers shaping those patterns. Based on freshwater fish occurrence records in 290 European river catchments, we computed richness and evenness for morphological, ecological and phylogenetic diversity using standardized effect sizes for each diversity index. We then used linear models including climatic, geo‐morphological, biotic and human‐related factors to determine the key drivers shaping freshwater fish biodiversity patterns across Europe. We found a weak spatial congruence between facets of diversity. Patterns of diversity were mainly driven by elevation range, climatic seasonality and species richness while other factors played a minor role. Finally, we found that non‐native species introductions significantly affected diversity patterns and influenced the effects of some environmental drivers. Morphological, ecological and phylogenetic diversity constitute complementary facets of fish diversity rather than surrogates, testifying that they deserve to be considered altogether to properly assess biodiversity. Although the same environmental and anthropogenic factors overall explained those diversity facets, their relative influence varied. In the current context of global change, non‐native species introductions may also lead to important reshuffling of assemblages resulting in profound changes of diversity patterns.