Baisong Zheng

Analysis of enterovirus 68 strains from the 2014 North American outbreak reveals a new clade, indicating viral evolution

Enterovirus 68 (EVD68) causes respiratory illness, mostly in children. Despite a reported low-level of transmission, the occurrence of several recent outbreaks worldwide including the 2014 outbreak in North America has raised concerns regarding the pathogenesis and evolution of EVD68. To elucidate the phylogenetic features of EVD68 and possible causes for the 2014 outbreak, 216 EVD68 strain sequences were retrieved from Genbank, including 22 from the 2014 outbreak. Several geographic and genotypic origins were established for these 22 strains, 19 of which were classified as Clade B. Of these 19 strains, 17 exhibited subsequent clustering and variation in protein residues involved in host-receptor interaction and/or viral antigenicity. Approximately 18 inter-clade variations were detected in VP1, which led to the identification of a new Clade D in EVD68 strains. The classification of this new clade was also verified by the re-construction of a Neighbor-Joining tree during the phylogenetic analysis. In addition, our results indicate that members of Clade B containing highly specific alterations in VP1 protein residues were the foremost contributors to the 2014 outbreak in the US. Altered host-receptor interaction and/or host immune recognition may explain the evolution of EVD68 as well as the global emergence and ongoing adaptation of this virus.

Crystal Structure of Hyperthermophilic Endo-β-1,4-glucanase IMPLICATIONS FOR CATALYTIC MECHANISM AND THERMOSTABILITY

Endo-β-1,4-glucanase from thermophilic Fervidobacterium nodosum Rt17-B1 (FnCel5A), a new member of glycosyl hydrolase family 5, is highly thermostable and exhibits the highest activity on carboxymethylcellulose among the reported homologues. To understand the structural basis for the thermostability and catalytic mechanism, we report here the crystal structures of FnCel5A and the complex with glucose at atomic resolution. FnCel5A exhibited a (β/α)8-barrel structure typical of clan GH-A of the glycoside hydrolase families with a large and deep catalytic pocket located in the C-terminal end of the β-strands that may permit substrate access. A comparison of the structure of FnCel5A with related structures from thermopile Clostridium thermocellum, mesophile Clostridium cellulolyticum, and psychrophile Pseudoalteromonas haloplanktis showed significant differences in intramolecular interactions (salt bridges and hydrogen bonds) that may account for the difference in their thermostabilities. The substrate complex structure in combination with a mutagenesis analysis of the catalytic residues implicates a distinctive catalytic module Glu167-His226-Glu283, which suggests that the histidine may function as an intermediate for the electron transfer network between the typical Glu-Glu catalytic module. Further investigation suggested that the aromatic residues Trp61, Trp204, Phe231, and Trp240 as well as polar residues Asn51, His127, Tyr228, and His235 in the active site not only participated in substrate binding but also provided a unique microenvironment suitable for catalysis. These results provide substantial insight into the unique characteristics of FnCel5A for catalysis and adaptation to extreme temperature.

Cloning, Expression and Characterization of a Novel Thermophilic Polygalacturonase from Caldicellulosiruptor bescii DSM 6725

We cloned the gene ACM61449 from anaerobic, thermophilic Caldicellulosiruptor bescii, and expressed it in Escherichia coli origami (DE3). After purification through thermal treatment and Ni-NTA agarose column extraction, we characterized the properties of the recombinant protein (CbPelA). The optimal temperature and pH of the protein were 72 °C and 5.2, respectively. CbPelA demonstrated high thermal-stability, with a half-life of 14 h at 70 °C. CbPelA also showed very high activity for polygalacturonic acid (PGA), and released monogalacturonic acid as its sole product. The Vmax and Km of CbPelA were 384.6 U·mg−1 and 0.31 mg·mL−1, respectively. CbPelA was also able to hydrolyze methylated pectin (48% and 10% relative activity on 20%–34% and 85% methylated pectin, respectively). The high thermo-activity and methylated pectin hydrolization activity of CbPelA suggest that it has potential applications in the food and textile industry.

Chimeric Cellulase Matrix for Investigating Intramolecular Synergism between Non-hydrolytic Disruptive Functions of Carbohydrate-binding Modules and Catalytic Hydrolysis

Background: Disruptive functions of carbohydrate-binding modules (CBMs) toward crystalline cellulose and their synergism with hydrolases are important. Results: Chimeric cellulases derived from the cellulase matrix have activities toward crystalline cellulose. Conclusion: Analysis of chimeric cellulase activities allows quantification of the disruptive functions of CBMs. Significance: An efficient strategy was established to investigate non-hydrolytic disruptive functions and their synergism with hydrolysis. The conversion of renewable cellulosic biomass is of considerable interest for the production of biofuels and materials. The bottleneck in the efficient conversion is the compactness and resistance of crystalline cellulose. Carbohydrate-binding modules (CBMs), which disrupt crystalline cellulose via non-hydrolytic mechanisms, are expected to overcome this bottleneck. However, the lack of convenient methods for quantitative analysis of the disruptive functions of CBMs have hindered systematic studies and molecular modifications. Here we established a practical and systematic platform for quantifying and comparing the non-hydrolytic disruptive activities of CBMs via the synergism of CBMs and a catalytic module within designed chimeric cellulase molecules. Bioinformatics and computational biology were also used to provide a deeper understanding. A convenient vector was constructed to serve as a cellulase matrix into which heterologous CBM sequences can be easily inserted. The resulting chimeric cellulases were suitable for studying disruptive functions, and their activities quantitatively reflected the disruptive functions of CBMs on crystalline cellulose. In addition, this cellulase matrix can be used to construct novel chimeric cellulases with high hydrolytic activities toward crystalline cellulose.

Active Site Loop Conformation Regulates Promiscuous Activity in a Lactonase from Geobacillus kaustophilus HTA426

Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL) from Geobacillus kaustophilus HTA426 (GkaP) exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a “hot spot” in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (kcat/Km) toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity.

Crystallization and preliminary crystallographic analysis of the phosphotriesterase-like lactonase from Geobacillus kaustophilus

GK1506 from the thermophilic bacterium Geobacillus kaustophilus is a member of the phosphotriesterase-like lactonases, which can catalyze the hydrolysis of a broad range of compounds with different chemical properties. It is of particular interest because of its high thermostability and its dual activity towards organophosphate compounds and some lactones. These properties make GK1506 an attractive target for future enzyme engineering and use in practical applications. In order to resolve the crystal structure of GK1506 and to gain a better understanding of its biological function, recombinant GK1506 was expressed, purified and crystallized using 0.1 M HEPES pH 7.6, 12%(w/v) PEG 8000, 8%(v/v) ethylene glycol at 291 K. A 2.6 Å resolution native data set was collected from a single flash-cooled crystal (100 K) using 10%(v/v) glycerol as a cryoprotectant. These crystals belonged to space group P2(1), with unit-cell parameters a=51.444, b=80.453, c=92.615 Å, β=99.29°. Two molecules were assumed to be present per asymmetric unit, which gives a Matthews coefficient of 2.7 Å3 Da(-1).

Influence of the N-terminal peptide on the cocrystallization of a thermophilic endo-β-1,4-glucanase with polysaccharide substrates.

It is well known that protein cocrystallization is affected by several parameters such as the ratio of the protein to the ligand, the reservoir solution, the pH and the temperature. Previously, spatial blocking by the N-terminus was observed in the active site in the crystal structure of the native protein of a thermostable endoglucanase from the thermophilic bacterium Fervidobacterium nodosum Rt17-B1 (FnCel5A). It was speculated that the N-terminal α-helix might form interactions with the substrate-binding residues and it was believed that this spatial block is special to some extent. In order to confirm the effect on cocrystallization, two N-terminally truncated variants of FnCel5A were constructed, purified and cocrystallized at 291 K. A crystal of FnCel5AND_12-343 in complex with cellobiose was obtained using PEG 8000 as a precipitant. A 2.2 Å resolution data set was collected. This crystal form (space group P4(1)2(1)2, unit-cell parameters a = b = 47.3, c = 271.4 Å) differed from that of the native protein. One molecule is assumed to be present per asymmetric unit, which gives a Matthews coefficient of 2.05 Å(3) Da(-1).

Jembrana disease virus Vif antagonizes the inhibition of bovine APOBEC3 proteins through ubiquitin-mediate protein degradation

Viral infectivity factor (Vif) encoded by lentiviruses is essential for viral replication and escaping from antiviral activity of host defensive factors APOBEC3. Jembrana disease virus (JDV) causes an acute disease syndrome with approximately 20% case fatality rate in Bali cattle. However, the interplay mechanism between JDV Vif and Bos taurus APOBEC3 (btA3) is poorly understood. In this study, we determined that JDV Vif recruits ElonginB, ElonginC(ELOB/C), Cul2 and RBX1 but without the need of CBF-β to form E3 ubiquitin ligase and induces the degradation of btA3 proteins. Further investigation identified BC-box (T149LQ151) motif required for ELOB/C binding, Cul2 box (Y167xxxxV/X172) and a zinc-binding motif (H95-C113-H115-C133) required for Cul2 binding in JDV Vif. The precise mechanism of JDV Vif overcoming the antiviral activity of btA3 proteins is helpful for the application of the broad spectrum antiviral drug targeting conserved functional domains of various species Vif proteins in the future.

Enterovirus 71 antagonizes the inhibition of the host intrinsic antiviral factor A3G

Abstract Although the host restriction factor APOBEC3G (A3G) has broad spectrum antiviral activity, whether A3G inhibits enterovirus 71 (EV71) has been unclear until now. In this study, we demonstrated for the first time that A3G could inhibit EV71 virus replication. Silencing A3G in H9 cells enhanced EV71 replication, and EV71 replication was lower in H9 cells expressing A3G than in Jurkat cells without A3G expression, indicating that the EV71 inhibition was A3G-specific. Further investigation revealed that A3G inhibited the 5′UTR activity of EV71 by competitively binding to the 5′UTR through its nucleic acid binding activity. This binding impaired the interaction between the 5′UTR and the host protein poly(C)-binding protein 1 (PCBP1), which is required for the synthesis of EV71 viral proteins and RNA. On the other hand, we found that EV71 overcame A3G suppression through its non-structural protein 2C, which induced A3G degradation through the autophagy–lysosome pathway. Our research provides new insights into the interplay mechanisms of A3G and single-stranded positive RNA viruses.

Coxsackievirus A6 Induces Cell Cycle Arrest in G0/G1 Phase for Viral Production

Recent epidemiological data indicate that outbreaks of hand, foot, and mouth disease (HFMD), which can be categorized according to its clinical symptoms as typical or atypical, have markedly increased worldwide. A primary causative agent for typical HFMD outbreaks, enterovirus 71 (EV71), has been shown to manipulate the cell cycle in S phase for own replication; however, it is not clear whether coxsackievirus (CVA6), the main agent for atypical HFMD, also regulates the host cell cycle. In this study, we demonstrate for the first time that CVA6 infection arrests the host cell cycle in G0/G1-phase. Furthermore, synchronization in G0/G1 phase, but not S phase or G2/M phase, promotes viral production. To investigate the mechanism of cell cycle arrest induced by CVA6 infection, we analyzed cell cycle progression after cell cycle synchronization at G0/G1 or G2/M. Our results demonstrate that CVA6 infection promotes G0/G1 phase entry from G2/M phase, and inhibits G0/G1 exit into S phase. In line with its role to arrest cells in G0/G1 phase, the expression of cyclinD1, CDK4, cyclinE1, CDK2, cyclinB1, CDK1, P53, P21, and P16 is regulated by CVA6. Finally, the non-structural proteins of CVA6, RNA-dependent RNA polymerase 3D and protease 3C , are demonstrated to be responsible for the G0/G1-phase arrest. These findings suggest that CVA6 infection arrested cell cycle in G0/G1-phase via non-structural proteins 3D and 3C, which may provide favorable environments for virus production.