Dystrophin Dp71 is Critical for Stability of the DAPs in the Nucleus of PC12 Cells

We have adopted the PC12 cell line as in vitro cell model for studying Dp71 function in neuronal cells. These cells express a cytoplasmic (Dp71f) and a nuclear (Dp71d) isoform of Dp71 as well as various dystrophin-associated proteins (DAPs). In this study, we revealed by confocal microscopy analysis and Western blotting evaluation of cell fractions the presence of different DAPs (β-dystroglycan, β-dystrobrevin, ε-sarcoglycan and γ1-syntrophin) in the nucleus of PC12 cells. Furthermore, we established by immunoprecipitation assays that Dp71d and the DAPs form a dystrophin-associated protein complex (DAPC) in the nucleus. Interestingly, depletion of Dp71 by antisense treatment (antisense-Dp71 cells) provoked a drastic reduction of nuclear DAPs, which indicates that Dp71d is critical for DAPs stability within the nucleus. Although Up71, the utrophin gene product homologous to Dp71, exhibited increased expression in the antisense-Dp71 cells, its scarce nuclear levels makes unlikely that could compensate for Dp71 nuclear deficiency.     

Structure of O-linked GlcNAc transferase: mediator of glycan-dependent signaling

In the brain, Dp71 is the most abundant protein product of the DMD gene and by alternative splicing of exon 78 two isoforms can be expressed, Dp71d and Dp71f. To explore the subcellular distribution of these Dp71 isoforms, specific monoclonal antibodies were used. Dp71d (with exon 78) was found in microsomes, while Dp71f (without exon 78) was detected in mitochondria. To determine the alterations which the absence of dystrophin proteins induces, we compared the expression of Dp71d in microsomes and Dp71f in mitochondria from mdx and mdx(3CV) mice. Dp71d in microsomes of mdx was similar to that of wild-type mice and, as expected, in mdx(3CV) this protein was undetectable. However, in mitochondria from mdx(3CV), Dp71f was overexpressed in comparison to mitochondria from mdx mice. Because in mdx(3CV) mice all the dystrophin proteins are mutated or diminished, we concluded that the protein detected in mitochondria is not a Dp71f but a novel product named Dp71f-like protein.     

Expression and purification of enterovirus type 71 polyprotein P1 using Pichia pastoris system

Enterovirus type 71(EV71) causes severe hand-foot-and-mouth disease (HFMD) resulting in hundreds of deaths of children every year; However, currently, there is no effective treatment for EV71. In this study, the EV71 poly-protein (EV71-P1 protein) gene was processed and cloned into the eukaryotic expression vector pPIC9k and then expressed in Pichia pastoris strain GS115. The EV71 P1 protein with a molecular weight of 100 kD was produced and secreted into the medium. The soluble EV71 P1 protein was purified by column chromatography with a recovery efficiency of 70%. The result of the immunological analysis showed that the EV71 P1 protein had excellent immunogenicity and could stimulate the production of EV71-VP1 IgG antibody in injected rabbits. We suggest that EV71-P1 protein is an ideal candidate for an EV71 vaccine to prevent EV71 infection.     

Synthesis and biological evaluation of a 3-positon epimer of 1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D3 (ED-71)

1alpha,25-Dihydroxy-2beta-(3-hydroxypropoxy)vitamin D(3) (ED-71), an analog of active vitamin D(3), 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], possesses a hydroxypropoxy substituent at the 2beta-position of 1,25(OH)(2)D(3). ED-71 has potent biological effects on bone and is currently under phase III clinical studies for bone fracture prevention. It is well-known that the synthesis and secretion of parathyroid hormone (PTH) is regulated by 1,25(OH)(2)D(3). Interestingly, during clinical development of ED-71, serum intact PTH in osteoporotic patients did not change significantly upon treatment with ED-71. The reason remains unclear, however. Brown et al. reported that 3-epi-1,25(OH)(2)D(3), an epimer of 1,25(OH)(2)D(3) at the 3-position, shows equipotent and prolonged activity compared to 1,25(OH)(2)D(3) at suppressing PTH secretion. Since ED-71 has a bulky hydroxypropoxy substituent at the 2-position, epimerization at the adjacent and sterically hindered 3-position might be prevented, which may account for its weak potency in PTH suppression observed in clinical studies. We have significant interest in ED-71 epimerization at the 3-position and the biological potency of 3-epi-ED-71 in suppressing PTH secretion. In the present studies, synthesis of 3-epi-ED-71 and investigations of in vitro suppression of PTH using bovine parathyroid cells are described. The inhibitory potency of vitamin D(3) analogs were found to be 1,25(OH)(2)D(3)>ED-71> or =3-epi-1,25(OH)(2)D(3)>3-epi-ED-71. ED-71 and 3-epi-ED-71 showed weak activity towards PTH suppression in our assays.     

Monoclonal neutralizing antibodies against EV71 screened from mice immunized with yeast-produced virus-like particles

Periodic outbreaks of hand, foot and mouth disease(HFMD) occur in children under 5 years old, and can cause death in some cases. The C4 strain of enterovirus 71(EV71) is the main pathogen that causes HFMD in China. Although no drugs against EV71 are available, some studies have shown that candidate vaccines or viral capsid proteins can produce anti-EV71 immunity. In this study, female BABL/c mice(6–8 weeks old) were immunized with virus-like particles(VLPs) of EV71 produced in yeast to screen for anti-EV71 antibodies. Two hybridomas that could produce neutralizing antibodies against EV71 were obtained. Both neutralizing m Abs(D4 and G12) were confirmed to bind the VP1 capsid protein of EV71, and could protect 95% cells from 100 TCID50 EV71 infection at 25 μg/m L solution(lowest concentration). Those two neutralizing m Abs identified in the study may be promising candidates in development for m Abs to treat EV71 infection, and utilized as suitable reagents for use in diagnostic tests and biological studies.     

Enterovirus-71 Virus-Like Particles Induce the Activation and Maturation of Human Monocyte-Derived Dendritic Cells through TLR4 Signaling

Enterovirus 71 (EV71) causes seasonal epidemics of hand-foot-and-mouth disease and has a high mortality rate among young children. We recently demonstrated potent induction of the humoral and cell-mediated immune response in monkeys immunized with EV71 virus-like particles (VLPs), with a morphology resembling that of infectious EV71 virions but not containing a viral genome, which could potentially be safe as a vaccine for EV71. To elucidate the mechanisms through which EV71 VLPs induce cell-mediated immunity, we studied the immunomodulatory effects of EV71 VLPs on human monocyte-derived dendritic cells (DCs), which bind to and incorporate EV71 VLPs. DC treatment with EV71 VLPs enhanced the expression of CD80, CD86, CD83, CD40, CD54, and HLA-DR on the cell surface; increased the production of interleukin (IL)-12 p40, IL-12 p70, and IL-10 by DCs; and suppressed the capacity of DCs for endocytosis. Treatment with EV71 VLPs also enhanced the ability of DCs to stimulate naïve T cells and induced secretion of interferon (IFN)-γ by T cells and Th1 cell responses. Neutralization with antibodies against Toll-like receptor (TLR) 4 suppressed the capacity of EV71 VLPs to induce the production of IL-12 p40, IL-12 p70, and IL-10 by DCs and inhibited EV71 VLPs binding to DCs. Our study findings clarified the important role for TLR4 signaling in DCs in response to EV71 VLPs and showed that EV71 VLPs induced inhibitor of kappaB alpha (IκBα) degradation and nuclear factor of kappaB (NF-κB) activation.     

An attenuated strain of enterovirus 71 belonging to genotype a showed a broad spectrum of antigenicity with attenuated neurovirulence in cynomolgus monkeys

Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease and is also sometimes associated with serious neurological disorders. In this study, we characterized the antigenicity and tissue specificity of an attenuated strain of EV71 [EV71(S1-3')], which belongs to genotype A, in a monkey infection model. Three cynomolgus monkeys were inoculated with EV71(S1-3'), followed by lethal challenge with the parental virulent strain EV71(BrCr-TR) via an intravenous route on day 45 postinoculation of EV71(S1-3'). Monkeys inoculated with EV71(S1-3') showed a mild neurological symptom (tremor) but survived lethal challenge by virulent EV71(BrCr-TR) without exacerbation of the symptom. The immunized monkey sera showed a broad spectrum of neutralizing activity against different genotypes of EV71, including genotypes A, B1, B4, C2, and C4. For the strains examined, the sera showed the highest neutralization activity against the homotype (genotype A) and the lowest neutralization activity against genotype C2. The order of decreasing neutralization activity of sera was as follows: A > B1 > C4 > B4 > C2. To examine the tissue specificity of EV71(S1-3'), two monkeys were intravenously inoculated with EV71(S1-3'), followed by examination of virus distribution in the central nervous system (CNS) and extraneural tissues. In the CNS, EV71(S1-3') was isolated only from the spinal cord. These results indicate that EV71(S1-3') acts as an effective antigen, although this attenuated strain was still neurotropic when inoculated via the intravenous route.     

Caveolar Endocytosis Is Required for Human PSGL-1-Mediated Enterovirus 71 Infection

Enterovirus 71 (EV71) causes hand, foot, and mouth disease and severe neurological disorders in children. Human scavenger receptor class B member 2 (hSCARB2) and P-selectin glycoprotein ligand-1 (PSGL-1) are identified as receptors for EV71. The underling mechanism of PSGL-1-mediated EV71 entry remains unclear. The endocytosis required for EV71 entry were investigated in Jurkat T and mouse L929 cells constitutively expressing human PSGL-1 (PSGL-1-L929) or human rhabdomyosarcoma (RD) cells displaying high SCARB2 but no PSGL-1 by treatment of specific inhibitors or siRNA. We found that disruption of clathrin-dependent endocytosis prevented EV71 infection in RD cells, while there was no influence in Jurkat T and PSGL-1-L929 cells. Disturbing caveolar endocytosis by specific inhibitor or caveolin-1 siRNA in Jurkat T and PSGL-1-L929 cells significantly blocked EV71 infection, whereas it had no effect on EV71 infection in RD cells. Confocal immunofluorescence demonstrated caveola, and EV71 was directly colocalized. pH-dependent endosomal acidification and intact membrane cholesterol were important for EV71 infection, as judged by the pretreatment of inhibitors that abrogated the infection. A receptor-dominated endocytosis of EV71 infection was observed: PSGL-1 initiates caveola-dependent endocytosis and hSCARB2 activates clathrin-dependent endocytosis.     

Antiviral effects of two Ganoderma lucidum triterpenoids against enterovirus 71 infection

Enterovirus 71 (EV71) is a major causative agent for hand, foot and mouth disease (HFMD), and fatal neurological and systemic complications in children. However, there is currently no clinical approved antiviral drug available for the prevention and treatment of the viral infection. Here, we evaluated the antiviral activities of two Ganoderma lucidum triterpenoids (GLTs), Lanosta-7,9(11),24-trien-3-one,15;26-dihydroxy (GLTA) and Ganoderic acid Y (GLTB), against EV71 infection. The results showed that the two natural compounds display significant anti-EV71 activities without cytotoxicity in human rhabdomyosarcoma (RD) cells as evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay. The mechanisms by which the two compounds affect EV71 infection were further elucidated by three action modes using Ribavirin, a common antiviral drug, as a positive control. The results suggested that GLTA and GLTB prevent EV71 infection through interacting with the viral particle to block the adsorption of virus to the cells. In addition, the interactions between EV71 virion and the compounds were predicated by computer molecular docking, which illustrated that GLTA and GLTB may bind to the viral capsid protein at a hydrophobic pocket (F site), and thus may block uncoating of EV71. Moreover, we demonstrated that GLTA and GLTB significantly inhibit the replication of the viral RNA (vRNA) of EV71 replication through blocking EV71 uncoating. Thus, GLTA and GLTB may represent two potential therapeutic agents to control and treat EV71 infection.     

Novel Nuclear Protein Complexes of Dystrophin 71 Isoforms in Rat Cultured Hippocampal GABAergic and Glutamatergic Neurons

The precise functional role of the dystrophin 71 in neurons is still elusive. Previously, we reported that dystrophin 71d and dystrophin 71f are present in nuclei from cultured neurons. In the present work, we performed a detailed analysis of the intranuclear distribution of dystrophin 71 isoforms (Dp71d and Dp71f), during the temporal course of 7-day postnatal rats hippocampal neurons culture for 1h, 2, 4, 10, 15 and 21 days in vitro (DIV). By immunofluorescence assays, we detected the highest level of nuclear expression of both dystrophin Dp71 isoforms at 10 DIV, during the temporal course of primary culture. Dp71d and Dp71f were detected mainly in bipolar GABAergic (≥60%) and multipolar Glutamatergic (≤40%) neurons, respectively. We also characterized the existence of two nuclear dystrophin-associated protein complexes (DAPC): dystrophin 71d or dystrophin 71f bound to β-dystroglycan, α1-, β-, α2-dystrobrevins, α-syntrophin, and syntrophin-associated protein nNOS (Dp71d-DAPC or Dp71f-DAPC, respectively), in the hippocampal neurons. Furthermore, both complexes were localized in interchromatin granule cluster structures (nuclear speckles) of neuronal nucleoskeleton preparations. The present study evinces that each Dp71’s complexes differ slightly in dystrobrevins composition. The results demonstrated that Dp71d-DAPC was mainly localized in bipolar GABAergic and Dp71f-DAPC in multipolar Glutamatergic hippocampal neurons. Taken together, our results show that dystrophin 71d, dystrophin 71f and DAP integrate protein complexes, and both complexes were associated to nuclear speckles structures.     

Specificity of olfactory receptor interactions with other G protein-coupled receptors

Studies on olfactory receptor (OR) pharmacology have been hindered by the poor plasma membrane localization of most ORs in heterologous cells. We previously reported that association with the beta(2)-adrenergic receptor (beta(2)-AR) facilitates functional expression of the OR M71 at the plasma membrane of HEK-293 cells. In the present study, we examined the specificity of M71 interactions with other G protein-coupled receptors (GPCRs). M71 was co-expressed in HEK-293 cells with 42 distinct GPCRs, and the vast majority of these receptors had no significant effect on M71 surface expression. However, co-expression with three subtypes of purinergic receptor (P2Y(1)R, P2Y(2)R, and A(2A)R) resulted in markedly enhanced plasma membrane localization of M71. Agonist stimulation of M71 co-expressed with P2Y(1)R and P2Y(2)R activated the mitogen-activated protein kinase pathway via coupling of M71 to Galpha(o). We also examined the ability of beta(2)-AR, P2Y(1)R, P2Y(2)R, and A(2A)Rto interact with and regulate ORs beyond M71. We found that co-expression of beta(2)-AR or the purinergic receptors enhanced the surface expression for an M71 subfamily member but not for several other ORs from different subfamilies. In addition, through chimeric receptor studies, we determined that the second transmembrane domain of beta(2)-AR is necessary for beta(2)-AR facilitation of M71 plasma membrane localization. These studies shed light on the specificity of OR interactions with other GPCRs and the mechanisms governing olfactory receptor trafficking.     

Phosphorylation of dystrophin Dp71d by Ca2+/calmodulin-dependent protein kinase II modulates the Dp71d nuclear localization in PC12 cells

We have shown that the splicing isoform of Dp71 (Dp71d) localizes to the nucleus of PC12 cells, an established cell line derived from a rat pheochromocytoma; however, the mechanisms governing its nuclear localization are unknown. As protein phosphorylation modulates the nuclear import of proteins, and as Dp71d presents several potential sites for phosphorylation, we analyzed whether Dp71d is phosphorylated in PC12 cells and the role of phosphorylation on its nuclear localization. We demonstrated that Dp71d is phosphorylated under basal conditions at serine and threonine residues by endogenous protein kinases. Dp71d phosphorylation was activated by 2-O-tetradecanoyl phorbol 13-acetate (TPA), but this effect was blocked by EGTA. Supporting the role of intracellular calcium on Dp71d phosphorylation, we observed that the stimulation of calcium influx by cell depolarization increased Dp71d phosphorylation, and that the calcium-calmodulin inhibitor N-(6-aminohexyl)-1-naphthalenesulfonamide (W-7) blocked such induction. The blocking action of bisindolylmaleimide I (Bis I), a specific inhibitor for Ca2+/diacylglicerol-dependent protein kinase (PKC), on Dp71d phosphorylation suggested the participation of PKC in this event. In addition, transfection experiments with Ca2+/calmodulin-dependent protein kinase II (CaMKII) expression vectors as well as the use of KN-62, a CaMKII-specific inhibitor, demonstrated that CaMKII is also involved in Dp71d phosphorylation. Stimulation of Dp71d phosphorylation by cell depolarization and/or the overexpression of CaMKII favored the Dp71d nuclear accumulation. Overall, our results indicate that CAMKII-mediated Dp71d phosphorylation modulates its nuclear localization.     

Proteomic and metabonomic analysis uncovering Enterovirus A71 reprogramming host cell metabolic pathway

Enterovirus A71 (EV71) infection can cause hand, foot, and mouth disease (HFMD) and severe neurological complications in children. However, the biological processes regulated by EV71 remain poorly understood. Herein, proteomics and metabonomics studies were conducted to uncover the mechanism of EV71 infection in rhabdomyosarcoma (RD) cells and identify potential drug targets. Differential expressed proteins from enriched membrane were analyzed by isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics technology. Twenty-six differential proteins with 1.5-fold (p < 0.05) change were detected, including 14 upregulated proteins and 12 downregulated proteins. The upregulated proteins are mainly involved in metabolic process, especially in the glycolysis pathway. Alpha-enolase (ENO1) protein was found to increase with temporal dependence following EV71 infection. The targeted metabolomics analysis revealed that glucose absorption and glycolysis metabolites were increased after EV71 infection. The glycolysis pathway was inhibited by knocking down ENO1 or the use of a glycolysis inhibitor (dichloroacetic acid [DCA]); and we found that EV71 infection was inhibited by depleting ENO1 or using DCA. Our study indicates that EV71 may reprogram glucose metabolism by activating glycolysis, and EV71 infection can be inhibited by interrupting the glycolysis pathway. ENO1 may be a potential target against EV71, and DCA could act as an inhibitor of EV71.     

Enterovirus71 (EV71) Utilise Host microRNAs to Mediate Host Immune System Enhancing Survival during Infection

Hand, Foot and Mouth Disease (HFMD) is a self-limiting viral disease that mainly affects infants and children. In contrast with other HFMD causing enteroviruses, Enterovirus71 (EV71) has commonly been associated with severe clinical manifestation leading to death. Currently, due to a lack in understanding of EV71 pathogenesis, there is no antiviral therapeutics for the treatment of HFMD patients. Therefore the need to better understand the mechanism of EV71 pathogenesis is warranted. We have previously reported a human colorectal adenocarcinoma cell line (HT29) based model to study the pathogenesis of EV71. Using this system, we showed that knockdown of DGCR8, an essential cofactor for microRNAs biogenesis resulted in a reduction of EV71 replication. We also demonstrated that there are miRNAs changes during EV71 pathogenesis and EV71 utilise host miRNAs to attenuate antiviral pathways during infection. Together, data from this study provide critical information on the role of miRNAs during EV71 infection.     

Enterovirus 71-induced autophagy increases viral replication and pathogenesis in a suckling mouse model

Background

We previously reported that Enterovirus 71 (EV71) infection activates autophagy, which promotes viral replication both in vitro and in vivo. In the present study we further investigated whether EV71 infection of neuronal SK-N-SH cells induces an autophagic flux. Furthermore, the effects of autophagy on EV71-related pathogenesis and viral load were evaluated after intracranial inoculation of mouse-adapted EV71 (MP4 strain) into 6-day-old ICR suckling mice.

Results

We demonstrated that in EV71-infected SK-N-SH cells, EV71 structural protein VP1 and nonstructural protein 2C co-localized with LC3 and mannose-6-phosphate receptor (MPR, endosome marker) proteins by immunofluorescence staining, indicating amphisome formation. Together with amphisome formation, EV71 induced an autophagic flux, which could be blocked by NH₄Cl (inhibitor of acidification) and vinblastine (inhibitor of fusion), as demonstrated by Western blotting. Suckling mice intracranially inoculated with EV71 showed EV71 VP1 protein expression (representing EV71 infection) in the cerebellum, medulla, and pons by immunohistochemical staining. Accompanied with these infected brain tissues, increased expression of LC3-II protein as well as formation of LC3 aggregates, autophagosomes and amphisomes were detected. Amphisome formation, which was confirmed by colocalization of EV71-VP1 protein or LC3 puncta and the endosome marker protein MPR. Thus, EV71-infected suckling mice (similar to EV71-infected SK-N-SH cells) also show an autophagic flux. The physiopathological parameters of EV71-MP4 infected mice, including body weight loss, disease symptoms, and mortality were increased compared to those of the uninfected mice. We further blocked EV71-induced autophagy with the inhibitor 3-methyladenine (3-MA), which attenuated the disease symptoms and decreased the viral load in the brain tissues of the infected mice.

Conclusions

In this study, we reveal that EV71 infection of suckling mice induces an amphisome formation accompanied with the autophagic flux in the brain tissues. Autophagy induced by EV71 promotes viral replication and EV71-related pathogenesis.     

miR-545 promoted enterovirus 71 replication via directly targeting phosphatase and tensin homolog and tumor necrosis factor receptor-associated factor 6

Enterovirus 71 (EV71) is a small, nonenveloped icosahedral RNA virus and is the predominant causative pathogen of hand-foot-and-mouth disease. Recently, microRNAs (miRNAs) are reported to play important roles in the pathogenesis of EV71 replication. This study investigated the role of miR-545 in the EV71 replication and explored the underlying molecular mechanisms. We showed that miR-545 was upregulated in the EV71-infected human embryonic kidney (HEK) 293 cells and rhabdomyosarcoma (RD) cells. Overexpression of miR-545 promoted the viral replication of EV71 and attenuated the inhibitory effects of EV71 on cell viability in HEK293 and RD cells; while knockdown of miR-545 significantly suppressed the EV71 replication in these two cell lines. Bioinformatics analysis and luciferase reporter assay showed that miR-545 directly targeted the 3′untranslated region of phosphatase and tensin homolog (PTEN) and tumor necrosis factor receptor-associated factor 6 (TRAF6) in HEK293 cells. Furthermore, miR-545 negatively regulated the messenger RNA (mRNA) and protein expression of PTEN and TRAF6. The mRNA and protein expression of PTEN and TRAF6 was also suppressed by EV71 infection, which was attenuated by miR-545 knockdown in HEK293 cells. Overexpression of PTEN and TRAF6 both suppressed the EV71 replication in HKE293 cells, and also attenuated the enhanced effects of miR-545 overexpression on the EV71 replication in HEK293 cells. Collectively, our study for the first time showed that miR-545 had an enhanced effect on the EV71 replication in HEK293 and RD cells. Further mechanistic results indicated that miR-545 promoted EV71 replication at least partly via targeting PTEN and TRAF6.     

Recent Progress on Functional Genomics Research of Enterovirus 71

Enterovirus 71(EV71) is one of the main pathogens that causes hand-foot-and-mouth disease(HFMD). HFMD caused by EV71 infection is mostly self-limited; however, some infections can cause severe neurological diseases, such as aseptic meningitis, brain stem encephalitis, and even death. There are still no effective clinical drugs used for the prevention and treatment of HFMD. Studying EV71 protein function is essential for elucidating the EV71 replication process and developing anti-EV71 drugs and vaccines. In this review, we summarized the recent progress in the studies of EV71 noncoding regions(50 UTR and 30 UTR) and all structural and nonstructural proteins, especially the key motifs involving in viral infection, replication, and immune regulation. This review will promote our understanding of EV71 virus replication and pathogenesis, and will facilitate the development of novel drugs or vaccines to treat EV71.     

Crystal structures of enterovirus 71 3C protease complexed with rupintrivir reveal the roles of catalytically important residues

EV71 is the primary pathogenic cause of hand-foot-mouth disease (HFMD), but an effective antiviral drug currently is unavailable. Rupintrivir, an inhibitor against human rhinovirus (HRV), has potent antiviral activities against EV71. We determined the high-resolution crystal structures of the EV71 3C(pro)/rupintrivir complex, showing that although rupintrivir interacts with EV71 3C(pro) similarly to HRV 3C(pro), the C terminus of the inhibitor cannot accommodate the leaving-group pockets of EV71 3C(pro). Our structures reveal that EV71 3C(pro) possesses a surface-recessive S2' pocket that is not present in HRV 3C(pro) that contributes to the additional substrate binding affinity. Combined with mutagenic studies, we demonstrated that catalytic Glu71 is irreplaceable for maintaining the overall architecture of the active site and, most importantly, the productive conformation of catalytic His40. We discovered the role of a previously uncharacterized residue, Arg39 of EV71 3C(pro), that can neutralize the negative charge of Glu71, which may subsequently assist deprotonation of His40 during proteolysis.     

Phylogenetic Analysis of Enterovirus 71 Circulating in Beijing,China from 2007 to 2009

The major pathogens of hand, foot and mouth disease (HFMD) in Beijing, China from 2007 to 2009 were identified in this study. A total of 186 HFMD cases were included, and 136 cases (73%) were positive for enterovirus (EV). In 2007, 75% (27/36) were Coxsackievirus A16 (CA16) positive and 19% (7/36) were Enterovirus 71 (EV71) positive cases. However, EV71 was the predominant virus in 2008, when 56% (31/55) of the cases were positive for EV71 and 22% (12/55) were positive for CA16. In 2009, EV71 and CA16, with positive rates of 36% (16/45) and 29% (13/45), respectively, were still the major pathogens of HFMD. Phylogenetic analysis revealed that the dominant genotype of EV71 was C4, with co-circulation of genotype A in 2009. The prevalent cluster of the EV71 subgenotype C4 changed over time. A proposed new sublineage of EV71, C4a-2, was the predominant virus associated with the Beijing and nationwide HFMD outbreaks since 2008 and amino acid substitution, which possibly link to the central nervous system tropism of EV71, was found in genotype A viruses. Persistent surveillance of HFMD-associated pathogens is required for predicting potential emerging viruses and related disease outbreaks.