DWARF14 is a non-canonical hormone receptor for strigolactone.

Classical hormone receptors reversibly and non-covalently bind active hormone molecules, which are generated by biosynthetic enzymes, to trigger signal transduction. The α/ß hydrolase DWARF14 (D14), which hydrolyses the plant branching hormone strigolactone and interacts with the F-box protein D3/MAX2, is probably involved in strigolactone detection. However, the active form of strigolactone has yet to be identified and it is unclear which protein directly binds the active form of strigolactone, and in which manner, to act as the genuine strigolactone receptor. Here we report the crystal structure of the strigolactone-induced AtD14-D3-ASK1 complex, reveal that Arabidopsis thaliana (At)D14 undergoes an open-to-closed state transition to trigger strigolactone signalling, and demonstrate that strigolactone is hydrolysed into a covalently linked intermediate molecule (CLIM) to initiate a conformational change of AtD14 to facilitate interaction with D3. Notably, analyses of a highly branched Arabidopsis mutant d14-5 show that the AtD14(G158E) mutant maintains enzyme activity to hydrolyse strigolactone, but fails to efficiently interact with D3/MAX2 and loses the ability to act as a receptor that triggers strigolactone signalling in planta. These findings uncover a mechanism underlying the allosteric activation of AtD14 by strigolactone hydrolysis into CLIM, and define AtD14 as a non-canonical hormone receptor with dual functions to generate and sense the active form of strigolactone.

Seneca Valley virus attachment and uncoating mediated by its receptor anthrax toxin receptor 1.

Seneca Valley virus (SVV) is an oncolytic picornavirus with selective tropism for neuroendocrine cancers. SVV mediates cell entry by attachment to the receptor anthrax toxin receptor 1 (ANTXR1). Here we determine atomic structures of mature SVV particles alone and in complex with ANTXR1 in both neutral and acidic conditions, as well as empty "spent" particles in complex with ANTXR1 in acidic conditions by cryoelectron microscopy. SVV engages ANTXR1 mainly by the VP2 DF and VP1 CD loops, leading to structural changes in the VP1 GH loop and VP3 GH loop, which attenuate interprotomer interactions and destabilize the capsid assembly. Despite lying on the edge of the attachment site, VP2 D146 interacts with the metal ion in ANTXR1 and is required for cell entry. Though the individual substitution of most interacting residues abolishes receptor binding and virus propagation, a serine-to-alanine mutation at VP2 S177 significantly increases SVV proliferation. Acidification of the SVV-ANTXR1 complex results in a major reconfiguration of the pentameric capsid assemblies, which rotate ∼20° around the icosahedral fivefold axes to form a previously uncharacterized spent particle resembling a potential uncoating intermediate with remarkable perforations at both two- and threefold axes. These structures provide high-resolution snapshots of SVV entry, highlighting opportunities for anticancer therapeutic optimization.

Site-Specific Incorporation of Chemical Fluorescence on Live Enterovirus-71 Virion by Using an Organometallic Palladium Reagent To Monitor Virus Entry.

Imaging live virus to monitor the viral entry process is essential to understand virus-host interactions during pathogen infection. However, methods for efficient labeling of live viruses, in particular labeling non-enveloped viruses and tracing virus entry processes, remain limited. Recently, labeling by using organometallic palladium reagents has provided a highly efficient and selective way to bioconjugate cysteines of virus proteins. Here, site-specific bioorthogonal labeling mediated by an organometallic palladium reagent on the surface of live enterovirus-71 (EV71) was used to visualize its entry into live cells. In contrast to currently used immunofluorescence and membrane-anchored dyes, this site-specific and quantitative labeling of live EV71 allows temporal imaging of its entry into host cell membranes on the timescale of seconds with little negative impact on its virulence. This method revealed details of EV71 virus entry and has broad applicability for monitoring virus entry that is difficult to assess by using conventional protein-labeling approaches.

Distinct Mechanism for the Formation of the Ribonucleoprotein Complex of Tomato Spotted Wilt Virus.

The Tomato spotted wilt virus (TSWV) belongs to the Tospovirus genus of the Bunyaviridae family and represents the sole plant-infecting group within bunyavirus. TSWV encodes a nucleocapsid protein (N) which encapsidates the RNA genome to form a ribonucleoprotein complex (RNP). In addition, the N has multiple roles during the infection of plant cells. Here, we report the crystal structure of the full-length TSWV N. The N features a body domain consisting of an N-lobe and a C-lobe. These lobes clamp a positively charged groove which may constitute the RNA binding site. Furthermore, the body domains are flanked by N- and C-terminal arms which mediate homotypic interactions to the neighboring subunits, resulting in a ring-shaped N trimer. Interestingly, the C terminus of one protomer forms an additional interaction with the protomer of an adjacent trimer in the crystal, which may constitute a higher-order oligomerization contact. In this way, this study provides insights into the structure and trimeric assembly of TSWV N, which help to explain previous functional findings, but also suggests distinct N interactions within a higher-order RNP.IMPORTANCE TSWV is one of the most devastating plant pathogens that cause severe diseases in numerous agronomic and ornamental crops worldwide. TSWV is also the prototypic member of the Tospovirus genus, which is the sole group of plant-infecting viruses in the bunyavirus family. This study determined the structure of full-length TSWV N in an oligomeric state. The structural observations explain previously identified biological properties of TSWV N. Most importantly, the additional homotypic interaction between the C terminus of one protomer with another protomer indicates that there is a distinct mechanism of RNP formation in the bunyavirus family, thereby enhancing the current knowledge of negative-sense single-stranded RNA virus-encoded N. TSWV N is the last remaining representative N with an unknown structure in the bunyavirus family. Combined with previous studies, the structure of TSWV N helps to build a complete picture of the bunyavirus-encoded N family and reveals a close evolutionary relationship between orthobunyavirus, phlebovirus, hantavirus, and tospovirus.

Using steered molecular dynamics to study the interaction between ADP and the nucleotide-binding domain of yeast Hsp70 protein Ssa1.

Genetics experiments have identified six mutations located in the subdomain IA (A17V, R23H, G32D, G32S, R34K, V372I) of Ssa1 that influence propagation of the yeast [PSI+] prion. However, the underlining molecular mechanisms of these mutations are still unclear. The six mutation sites are present in the IA subdomain of the nucleotide-binding domain (NBD). The ATPase subdomain IA is a critical mediator of inter-domain allostery in Hsp70 molecular chaperones, so the mutation and changes in this subdomain may influence the function of the substrate-binding domain. In addition, ADP release is a rate-limiting step of the ATPase cycle and dysregulation of the ATPase cycle influences the propagation of the yeast [PSI+] prion. In this work, steered molecular dynamics (SMD) simulations were performed to explore the interaction between ADP and NBD. Results suggest that during the SMD simulations, hydrophobic interactions are predominant and variations in the binding state of ADP within the mutants is a potential reason for in vivo effects on yeast [PSI+] prion propagation. Additionally, we identify the primary residues in the ATPase domain that directly constitute the main hydrophobic interaction network and directly influence the ADP interaction state with the NBD of Ssa1. Furthermore, this in silico analysis reaffirms the importance of previously experimentally-determined residues in the Hsp70 ATPase domain involved in ADP binding and also identifies new residues potentially involved in this process.

Structural basis and functional analysis of the SARS coronavirus nsp14-nsp10 complex.

Nonstructural protein 14 (nsp14) of coronaviruses (CoV) is important for viral replication and transcription. The N-terminal exoribonuclease (ExoN) domain plays a proofreading role for prevention of lethal mutagenesis, and the C-terminal domain functions as a (guanine-N7) methyl transferase (N7-MTase) for mRNA capping. The molecular basis of both these functions is unknown. Here, we describe crystal structures of severe acute respiratory syndrome (SARS)-CoV nsp14 in complex with its activator nonstructural protein10 (nsp10) and functional ligands. One molecule of nsp10 interacts with ExoN of nsp14 to stabilize it and stimulate its activity. Although the catalytic core of nsp14 ExoN is reminiscent of proofreading exonucleases, the presence of two zinc fingers sets it apart from homologs. Mutagenesis studies indicate that both these zinc fingers are essential for the function of nsp14. We show that a DEEDh (the five catalytic amino acids) motif drives nucleotide excision. The N7-MTase domain exhibits a noncanonical MTase fold with a rare ß-sheet insertion and a peripheral zinc finger. The cap-precursor guanosine-P3-adenosine-5',5'-triphosphate and S-adenosyl methionine bind in proximity in a highly constricted pocket between two ß-sheets to accomplish methyl transfer. Our studies provide the first glimpses, to our knowledge, into the architecture of the nsp14-nsp10 complex involved in RNA viral proofreading.

Structure of the Enterovirus 71 3C Protease in Complex with NK-1.8k and Indications for the Development of Antienterovirus Protease Inhibitor.

Hand-foot-and-mouth disease (HFMD), caused by enterovirus, is a threat to public health worldwide. To date, enterovirus 71 (EV71) has been one of the major causative agents of HFMD in the Pacific-Asia region, and outbreaks with EV71 cause millions of infections. However, no drug is currently available for clinical therapeutics. In our previous works, we developed a set of protease inhibitors (PIs) targeting the EV71 3C protease (3Cpro). Among these are NK-1.8k and NK-1.9k, which have various active groups and high potencies and selectivities. In the study described here, we determined the structures of the PI NK-1.8k in complex with wild-type (WT) and drug-resistant EV71 3Cpro Comparison of these structures with the structure of unliganded EV71 3Cpro and its complex with AG7088 indicated that the mutation of N69 to a serine residue destabilized the S2 pocket. Thus, the mutation influenced the cleavage activity of EV71 3Cpro and the inhibitory activity of NK-1.8k in an in vitro protease assay and highlighted that site 69 is an additional key site for PI design. More information for the optimization of the P1' to P4 groups of PIs was also obtained from these structures. Together with the results of our previous works, these in-depth results elucidate the inhibitory mechanism of PIs and shed light to develop PIs for the clinical treatment of infections caused by EV71 and other enteroviruses.

Crystal Structure of the Core Region of Hantavirus Nucleocapsid Protein Reveals the Mechanism for Ribonucleoprotein Complex Formation.

UNLABELLED: Hantaviruses, which belong to the genus Hantavirus in the family Bunyaviridae, infect mammals, including humans, causing either hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS) in humans with high mortality. Hantavirus encodes a nucleocapsid protein (NP) to encapsidate the genome and form a ribonucleoprotein complex (RNP) together with viral polymerase. Here, we report the crystal structure of the core domains of NP (NPcore) encoded by Sin Nombre virus (SNV) and Andes virus (ANDV), which are two representative members that cause HCPS in the New World. The constructs of SNV and ANDV NPcore exclude the N- and C-terminal portions of full polypeptide to obtain stable proteins for crystallographic study. The structure features an N lobe and a C lobe to clamp RNA-binding crevice and exhibits two protruding extensions in both lobes. The positively charged residues located in the RNA-binding crevice play a key role in RNA binding and virus replication. We further demonstrated that the C-terminal helix and the linker region connecting the N-terminal coiled-coil domain and NPcore are essential for hantavirus NP oligomerization through contacts made with two adjacent protomers. Moreover, electron microscopy (EM) visualization of native RNPs extracted from the virions revealed that a monomer-sized NP-RNA complex is the building block of viral RNP. This work provides insight into the formation of hantavirus RNP and provides an understanding of the evolutionary connections that exist among bunyaviruses. IMPORTANCE: Hantaviruses are distributed across a wide and increasing range of host reservoirs throughout the world. In particular, hantaviruses can be transmitted via aerosols of rodent excreta to humans or from human to human and cause HFRS and HCPS, with mortalities of 15% and 50%, respectively. Hantavirus is therefore listed as a category C pathogen. Hantavirus encodes an NP that plays essential roles both in RNP formation and in multiple biological functions. NP is also the exclusive target for the serological diagnoses. This work reveals the structure of hantavirus NP, furthering the knowledge of hantavirus RNP formation, revealing the relationship between hantavirus NP and serological specificity and raising the potential for the development of new diagnosis and therapeutics targeting hantavirus infection.

[Comparative study between cardiac catheterization intervention therapy and transthoracic small incision surgery for closure of congenital atrial septal defect by domestic occluder with echocardiographic monitoring].

OBJECTIVE: To evaluate the safety of cardiac catheterization intervention therapy and transthoracic small incision surgery in the occlusion bydomestic occluder under echocardiography guiding in patients with atrial septal defect (ASD).
 Methods: A total of 1 080 patients with ASD in the occlusion by domestic occluder were analyzed retrospectively, and the interventional treatment were performed in 734 cases through cardiac catheterization intervention therapy and 346 cases through transthoracic small incision surgery. The patients undergone cardiac catheterization intervention therapy were guided under the digital substraction angiography (DSA) and were monitored by transthoracic echocardiography (TTE) in the whole interventional process, and the efficacy was evaluated with TTE. The occlusion of transthoracic small incision surgery was guided under the transesophageal echocardiography (TEE), which was used to monitor the position of occluder and evaluate the efficacy immediately.
 Results: Two kinds of intervention in the occlusion by domestic occluder had achieved satisfactory results in patients with ASD. There was no statistically difference in the longest size of ASD between the 2 intervention methods, while there were statistically differences in the ratio between ASD longest diameter and atrial septal length, and the size of the occlusion, and the disparity between the size of the occluder and ASD longest diameter (D value), respectively (all P<0.05). When the size of arithmetic mean of the ASD was <30 mm, the success rate of the 2 methods was both 100%. When the size of arithmetic mean of the ASD was ≥30 mm, the success rate was 100% in the transthoracic small incision surgery and 50% in the cardiac catheterization intervention therapy.
 Conclusion: Domestic occluder is safe. Compared with the imported one, its cost is lower. When the size of the defects is same, the occlusion is smaller in the transthoracic small incision surgery compared with that in the cardiac catheterization intervention therapy. When the size of arithmetic mean of the ASD is ≥30 mm, the success rate of the transthoracic small incision surgery is higher compared with the cardiac catheterization intervention therapy. When the cardiac catheterization intervention therapy fails, the transthoracic small incision surgery may be a better choice.

Molecular basis for the formation of ribonucleoprotein complex of Crimean-Congo hemorrhagic fever virus.

Negative-sense single-strand RNA (-ssRNA) viruses comprise a large family of pathogens that cause severe human infectious diseases. All -ssRNA viruses encode a nucleocapsid protein (NP) to encapsidate the viral genome, which, together with polymerase, forms a ribonucleoprotein complex (RNP) that is packaged into virions and acts as the template for viral replication and transcription. In our previous work, we solved the monomeric structure of NP encoded by Crimean-Congo hemorrhagic fever virus (CCHFV), which belongs to the Nairovirus genus within the Bunyaviridae family, and revealed its unusual endonuclease activity. However, the mechanism of CCHFV RNP formation remains unclear, due to the difficulty in reconstructing the oligomeric CCHFV NP-RNA complex. Here, we identified and isolated the oligomeric CCHFV NP-RNA complex that formed in expression cells. Sequencing of RNA extracted from the complex revealed sequence specificity and suggested a potential encapsidation signal facilitating the association between NP and viral genome. A cryo-EM reconstruction revealed the ring-shaped architecture of the CCHFV NP-RNA oligomer, thus defining the interaction between the head and stalk domains that results in NP multimerization. This structure also suggested a modified gating mechanism for viral genome encapsidation, in which both the head and stalk domains participate in RNA binding. This work provides insight into the distinct mechanism underlying CCHFV RNP formation compared to other -ssRNA viruses.

A Conserved Inhibitory Mechanism of a Lycorine Derivative against Enterovirus and Hepatitis C Virus.

Enterovirus 71 (EV71) (Picornaviridae family) and hepatitis C virus (HCV) (Flaviviridae family) are the causative agents of human hand, foot, and mouth disease (HFMD) and hepatitis C, resulting in a severe pandemic involving millions of infections in the Asia-Pacific region and worldwide. The great impact of EV71 and HCV on public health highlights the need to further our understanding of the biology of these two viruses and develop effective therapeutic antivirals. Here, we evaluated a total of 32 lycorine derivatives and demonstrated that 1-acetyllycorine suppressed the proliferation of multiple strains of EV71 in various cells. The results of the drug resistance analysis revealed that 1-acetyllycorine targeted a phenylalanine (F76) in EV71 2A protease (2A(pro)) to stabilize the conformation of a unique zinc finger. Most interestingly, the zinc binding site in EV71 2A(pro) is the exclusive homolog of HCV NS3 among all viruses. Further analysis revealed that 1-acetyllycorine also inhibits HCV with high efficacy, and the mutation on R118 in HCV NS3, which corresponds to F76 in EV71 2A(pro), confers the resistance of HCV to 1-acetyllycorine. These results revealed a conserved mechanism of 1-acetyllycorine against EV71 and HCV through targeting viral proteases. We also documented the significant synergistic anti-EV71 and anti-HCV effects of 1-acetyllycorine with reported inhibitors, supporting potential combination therapy for the treatment of EV71 and HCV infections.

Cyclophilin A associates with enterovirus-71 virus capsid and plays an essential role in viral infection as an uncoating regulator.

Viruses utilize host factors for their efficient proliferation. By evaluating the inhibitory effects of compounds in our library, we identified inhibitors of cyclophilin A (CypA), a known immunosuppressor with peptidyl-prolyl cis-trans isomerase activity, can significantly attenuate EV71 proliferation. We demonstrated that CypA played an essential role in EV71 entry and that the RNA interference-mediated reduction of endogenous CypA expression led to decreased EV71 multiplication. We further revealed that CypA directly interacted with and modified the conformation of H-I loop of the VP1 protein in EV71 capsid, and thus regulated the uncoating process of EV71 entry step in a pH-dependent manner. Our results aid in the understanding of how host factors influence EV71 life cycle and provide new potential targets for developing antiviral agents against EV71 infection.

Bunyamwera virus possesses a distinct nucleocapsid protein to facilitate genome encapsidation.

Bunyamwera virus (BUNV), which belongs to the genus Orthobunyavirus, is the prototypical virus of the Bunyaviridae family. Similar to other negative-sense single-stranded RNA viruses, bunyaviruses possess a nucleocapsid protein (NP) to facilitate genomic RNA encapsidation and virus replication. The structures of two NPs of members of different genera within the Bunyaviridae family have been reported. However, their structures, RNA-binding features, and functions beyond RNA binding significantly differ from one another. Here, we report the crystal structure of the BUNV NP-RNA complex. The polypeptide of the BUNV NP was found to possess a distinct fold among viral NPs. An N-terminal arm and a C-terminal tail were found to interact with neighboring NP protomers to form a tetrameric ring-shaped organization. Each protomer bound a 10-nt RNA molecule, which was acquired from the expression host, in the positively charged crevice between the N and C lobes. Inhomogeneous oligomerization was observed for the recombinant BUNV NP-RNA complex, which was similar to the Rift Valley fever virus NP-RNA complex. This result suggested that the flexibility of one NP protomer with adjacent protomers underlies the BUNV ribonucleoprotein complex (RNP) formation. Electron microscopy revealed that the monomer-sized NP-RNA complex was the building block of the natural BUNV RNP. Combined with previous results indicating that mutagenesis of the interprotomer or protein-RNA interface affects BUNV replication, our structure provides a great potential for understanding the mechanism underlying negative-sense single-stranded RNA RNP formation and enables the development of antiviral therapies targeting BUNV RNP formation.

Peptidyl aldehyde NK-1.8k suppresses enterovirus 71 and enterovirus 68 infection by targeting protease 3C.

Enterovirus (EV) is one of the major causative agents of hand, foot, and mouth disease in the Pacific-Asia region. In particular, EV71 causes severe central nervous system infections, and the fatality rates from EV71 infection are high. Moreover, an outbreak of respiratory illnesses caused by an emerging EV, EV68, recently occurred among over 1,000 young children in the United States and was also associated with neurological infections. Although enterovirus has emerged as a considerable global public health threat, no antiviral drug for clinical use is available. In the present work, we screened our compound library for agents targeting viral protease and identified a peptidyl aldehyde, NK-1.8k, that inhibits the proliferation of different EV71 strains and one EV68 strain and that had a 50% effective concentration of 90 nM. Low cytotoxicity (50% cytotoxic concentration, >200 µM) indicated a high selective index of over 2,000. We further characterized a single amino acid substitution inside protease 3C (3C(pro)), N69S, which conferred EV71 resistance to NK-1.8k, possibly by increasing the flexibility of the substrate binding pocket of 3C(pro). The combination of NK-1.8k and an EV71 RNA-dependent RNA polymerase inhibitor or entry inhibitor exhibited a strong synergistic anti-EV71 effect. Our findings suggest that NK-1.8k could potentially be developed for anti-EV therapy.

Biochemical characterization of recombinant Enterovirus 71 3C protease with fluorogenic model peptide substrates and development of a biochemical assay.

Enterovirus 71 (EV71), a primary pathogen of hand, foot, and mouth disease (HFMD), affects primarily infants and children. Currently, there are no effective drugs against HFMD. EV71 3C protease performs multiple tasks in the viral replication, which makes it an ideal antiviral target. We synthesized a small set of fluorogenic model peptides derived from cleavage sites of EV71 polyprotein and examined their efficiencies of cleavage by EV71 3C protease. The novel peptide P08 [(2-(N-methylamino)benzoyl) (NMA)-IEALFQGPPK(DNP)FR] was determined to be the most efficiently cleaved by EV71 3C protease, with a kinetic constant kcat/Km of 11.8 ± 0.82 mM(-1) min(-1). Compared with literature reports, P08 gave significant improvement in the signal/background ratio, which makes it an attractive substrate for assay development. A Molecular dynamics simulation study elaborated the interactions between substrate P08 and EV71 3C protease. Arg39, which is located at the bottom of the S2 pocket of EV71 3C protease, may participate in the proteolysis process of substrates. With an aim to evaluate EV71 3C protease inhibitors, a reliable and robust biochemical assay with a Z' factor of 0.87 ± 0.05 was developed. A novel compound (compound 3) (50% inhibitory concentration [IC50] = 1.89 ± 0.25 µM) was discovered using this assay, which effectively suppressed the proliferation of EV 71 (strain Fuyang) in rhabdomyosarcoma (RD) cells with a highly selective index (50% effective concentration [EC50] = 4.54 ± 0.51 µM; 50% cytotoxic concentration [CC50] > 100 µM). This fast and efficient assay for lead discovery and optimization provides an ideal platform for anti-EV71 drug development targeting 3C protease.

Crimean-Congo hemorrhagic fever virus nucleoprotein reveals endonuclease activity in bunyaviruses.

Crimean-Congo hemorrhagic fever virus (CCHFV), a virus with high mortality in humans, is a member of the genus Nairovirus in the family Bunyaviridae, and is a causative agent of severe hemorrhagic fever (HF). It is classified as a biosafety level 4 pathogen and a potential bioterrorism agent due to its aerosol infectivity and its ability to cause HF outbreaks with high case fatality (∼30%). However, little is known about the structural features and function of nucleoproteins (NPs) in the Bunyaviridae, especially in CCHFV. Here we report a 2.3-Šresolution crystal structure of the CCHFV nucleoprotein. The protein has a racket-shaped overall structure with distinct "head" and "stalk" domains and differs significantly with NPs reported so far from other negative-sense single-stranded RNA viruses. Furthermore, CCHFV NP shows a distinct metal-dependent DNA-specific endonuclease activity. Single residue mutations in the predicted active site resulted in a significant reduction in the observed endonuclease activity. Our results present a new folding mechanism and function for a negative-strand RNA virus nucleoprotein, extend our structural insight into bunyavirus NPs, and provide a potential target for antiviral drug development to treat CCHFV infection.

Crystal structure of enterovirus 71 RNA-dependent RNA polymerase complexed with its protein primer VPg: implication for a trans mechanism of VPg uridylylation.

Picornavirus RNA replication is initiated by VPg uridylylation, during which the hydroxyl group of the third tyrosine residue of the virally encoded protein VPg is covalently linked to two UMP molecules by RNA-dependent RNA polymerase (RdRp; also known as 3D(pol)). We previously identified site 311, located at the base of the palm domain of the enterovirus 71 (EV71) RdRp, to be the site for EV71 VPg binding and uridylylation. Here we report the crystal structure of EV71 3D(pol) complexed with VPg. VPg was anchored at the bottom of the palm domain of the 3D(pol) molecule and exhibited an extended V-shape conformation. The corresponding interface on 3D(pol) was mainly formed by residues within site 311 and other residues in the palm and finger domains. Mutations of the amino acids of 3D(pol) involved in the VPg interaction (3DL319A, 3DD320A, and 3DY335A) significantly disrupted VPg binding to 3D(pol), resulting in defective VPg uridylylation. In contrast, these mutations did not affect the RNA elongation activity of 3D(pol). In the context of viral genomic RNA, mutations that abolished VPg uridylylation activity were lethal for EV71 replication. Further in vitro analysis showed that the uridylylation activity was restored by mixing VPg-binding-defective and catalysis-defective mutants, indicating a trans mechanism for EV71 VPg uridylylation. Our results, together with previous results of other studies, demonstrate that different picornaviruses use distinct binding sites for VPg uridylylation.

Enterovirus 71 VPg uridylation uses a two-molecular mechanism of 3D polymerase.

VPg uridylylation is essential for picornavirus RNA replication. The VPg uridylylation reaction consists of the binding of VPg to 3D polymerase (3D(pol)) and the transfer of UMP by 3D(pol) to the hydroxyl group of the third amino acid Tyr of VPg. Previous studies suggested that different picornaviruses employ distinct mechanisms during VPg binding and uridylylation. Here, we report a novel site (Site-311, located at the base of the palm domain of EV71 3D(pol)) that is essential for EV71 VPg uridylylation as well as viral replication. Ala substitution of amino acids (T313, F314, and I317) at Site-311 reduced the VPg uridylylation activity of 3D(pol) by >90%. None of the Site-311 mutations affected the RNA elongation activity of 3D(pol), which indicates that Site-311 does not directly participate in RNA polymerization. However, mutations that abrogated VPg uridylylation significantly reduced the VPg binding ability of 3D(pol), which suggests that Site-311 is a potential VPg binding site on enterovirus 71 (EV71) 3D(pol). Mutation of a polymerase active site in 3D(pol) and Site-311 in 3D(pol) remarkably enables trans complementation to restore VPg uridylylation. In contrast, two distinct Site-311 mutants do not cause trans complementation in vitro. These results indicate that Site-311 is a VPg binding site that stabilizes the VPg molecule during the VPg uridylylation process and suggest a two-molecule model for 3D(pol) during EV71 VPg uridylylation, such that one 3D(pol) presents the hydroxyl group of Tyr3 of VPg to the polymerase active site of another 3D(pol), which in turn catalyzes VPg→VPg-pU conversion. For genome-length RNA, the Site-311 mutations that reduced VPg uridylylation were lethal for EV71 replication, which indicates that Site-311 is a potential antiviral target.

Crystallization and preliminary crystallographic analysis of Arabidopsis thaliana BRI1-associated kinase 1 (BAK1) cytoplasmic domain.

BRI1-associated kinase 1 (BAK1) is a member of the plant receptor-like kinase (RLK) superfamily. BAK1 has been shown to initiate brassinosteroid (BR) signalling and innate immune responses in plants by forming receptor complexes with both brassinosteroid-insensitive 1 (BRI1) and flagellin-sensing 2 (FLS2). To gain a better understanding of the structural details and the mechanism of action of the BAK1 kinase domain, recombinant BAK1 cytoplasmic domain has been expressed, purified and crystallized at 291 K using PEG 3350 as a precipitant. A 2.6 Å resolution data set was collected from a single flash-cooled crystal at 100 K. This crystal belonged to space group C2, with unit-cell parameters a = 70.3, b = 75.6, c = 71.9 Å, ß = 93.1°. Assuming the presence of one molecule in the asymmetric unit, the Matthews coefficient was 2.6 Å(3) Da(-1).

Purification, crystallization and preliminary X-ray crystallographic analysis of Arabidopsis thaliana dynamin-related protein 1A GTPase-GED fusion protein.

Plant-specific dynamin-related proteins play crucial roles in cell-plate formation, endocytosis or exocytosis, protein sorting to the vacuole and plasma membrane and the division of mitochondria and chloroplasts. In order to determine the crystal structure and thus to obtain a better understanding of the biological functions and mechanisms of dynamin-related proteins in plant cells, the GTPase domain of Arabidopsis thaliana dynamin-related protein 1A (AtDRP1A) fused to its GTPase effector domain (GED) was crystallized in a nucleotide-associated form using polyethylene glycol 3350 as precipitant. The hexagonal crystals (space group P6(1)) had unit-cell parameters a = b = 146.2, c = 204.3 Å, and diffraction data were collected to 3.6 Å resolution using synchrotron radiation. Four molecules, comprising two functional dimers, are assumed per asymmetric unit, corresponding to a Matthews coefficient of 3.9 Å(3) Da(-1) according to the molecular weight of 39 kDa.