Statement on the consideration of biodefence and biosecurity.
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Bioterrorism↗
Biomedical subjects
Eckard Wimmer
Publications and source records attributed to Eckard Wimmer.
Uncensored exchange of scientific results.
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Information Services↗
Analysis of the cloverleaf element in a human rhinovirus type 14/poliovirus chimera: correlation of subdomain D structure, ternary protein complex formation and virus replication.
RNA genomes of enteroviruses and rhinoviruses contain a 5'-terminal structure, the cloverleaf (CL), which serves as signal in RNA synthesis. Substitution of the poliovirus [PV1(M)] CL with that of human rhinovirus type 2 (HRV2) was shown previously to produce a viable chimeric PV, whereas substitution with the HRV14 CL produced a null phenotype. Fittingly, the HRV14 CL failed to form a complex with PV-specific proteins 3CD(pro)-3AB or 3CD(pro)-PCBP2, considered essential for RNA synthesis. It was reported previously (Rohll et al., J Virol 68, 4384-4391, 1994) that the major determinant for the null phenotype of a PV/HRV14 chimera resides in subdomain Id of the HRV14 CL. Using a chimeric PV/HRV14 CL in the context of the PV genome, stem-loop Id of HRV14 CL was genetically dissected. It contains the sequence C(57)UAU(60)-G, the underlined nucleotides forming the loop that is shorter by 1 nt when compared to the corresponding PV structure (UUGC(60)GG). Insertion of a G nucleotide to form a tetra loop (C(57)UAU(60)GG(61)) did not rescue replication of the chimera. However, an additional mutation at position 60 (C(57)UAC(60)GG(61)) yielded a replicating genome. Only the mutant PV/HRV14 CL with the UAC(60)G tetra loop formed ternary complexes efficiently with either PV proteins 3CD(pro)-3AB or 3CD(pro)-PCBP2. Thus, in the context of PV RNA synthesis, the presence of a tetra loop in subdomain D of the CL per se is not sufficient for function. The sequence and, consequently, the structure of the tetra loop plays an essential role. Biochemical assays demonstrated that the function of the CL element and the function of the cis-acting replication element in the 3D(pol)-3CD(pro)-dependent uridylylation of VPg are not linked.
Base Sequence↗
Complexes of poliovirus serotypes with their common cellular receptor, CD155.
Structures of all three poliovirus (PV) serotypes (PV1, PV2, and PV3) complexed with their cellular receptor, PV receptor (PVR or CD155), were determined by cryoelectron microscopy. Both glycosylated and fully deglycosylated CD155 exhibited similar binding sites and orientations in the viral canyon for all three PV serotypes, showing that all three serotypes use a common mechanism for cell entry. Difference maps between the glycosylated and deglycosylated CD155 complexes determined the sites of the carbohydrate moieties that, in turn, helped to verify the position of the receptor relative to the viral surface. The proximity of the CD155 carbohydrate site at Asn105 to the viral surface in the receptor-virus complex suggests that it might interfere with receptor docking, an observation consistent with the properties of mutant CD155. The footprints of CD155 on PV surfaces indicate that the south rim of the canyon dominates the virus-receptor interactions and may correspond to the initial CD155 binding state of the receptor-mediated viral uncoating. In contrast, the interaction of CD155 with the north rim of the canyon, especially the region immediately outside the viral hydrophobic pocket that normally binds a cellular "pocket factor," may be critical for the release of the pocket factor, decreasing the virus stability and hence initiating uncoating. The large area of the CD155 footprint on the PV surface, in comparison with other picornavirus-receptor interactions, could be a potential limitation on the viability of PV escape mutants from antibody neutralization. Many of these are likely to have lost their ability to bind CD155, resulting in there being only three PV serotypes.
Amino Acid Sequence↗
Functional dissection of a poliovirus cis-acting replication element [PV-cre(2C)]: analysis of single- and dual-cre viral genomes and proteins that bind specifically to PV-cre RNA.
The role of the cis replication element (cre) in the 2C(ATPase) coding region of the poliovirus (PV) genome has been studied with a series of mutants derived from either a PV1 full-length genome or a replicon (P/L) containing the firefly luciferase reporter gene in place of the capsid region. Using the P/L replicon we have inserted cre elements at three different locations in the genome including the 5' nontranslated region and within the open reading frame. The successful recovery of replication of a nonviable P/L (A(5)C) mutant replicon with an artificial cre element as "rescuer," in addition to the results of site-directed mutagenesis and experiments with truncated forms of PV-cre(2C), indicated that (i) the sequence within the upper stem and loop regions contains the minimal cre RNA required for VPg uridylylation in vitro, (ii) the location of the cre RNA in the poliovirus genome is not relevant to RNA infectivity, and (iii) specific binding of 3CD(pro) to PV-cre(2C) occurs within the upper stem region and probably involves several contact residues. The role of a 14-nucleotide conserved "core" sequence among known cre structures in picornaviruses was examined by site-directed mutagenesis of individual nucleotides. In addition to a conserved AAA (4472 to 4474) triplet previously shown to be the primary RNA template for VPg uridylylation by the PV RNA polymerase 3D(pol) (E. Rieder, A. V. Paul, D. W. Kim, J. H. van Boom, and E. Wimmer, J. Virol. 74:10371-10380, 2000), we have now shown that important residues (G(4468) and A(4481)) are contained in a predicted internal bulge at the upper stem-loop of PV-cre(2C). We have further demonstrated that the viral proteins 3CD(pro) and 3C(pro) form stable complexes with a transcript PV-cre(2C) RNA that can be considered critical for VPg uridylylation.
Base Sequence↗
Vaccines should be kept even if polio is wiped out.
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Humans↗
Immunofluorescence analysis of poliovirus receptor expression in Peyer's patches of humans, primates, and CD155 transgenic mice: implications for poliovirus infection.
Oral transmission of poliovirus is restricted to humans and certain primate species. The expression of the human poliovirus receptor (CD155) within gastrointestinal-associated lymphoid tissues from species that are susceptible (human) or resistant (rhesus macaque and CD155 transgenic [Tg] mice) to oral poliovirus infection was examined. Sensitivity to oral infection correlated with CD155 expression not only in the intestinal epithelium, including the follicle-associated epithelium (FAE) and microfold (M) cells of Peyer's patches, but also in germinal centers within the Peyer's patches. CD155 expression in rhesus macaques was reduced in FAE and, significantly, absent in germinal centers. In CD155 Tg mice, CD155 expression was barely observable in the intestinal epithelium, absent in germinal centers, but prominent in the tunica muscularis. This suggests that productive poliovirus infection of the gut is dependent on the expression of CD155 within the FAE, including the M cells, and on cells within Peyer's patches, most likely within germinal centers.
Animals↗
Chemical synthesis of poliovirus cDNA: generation of infectious virus in the absence of natural template.
Full-length poliovirus complementary DNA (cDNA) was synthesized by assembling oligonucleotides of plus and minus strand polarity. The synthetic poliovirus cDNA was transcribed by RNA polymerase into viral RNA, which translated and replicated in a cell-free extract, resulting in the de novo synthesis of infectious poliovirus. Experiments in tissue culture using neutralizing antibodies and CD155 receptor-specific antibodies and neurovirulence tests in CD155 transgenic mice confirmed that the synthetic virus had biochemical and pathogenic characteristics of poliovirus. Our results show that it is possible to synthesize an infectious agent by in vitro chemical-biochemical means solely by following instructions from a written sequence.
Animals↗
Nectin couples cell-cell adhesion and the actin scaffold at heterotypic testicular junctions.
Actin-based cell-cell adherens junctions (AJs) are crucial not only for mechanical adhesion but also for cell morphogenesis and differentiation. While organization of homotypic AJs is attributed mostly to classic cadherins, the adhesive mechanism of heterotypic AJs in more complex tissues remains to be clarified. Nectin, a member of a family of immunoglobulin-like adhesion molecules at various AJs, is a possible organizer of heterotypic AJs because of its unique heterophilic trans-interaction property. Recently, nectin-2 (-/-) mice have been shown to exhibit the defective sperm morphogenesis and the male-specific infertility, but the role of nectin in testicular AJs has not been investigated. We show here the heterotypic trans-interaction between nectin-2 in Sertoli cells and nectin-3 in spermatids at Sertoli-spermatid junctions (SspJs), heterotypic AJs in testes. Moreover, each nectin-based adhesive membrane domain exhibits one-to-one colocalization with each actin bundle underlying SspJs. Inactivation of the mouse nectin-2 gene causes not only impaired adhesion but also loss of the junctional actin scaffold at SspJs, resulting in aberrant morphogenesis and positioning of spermatids. Localization of afadin, an adaptor protein of nectin with the actin cytoskeleton, is also nectin-2 dependent at SspJs. These results indicate that the nectin-afadin system plays essential roles in coupling cell-cell adhesion and the cortical actin scaffold at SspJs and in subsequent sperm morphogenesis.
Actins↗
Expression of the human poliovirus receptor/CD155 gene is activated by sonic hedgehog.
The human poliovirus receptor/CD155 is a transmembrane glycoprotein belonging to the immunoglobulin superfamily. The ectodomain of CD155 mediates cell attachment to the extracellular matrix molecule vitronectin, while its intracellular domain interacts with the dynein light chain Tctex-1. CD155 is a primate-restricted gene that is expressed during development in mesenchymal tissues and ventrally derived structures within the CNS. Its function in adults is as yet unknown, but significantly, CD155 is aberrantly expressed in neuroectodermal tumors. We show that the expression of CD155 mRNA is up-regulated when human Ntera2 cells are treated with purified Sonic hedgehog (Shh) protein. Reporter gene expression driven by the CD155 core promoter is activated by Shh in transient co-transfection assays. Analysis of the CD155 core promoter indicates that an intact GLI binding site is required for Shh activation. In addition, overexpression of Gli1 or Gli3 potently activates reporter gene expression driven by the CD155 core promoter. These data identify the CD155 gene as a transcriptional target of Shh, a finding that has significance for the normal function of CD155 during development and the expression of CD155 in neuroectodermal tumors.
Animals↗
Sequence requirements for viral RNA replication and VPg uridylylation directed by the internal cis-acting replication element (cre) of human rhinovirus type 14.
Until recently, the cis-acting signals required for replication of picornaviral RNAs were believed to be restricted to the 5' and 3' noncoding regions of the genome. However, an RNA stem-loop in the VP1-coding sequence of human rhinovirus type 14 (HRV-14) is essential for viral minus-strand RNA synthesis (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998). The nucleotide sequence of the apical loop of this internal cis-acting replication element (cre) was critical for RNA synthesis, while secondary RNA structure, but not primary sequence, was shown to be important within the duplex stem. Similar cres have since been identified in other picornaviral genomes. These RNA segments appear to serve as template for the uridylylation of the genome-linked protein, VPg, providing the VPg-pUpU primer required for viral RNA transcription (A. V. Paul et al., J. Virol. 74:10359-10370, 2000). Here, we show that the minimal functional HRV-14 cre resides within a 33-nucleotide (nt) RNA segment that is predicted to form a simple stem-loop with a 14-nt loop sequence. An extensive mutational analysis involving every possible base substitution at each position within the loop segment defined the sequence that is required within this loop for efficient replication of subgenomic HRV-14 replicon RNAs. These results indicate that three consecutive adenosine residues (nt 2367 to 2369) within the 5' half of this loop are critically important for cre function and suggest that a common RNNNAARNNNNNNR loop motif exists among the cre sequences of enteroviruses and rhinoviruses. We found a direct, positive correlation between the capacity of mutated cres to support RNA replication and their ability to function as template in an in vitro VPg uridylylation reaction, suggesting that these functions are intimately linked. These data thus define more precisely the sequence and structural requirements of the HRV-14 cre and provide additional support for a model in which the role of the cre in RNA replication is to act as template for VPg uridylylation.
Base Sequence↗
Synchrotron radiation-induced X-ray emission to identify metal ions in preparations of purified protein.
The suitability of synchrotron radiation-induced X-ray emission (SRIXE) for the detection and identification of metal ions in preparations of purified, soluble proteins was tested. Glutathione S-transferase fused to the proximal zinc finger motif of human transcription factor IIIA or to the cysteine-rich motif of poliovirus protein 2C was expressed in bacteria and purified by affinity chromatography. Aqueous samples containing the purified proteins were analyzed with SRIXE, and trace amounts of zinc and iron were detected. Mutation of the zinc-coordinating residues in the cysteine-rich motif of poliovirus protein 2C resulted in the loss of the zinc-binding ability. Relative quantities of metal in the protein preparations as determined by SRIXE corresponded well with the metal:protein ratios calculated by using a 4-(2-pyridylazo)resorcinol-based assay. We conclude that SRIXE is an accurate, sensitive, and simple method for the detection and identification of protein-bound metal ions in small amounts of sample. Thus, SRIXE may have wide use as a particularly effective method for rapidly determining trace metals in microarray samples.
Amino Acid Sequence↗
Interaction of the poliovirus receptor CD155 with the dynein light chain Tctex-1 and its implication for poliovirus pathogenesis.
The cellular receptor for poliovirus CD155 (or PVR) is the founding member of a new class of membrane-associated immunoglobulin-like proteins, which include the mouse tumor-associated antigen E4 (Tage4) and three proteins termed "nectins." Using a yeast two-hybrid screen we have discovered that the cytoplasmic domain of CD155 associates strongly and specifically with Tctex-1, a light chain of the dynein motor complex, the latter representing the major driving force for retrograde transport of endocytic vesicles and membranous organelles. We confirmed the interaction biochemically and by co-immunoprecipitation, and we mapped the Tctex-1 binding site to a SKCSR motif within the juxtamembrane region of CD155. Tctex-1 immunoreactivity was detected in mouse sciatic nerve and spinal cord (two tissues of central importance for poliovirus pathogenesis) in punctate, possibly vesicular, patterns. We propose that the cytoplasmic domain may target CD155-containing endocytic vesicles to the microtubular network. Neurotropic viruses like poliovirus, herpesvirus, rabies virus, and pseudorabies virus all utilize neuronal retrograde transport to invade the central nervous system. Association with Tctex-1 and, hence, with the dynein motor complex may offer an explanation for how poliovirus hijacks the cellular transport machinery to retrogradely ascend along the axon to the neuronal cell body.
Amino Acid Motifs↗
The C-terminal residues of poliovirus proteinase 2A(pro) are critical for viral RNA replication but not for cis- or trans-proteolytic cleavage.
Poliovirus proteinase 2A(pro) is an essential enzyme involved in cleavages of viral and cellular proteins during the infectious cycle. Evidence has been obtained that 2A(pro) is also involved in genome replication. All enteroviruses have a negatively charged cluster of amino acids at their C terminus (E(E)/(D)(E)/(D)AMEQ-NH(2)), a common motif suggesting function. When aligned with enterovirus sequences, the 2A(pro) proteinase of human rhinovirus type 2 (HRV2) has a shorter C terminus (EE.Q:-NH(2)) and, indeed, the HRV2 2A(pro) cannot substitute for poliovirus 2A(pro) to yield a viable chimeric virus. Here evidence is provided that the C-terminal cluster of amino acids plays an unknown role in poliovirus genome replication. Deletion of the EEAME sequence from poliovirus 2A(pro) is lethal without significantly influencing proteinase function. On the other hand, addition of EAME to HRV2 2A(pro), yielding a C terminus of this enzyme of EEEAMEQ:, stimulated RNA replication of a poliovirus/HRV2 chimera 100-fold. The novel role of the C-terminal sequence motif is manifested at the level of protein function, since silent mutations in its coding region had no effect on virus proliferation. Poliovirus type 1 Mahoney 2A(pro) could be provided in trans to rescue the lethal deletion EEAME in the poliovirus variant. Encapsidation studies left open the question of whether the C terminus of poliovirus 2A(pro) is involved in particle formation. It is concluded that the C terminus of poliovirus 2A(pro) is an essential domain for viral RNA replication but is not essential for proteolytic processing.
Amino Acid Motifs↗
Cell-free synthesis of poliovirus: 14S subunits are the key intermediates in the encapsidation of poliovirus RNA.
In a cell-free system of uninfected HeLa cells, programmed with poliovirus RNA, extraneous radiolabelled 14S subunits assembled with endogenous 14S subunits and interacted with newly synthesized RNA to form virions (160S). This result suggests that 14S subunits are the key intermediates in the encapsidation of poliovirus RNA.
Cell-Free System↗