Two distinct binding affinities of poliovirus for its cellular receptor

To study the kinetics and equilibrium of poliovirus binding to the poliovirus receptor, we used surface plasmon resonance to examine the interaction of a soluble form of the receptor with poliovirus. Soluble receptor purified from mammalian cells is able to bind poliovirus, neutralize viral infectivity, and induce structural changes in the virus particle. Binding studies revealed that there are two binding sites for the receptor on the poliovirus type 1 capsid, with affinity constants at 20 degrees C of K(D)(1) = 0.67 microm and K(D)(2) = 0.11 microm. The relative abundance of the two binding sites varies with temperature. At 20 degrees C, the K(D)(2) site constitutes approximately 46% of the total binding sites on the sensor chip, and its relative abundance decreased with decreasing temperature such that at 5 degrees C, the relative abundance of the K(D)(2) site is only 12% of the total binding sites. Absolute levels of the K(D)(1) site remained relatively constant at all temperatures tested. The two binding sites may correspond to docking sites for domain 1 of the receptor on the viral capsid, as predicted by a model of the poliovirus-receptor complex. Alternatively, the binding sites may be a consequence of structural breathing, or could result from receptor-induced conformational changes in the virus.     

Analysis of poliovirus protein 3A interactions with viral and cellular proteins in infected cells

Poliovirus proteins 3A and 3AB are small, membrane-binding proteins that play multiple roles in viral RNA replication complex formation and function. In the infected cell, these proteins associate with other viral and cellular proteins as part of a supramolecular complex whose structure and composition are unknown. We isolated viable viruses with three different epitope tags (FLAG, hemagglutinin [HA], and c-myc) inserted into the N-terminal region of protein 3A. These viruses exhibited growth properties and characteristics very similar to those of the wild-type, untagged virus. Extracts prepared from the infected cells were subjected to immunoaffinity purification of the tagged proteins by adsorption to commercial antibody-linked beads and examined after elution for cellular and other viral proteins that remained bound to 3A sequences during purification. Viral proteins 2C, 2BC, 3D, and 3CD were detected in all three immunopurified 3A samples. Among the cellular proteins previously reported to interact with 3A either directly or indirectly, neither LIS1 nor phosphoinositol-4 kinase (PI4K) were detected in any of the purified tagged 3A samples. However, the guanine nucleotide exchange factor GBF1, which is a key regulator of membrane trafficking in the cellular protein secretory pathway and which has been shown previously to bind enteroviral protein 3A and to be required for viral RNA replication, was readily recovered along with immunoaffinity-purified 3A-FLAG. Surprisingly, we failed to cocapture GBF1 with 3A-HA or 3A-myc proteins. A model for variable binding of these 3A mutant proteins to GBF1 based on amino acid sequence motifs and the resulting practical and functional consequences thereof are discussed.     

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.     

The receptor function of galactosyltransferase during cellular interactions

Summary The molecular mechanisms that underly cellular interactions during development are still poorly understood. There is reason to believe that complex glycoconjugates participate in cellular interactions by binding to specific cell surface receptors. One class of carbohydrate binding proteins that could serve as receptors during cellular interactions are the glycosyltransferases. Glycosyltransferases have been detected on a variety of cell surfaces, and evidence suggests that they may participate during cellular interactions by binding their specific carbohydrate substrates on adjacent cells or in extracellular matrix (see Refs. 1–4 for review).This review will focus on the receptor function of galactosyltransferase, in particular, during fertilization, embryonic cell adhesion and migration, limb bud morphogenesis, immune recognition and growth control. In many of these systems, the galactosyltransferase substrate has been characterized as a novel, large molecular weight glycoconjugate composed of repeating N-acetyllactosamine residues. The function of surface galactosyl-transferase during cellular interactions has been examined with genetic and biochemical probes, including the T/t-complex morphogenetic mutants, enzyme inhibitors, enzyme modifiers, and competitive substrates. Collectively, these studies suggest that in the mouse, surface galactosyltransferase is under the genetic control of the T/t-complex, and participates in multiple cellular interactions during development by binding to its specific lactosaminoglycan substrate.     

Human poliovirus receptor gene expression and poliovirus tissue tropism in transgenic mice.

Expression of the human poliovirus receptor (PVR) in transgenic mice results in susceptibility to poliovirus infection. In the primate host, poliovirus infection is characterized by restricted tissue tropism. To determine the pattern of poliovirus tissue tropism in PVR transgenic mice, PVR gene expression and susceptibility to poliovirus infection were examined by in situ hybridization. PVR RNA is expressed in transgenic mice at high levels in neurons of the central and peripheral nervous system, developing T lymphocytes in the thymus, epithelial cells of Bowman's capsule and tubules in the kidney, alveolar cells in the lung, and endocrine cells in the adrenal cortex, and it is expressed at low levels in intestine, spleen, and skeletal muscle. After infection, poliovirus replication was detected only in neurons of the brain and spinal cord and in skeletal muscle. These results demonstrated that poliovirus tissue tropism is not governed solely by expression of the PVR gene nor by accessibility of cells to virus. Although transgenic mouse kidney tissue expressed poliovirus binding sites and was not a site of poliovirus replication, when cultivated in vitro, kidney cells developed susceptibility to infection. Identification of the changes in cultured kidney cells that permit poliovirus infection may provide information on the mechanism of poliovirus tissue tropism.     

Predictive bioinformatic identification of minor receptor group human rhinoviruses

Major group HRVs bind intercellular adhesion molecule 1 and minor group HRVs bind members of the low-density lipoprotein receptor (LDLR) family for cell entry. Whereas the former share common sequence motives in their viral capsid proteins (VPs), in the latter only a lysine residue within the binding epitope in VP1 is conserved; this lysine is also present in “K-type” major group HRVs that fail to use LDLR for infection. By using the available sequences three-dimensional models of VP1 of all HRVs were built and binding energies, with respect to module 3 of the very-low-density lipoprotein receptor, were calculated. Based on the predicted affinities K-type HRVs and minor group HRVs were correctly classified.     

Electromagnetic cellular interactions

Chemical and electrical interaction within and between cells is well established. Just the opposite is true about cellular interactions via other physical fields. The most probable candidate for an other form of cellular interaction is the electromagnetic field. We review theories and experiments on how cells can generate and detect electromagnetic fields generally, and if the cell-generated electromagnetic field can mediate cellular interactions. We do not limit here ourselves to specialized electro-excitable cells. Rather we describe physical processes that are of a more general nature and probably present in almost every type of living cell. The spectral range included is broad; from kHz to the visible part of the electromagnetic spectrum. We show that there is a rather large number of theories on how cells can generate and detect electromagnetic fields and discuss experimental evidence on electromagnetic cellular interactions in the modern scientific literature. Although small, it is continuously accumulating.     

Distinct cellular functions of MK2

Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2) is activated upon stress by p38 MAPK alpha and -beta, which bind to a basic docking motif in the C terminus of MK2 and which subsequently phosphorylate its regulatory sites. As a result of activation MK2 is exported from the nucleus to the cytoplasm and cotransports active p38 MAPK to this compartment. Here we show that the amount of p38 MAPK is significantly reduced in cells and tissues lacking MK2, indicating a stabilizing effect of MK2 for p38. Using a murine knockout model, we have previously shown that elimination of MK2 leads to a dramatic reduction of tumor necrosis factor (TNF) production in response to lipopolysaccharide. To further elucidate the role of MK2 in p38 MAPK stabilization and in TNF biosynthesis, we analyzed the ability of two MK2 isoforms and several MK2 mutants to restore both p38 MAPK protein levels and TNF biosynthesis in macrophages. We show that MK2 stabilizes p38 MAPK through its C terminus and that MK2 catalytic activity does not contribute to this stabilization. Importantly, we demonstrate that stabilizing p38 MAPK does not restore TNF biosynthesis. TNF biosynthesis is only restored with MK2 catalytic activity. We further show that, in MK2-deficient macrophages, formation of filopodia in response to extracellular stimuli is reduced. In addition, migration of MK2-deficient mouse embryonic fibroblasts (MEFs) and smooth muscle cells on fibronectin is dramatically reduced. Interestingly, reintroducing catalytic MK2 activity into MEFs alone is not sufficient to revert the migratory phenotype of these cells. In addition to catalytic activity, the proline-rich N-terminal region is necessary for rescuing the migratory phenotype. These data indicate that catalytic activity of MK2 is required for both cytokine production and cell migration. However, the proline-rich MK2 N terminus provides a distinct role restricted to cell migration.     

Sequence and structure of human rhinoviruses reveal the basis of receptor discrimination

The sequences of the capsid protein VP1 of all minor receptor group human rhinoviruses were determined. A phylogenetic analysis revealed that minor group HRVs were not more related to each other than to the nine major group HRVs whose sequences are known. Examination of the surface exposed amino acid residues of HRV1A and HRV2, whose X-ray structures are available, and that of three-dimensional models computed for the remaining eight minor group HRVs indicated a pattern of positively charged residues within the region, which, in HRV2, was shown to be the binding site of the very-low-density lipoprotein (VLDL) receptor. A lysine in the HI loop of VP1 (K224 in HRV2) is strictly conserved within the minor group. It lies in the middle of the footprint of a single repeat of the VLDL receptor on HRV2. Major group virus serotypes exhibit mostly negative charges at the corresponding positions and do not bind the negatively charged VLDL receptor, presumably because of charge repulsion.     

Distinct monoubiquitin signals in receptor endocytosis

Numerous cellular proteins are post-translationally modified by the addition of the small modifier protein ubiquitin (Ub). The functional consequences of the type of ubiquitination vary, such that polyubiquitinated proteins are targeted for degradation by the proteasome, whereas monoubiquitination is implicated in other cellular functions, including endocytic trafficking and DNA repair. The monoubiquitination of trafficking cargoes, such as receptors and associated proteins, as well as of endocytic accessory Ub-binding proteins, raises the question of the precise function of monoubiquitin signals in the endocytic route. Recent biochemical and genetic evidence shows that multiple monoubiquitination of epidermal growth factor and platelet-derived growth factor receptors provides trafficking and sorting tags that ensure receptor endocytosis and degradation, whereas monoubiquitination of accessory proteins might play a role in regulating their function as Ub-receptors in the endosome.     

The toll-like receptor 3-mediated antiviral response is important for protection against poliovirus infection in poliovirus receptor transgenic mice

RIG-I-like receptors and Toll-like receptors (TLRs) play important roles in the recognition of viral infections. However, how these molecules contribute to the defense against poliovirus (PV) infection remains unclear. We characterized the roles of these sensors in PV infection in transgenic mice expressing the PV receptor. We observed that alpha/beta interferon (IFN-α/β) production in response to PV infection occurred in an MDA5-dependent but RIG-I-independent manner in primary cultured kidney cells in vitro. These results suggest that, similar to the RNA of other picornaviruses, PV RNA is recognized by MDA5. However, serum IFN-α levels, the viral load in nonneural tissues, and mortality rates did not differ significantly between MDA5-deficient mice and wild-type mice. In contrast, we observed that serum IFN production was abrogated and that the viral load in nonneural tissues and mortality rates were both markedly higher in TIR domain-containing adaptor-inducing IFN-β (TRIF)-deficient and TLR3-deficient mice than in wild-type mice. The mortality rate of MyD88-deficient mice was slightly higher than that of wild-type mice. These results suggest that multiple pathways are involved in the antiviral response in mice and that the TLR3-TRIF-mediated signaling pathway plays an essential role in the antiviral response against PV infection.     

Modification of cellular autophagy protein LC3 by poliovirus

Poliovirus infection remodels intracellular membranes, creating a large number of membranous vesicles on which viral RNA replication occurs. Poliovirus-induced vesicles display hallmarks of cellular autophagosomes, including delimiting double membranes surrounding the cytosolic lumen, acquisition of the endosomal marker LAMP-1, and recruitment of the 18-kDa host protein LC3. Autophagy results in the covalent lipidation of LC3, conferring the property of membrane association to this previously microtubule-associated protein and providing a biochemical marker for the induction of autophagy. Here, we report that a similar modification of LC3 occurs both during poliovirus infection and following expression of a single viral protein, a stable precursor termed 2BC. Therefore, one of the early steps in cellular autophagy, LC3 modification, can be genetically separated from the induction of double-membraned vesicles that contain the modified LC3, which requires both viral proteins 2BC and 3A. The existence of viral inducers that promote a distinct aspect of the formation of autophagosome-like membranes both facilitates the dissection of this cellular process and supports the hypothesis that this branch of the innate immune response is directly subverted by poliovirus.     

Distinct cellular expression pattern of annexins in Hydra vulgaris

The annexins are a structurally related family of Ca2+ and phospholipid binding proteins whose function has not been clearly defined. Further investigations of annexin function may be enhanced by studying simpler organisms that express fewer annexin gene products. We previously characterized annexin XII from the freshwater cnidarian Hydra vulgaris (Schlaepfer, D. D., D. A. Fisher, M. E. Brandt, H. R. Bode, J. Jones, and H. T. Haigler. 1992. J. Biol. Chem. 267:9529-9539). In this report, we detected one other hydra annexin (40 kD) by screening hydra cell extracts with antibodies raised against peptides from highly conserved regions of known annexins. The 40-kD protein was expressed at less than 1% of annexin XII levels. These biochemical studies indicate that hydra contain a very limited number of annexin gene products. The cellular hydra annexin distribution was analyzed by indirect immunofluorescence. Using affinity-purified antibodies to annexin XII, the epithelial battery cells were stained throughout the tentacle. A lower level of annexin XII staining was detected in peduncle region epithelial cells. No other cell types showed detectable annexin XII staining. The anti-peptide antibody that specifically detected the 40-kD hydra annexin, maximally stained the cytoplasm of nematocytes. The immunofluorescent results showed that annexin XII and the 40-kD annexin were not co-expressed in the same cells. Since the hydra annexins localized to specific subsets of the total hydra cell types, it is likely that these proteins perform specialized biological roles, and not general "housekeeping" functions which are part of the essential molecular machinery of all cells.     

Stabilization of poliovirus polymerase by NTP binding and fingers-thumb interactions

The viral RNA-dependent RNA polymerases show a conserved structure where the fingers domain interacts with the top of the thumb domain to create a tunnel through which nucleotide triphosphates reach the active site. We have solved the crystal structures of poliovirus polymerase (3D^pol) in complex with all four NTPs, showing that they all bind in a common pre-insertion site where the phosphate groups are not yet positioned over the active site. The NTPs interact with both the fingers and palm domains, forming bridging interactions that explain the increased thermal stability of 3D^pol in the presence of NTPs. We have also examined the importance of the fingers-thumb domain interaction for the function and structural stability of 3D^pol. Results from thermal denaturation experiments using circular dichroism and 2-anilino-6-napthaline-sulfonate (ANS) fluorescence show that 3D^pol has a melting temperature of only ∼ 40 °C. NTP binding stabilizes the protein and increases the melting by 5-6 °C while mutations in the fingers-thumb domain interface destabilize the protein and reduce the melting point by as much as 6 °C. In particular, the burial of Phe30 and Phe34 from the tip of the index finger into a pocket at the top of the thumb and the presence of Trp403 on the thumb domain are key interactions required to maintain the structural integrity of the polymerase. The data suggest the fingers domain has significant conformational flexibility and exists in a highly dynamic molten globule state at physiological temperature. The role of the enclosed active site motif as a structural scaffold for constraining the fingers domain and accommodating conformational changes in 3D^pol and other viral polymerases during the catalytic cycle is discussed.