All-atom molecular dynamics calculation study of entire poliovirus empty capsids in solution.

Small viruses that belong, for example, to the Picornaviridae, such as poliovirus and foot-and-mouth disease virus, consist simply of capsid proteins and a single-stranded RNA (ssRNA) genome. The capsids are quite stable in solution to protect the genome from the environment. Here, based on long-time and large-scale 6.5 × 10(6) all-atom molecular dynamics calculations for the Mahoney strain of poliovirus, we show microscopic properties of the viral capsids at a molecular level. First, we found equilibrium rapid exchange of water molecules across the capsid. The exchange rate is so high that all water molecules inside the capsid (about 200,000) can leave the capsid and be replaced by water molecules from the outside in about 25 µs. This explains the capsid's tolerance to high pressures and deactivation by exsiccation. In contrast, the capsid did not exchange ions, at least within the present simulation time of 200 ns. This implies that the capsid can function, in principle, as a semipermeable membrane. We also found that, similar to the xylem of trees, the pressure of the solution inside the capsid without the genome was negative. This is caused by coulombic interaction of the solution inside the capsid with the capsid excess charges. The negative pressure may be compensated by positive osmotic pressure by the solution-soluble ssRNA and the counter ions introduced into it.

Research Note: Expression of IGF-1 and IGF-1 receptor proteins in skeletal muscle fiber types in chickens with hepatic fibrosis.

We investigated the expression of insulin-like growth factor 1 (IGF-1) and IGF-1 type 1 receptor (IGF-1R) in skeletal muscle fiber types in chickens with hepatic fibrosis induced by bile duct ligation (BDL). Eleven hens, approximately 104 weeks old, were randomly assigned to BDL (n = 4) and sham surgery (SHAM; n = 7) groups. In BDL hens, histopathology revealed marked bile duct proliferation and liver fibrosis. The cross-sectional area (CSA) of myofibers from both the pectoralis (PCT) muscles significantly decreased in the BDL group compared with the SHAM group (P < 0.01). In contrast, the CSA of myofibers from the femorotibialis lateralis (FTL) muscle did not decrease in the BDL group. Type I fibers were large, round, and hypertrophic. Elongated type IIA and IIB fibers were also present. For IGF-1 immunostaining, the immunoreaction intensity was higher in the PCT in the BDL group than the SHAM group. Within the BDL group, type I fibers from FTL had a stronger immunoreaction intensity than the type II fibers. For IGF-1R immunostaining, the intensity of the immunoreactions was similar within the PCT in the BDL group compared with the SHAM group. For FTL, type I fibers had stronger reactions to IGF-1R than type II fibers in the BDL group. These results suggest that type I fibers express both IGF-1 and IGF-1R and become hypertrophic in chickens with hepatic fibrosis.

Sarcopenic Dysphagia with Low Tongue Pressure Is Associated with Worsening of Swallowing, Nutritional Status, and Activities of Daily Living.

OBJECTIVES: According to the recently proposed diagnostic criteria for sarcopenic dysphagia, sarcopenic dysphagia can be classified as probable or possible based on tongue pressure. However, it is unclear whether patients with probable and possible sarcopenic dysphagia have different characteristics. Therefore, this study aimed to investigate whether patients with possible and probable sarcopenic dysphagia have different clinical characteristics. DESIGN: A cross-sectional study. SETTING: A rehabilitation hospital. PARTICIPANTS: In total, 129 patients aged ≥65 years with sarcopenic dysphagia were included. METHODS: A tongue pressure of <20 kPa was indicative of probable sarcopenic dysphagia, and a tongue pressure of ≥20 kPa was indicative of possible sarcopenic dysphagia. Kuchi-Kara Taberu (KT) index scores were compared between the probable or possible sarcopenic dysphagia groups. RESULTS: According to the tongue pressure, 76 and 53 patients were classified into the probable and possible sarcopenic dysphagia groups, respectively. In multiple linear regression analysis, the presence of probable sarcopenic dysphagia was independently associated with the total KT index score (standardized coefficient: -0.313, regression coefficient: -4.500, 95% confidence interval [CI], -6.920 to -2.080, P < 0.001). The presence of probable sarcopenic dysphagia was independently associated with some subitems of the KT index (willingness to eat, cognitive function while eating, oral preparatory and propulsive phase, severity of pharyngeal dysphagia, eating behavior, and daily living activities). CONCLUSIONS: Patients with probable sarcopenic dysphagia were characterized by poor overall eating-related conditions, especially poor swallowing ability, ability to perform activities of daily living, and nutritional status.

Poliovirus-specific CD4+ Th1 clones with both cytotoxic and helper activity mediate protective humoral immunity against a lethal poliovirus infection in transgenic mice expressing the human poliovirus receptor.

The current understanding of the function of CD4+ T helper (Th) cells in immunity to infectious diseases is that Th1 cells, which secrete interleukin (IL)-2 and interferon-gamma, induce cellular immune responses, whereas Th2 cells, which secrete IL-4, IL-5, IL-6, and IL-10, provide helper function for humoral immunity. We have used a panel of poliovirus-specific murine CD4+ T cell clones and mice transgenic for the human poliovirus receptor to evaluate the role of Th cell subpopulations in protective immunity to poliovirus. The majority of T cell clones, as well as polyclonal T cells generated from mice infected or immunized with poliovirus, secreted IL-2 and interferon-gamma, but not IL-4, IL-5, or IL-10, a profile typical of Th1 cells. The Th1 clones displayed major histocompatibility complex class II-restricted cytotoxic T lymphocyte activity against specific poliovirus peptide-pulsed target cells, but also provided help for antipoliovirus neutralizing antibody production. To examine the mechanism of immunity in vivo, we have used poliovirus receptor-transgenic mice on a BALB/c (H-2d) background. These animals developed a poliomyelitis-like disease when challenged intravenously with a virulent wild-type strain of poliovirus, but not with an attenuated vaccine strain. Furthermore, mice immunized with the vaccine strain were protected against a subsequent challenge with wild-type virus. Using an adoptive transfer technique, we demonstrated that it was possible to confer protection with primed B cells in the presence of polyclonal poliovirus-specific T cells, but not when transgenic mice received either B cells or T cells alone. Furthermore, protection was observed when mice received primed B cells in the presence of a VP4-specific Th1 clone. The findings demonstrate that Th1 cells can mediate a protective immune response against poliovirus infection in vivo through helper activity for humoral immunity and that CD4+ T cells, specific for the internal poliovirus capsid protein, VP4, can provide effective help for a protective antibody response directed against surface capsid proteins.

Nectin/PRR: an immunoglobulin-like cell adhesion molecule recruited to cadherin-based adherens junctions through interaction with Afadin, a PDZ domain-containing protein.

We have isolated a novel actin filament-binding protein, named afadin, localized at cadherin-based cell-cell adherens junctions (AJs) in various tissues and cell lines. Afadin has one PDZ domain, three proline-rich regions, and one actin filament-binding domain. We found here that afadin directly interacted with a family of the immunoglobulin superfamily, which was isolated originally as the poliovirus receptor-related protein (PRR) family consisting of PRR1 and -2, and has been identified recently to be the alphaherpes virus receptor. PRR has a COOH-terminal consensus motif to which the PDZ domain of afadin binds. PRR and afadin were colocalized at cadherin-based cell-cell AJs in various tissues and cell lines. In E-cadherin-expressing EL cells, PRR was recruited to cadherin-based cell-cell AJs through interaction with afadin. PRR showed Ca2+-independent cell-cell adhesion activity. These results indicate that PRR is a cell-cell adhesion molecule of the immunoglobulin superfamily which is recruited to cadherin-based cell-cell AJs through interaction with afadin. We rename PRR as nectin (taken from the Latin word "necto" meaning "to connect").

Antigenic variation and resistance to neutralization in poliovirus type 1.

Mutations have been identified in variants of poliovirus, type 1 (Mahoney) on the basis of their resistance to neutralization by individual monoclonal antibodies. The phenotypes of these variants were defined in terms of antibody binding; the pattern of epitopes expressed or able to be exploited for neutralization were complex. Single amino acid changes can have distant (in terms of linear sequence) and generalized effects on the antigenic structure of poliovirus and similarly constituted virions.

Comparative aspects on the role of polypyrimidine tract-binding protein in internal initiation of hepatitis C virus and picornavirus RNAs.

We compared the effects of polypyrimidine tract-binding protein (PTB) on hepatitis C virus (HCV genotype IIa), encephalomyocarditis virus (EMCV) and poliovirus internal ribosome entry site (IRES) activities in vitro. It bound strongly to EMCV IRES, but weakly to PV and HCV RNAs. PV IRES showed the strongest dependency to PTB and it showed less than one-tenth of IRES activity after the immuno-depletion of PTB from HeLa S10 lysate with pre-coated anti-PTB IgG beads, comparing to the normal IgG beads-treated S10 lysate. EMCV IRES activity was approximately 40% of that of normal control after PTB depletion. Especially, HCV IRES activity was approximately 95%, and most weekly affected by the depletion of PTB. Repletion of PTB to depleted S10 lysate restored activities of PV and EMCV IRESs. The data suggest that PTB plays an important role in picornaviral IRESs, but not in HCV IRES.

Integrating 3D Rotational Angiography into Gamma Knife Planning.

3D rotational angiography provides remarkable spatial resolution for cerebrovascular disorders; however, it cannot be integrated directly into gamma knife planning due to the discrepancy of DICOM "tag" information, and most physicians still cannot benefit from 3D rotational angiography. Here, we describe a simple and easy technique to enable the integration of 3D rotational angiography.

Regulation of the yeast Yap1p nuclear export signal is mediated by redox signal-induced reversible disulfide bond formation.

Yap1p, a crucial transcription factor in the oxidative stress response of Saccharomyces cerevisiae, is transported in and out of the nucleus under nonstress conditions. The nuclear export step is specifically inhibited by H(2)O(2) or the thiol oxidant diamide, resulting in Yap1p nuclear accumulation and induction of transcription of its target genes. Here we provide evidence for sensing of H(2)O(2) and diamide mediated by disulfide bond formation in the C-terminal cysteine-rich region (c-CRD), which contains 3 conserved cysteines and the nuclear export signal (NES). The H(2)O(2) or diamide-induced oxidation of the c-CRD in vivo correlates with induced Yap1p nuclear localization. Both were initiated within 1 min of application of oxidative stress, before the intracellular redox status of thioredoxin and glutathione was affected. The cysteine residues in the middle region of Yap1p (n-CRD) are required for prolonged nuclear localization of Yap1p in response to H(2)O(2) and are thus also required for maximum transcriptional activity. Using mass spectrometry analysis, the H(2)O(2)-induced oxidation of the c-CRD in vitro was detected as an intramolecular disulfide linkage between the first (Cys(598)) and second (Cys(620)) cysteine residues; this linkage could be reduced by thioredoxin. In contrast, diamide induced each pair of disulfide linkage in the c-CRD, but in this case the cysteine residues in the n-CRD appeared to be dispensable for the response. Our data provide evidence for molecular mechanisms of redox signal sensing through the thiol-disulfide redox cycle coupled with the thioredoxin system in the Yap1p NES.

Cross-family interaction between the bHLHZip USF and bZip Fra1 proteins results in down-regulation of AP1 activity.

Heterodimerization among the basic-leucine zipper (bZIP) proteins or among the basic-helix-loop-helix-leucine zipper (bHLHZip) proteins confers a multitude of combinational activities to these transcription factors. To further examine the function of the bHLHZip protein, USF, we screened for cellular proteins which could directly interact with USF using the yeast two-hybrid system. A bZip protein, Fra1, was found to efficiently interact with USF. USF specifically interacts with Fra1 but not with other closely related family members, c-Fos, Fra2, FosB, or with c-Jun. Both the bHLHZip and the N-terminal regions of Fra1 are required for efficient interaction with USF. In vivo association between USF and Fra1 has been demonstrated by co-immunoprecipitation. Expression of exogenous USF led to a decrease in AP1-dependent transcription in F9 cells. Co-expression of exogenous Fra1 restored the AP1 activity in a dose-dependent manner. These data show that USF and Fra1 physically and functionally interact demonstrating that cross-talk occurs between factors of distantly related transcription families.

Recent insights into poliovirus pathogenesis.

The development of a mouse model for poliomyelitis that is transgenic for the human poliovirus receptor (hPVR) has made it much easier to investigate the efficiency of the viral dissemination process in a whole organism. These studies have given an insight into the mechanisms of blood-brain barrier permeation and neural transport. Strain-specific neurovirulence levels, however, appear to depend mainly on the replicating capacity of the virus in the central nervous system rather than the dissemination efficiency. Studies of the poliovirus-induced cytopathic effects on neural cells and specific subcellular localization of hPVR isoforms might determine a new course of investigation of poliovirus pathogenesis.

Tissue tropism and species specificity of poliovirus infection.

Paralytic poliomyelitis is caused by the destruction of neurons in the central nervous system during the lytic replication of poliovirus. The human gene for the poliovirus receptor has been cloned recently, and transgenic mice generated that are susceptible to this virus. This has allowed the critical amino acids that determine the specificity of poliovirus to be identified.

The selective pathway and a high-density lipoprotein receptor (SR-BI) in ovarian granulosa cells of the mouse.

We recently reported that rat luteinized ovary tissue and primary cultures of rat ovarian granulosa cells reveal a remarkably tight functional correlation between expressed selective uptake of lipoprotein cholesteryl esters and the expression of an HDL receptor protein, scavenger receptor, class B, type I (SR-BI). In the current study, we examine these same processes in C57 mouse granulosa cells and report a different correlation. Unlike the rat cells, non-hormone stimulated mouse granulosa cells are able to effectively carry out their selective pathway functions and secrete HDL-derived progestins despite low levels of SR-BI and barely detectable levels of SR-BII (an isoform of SR-BI). Once stimulated with trophic hormones or Bt2cAMP, small (30-40%) increases are observed in selective pathway functions, but major (approximately 20-fold) increases are seen in SR-BI and SR-BII expression: thus, relatively little is gained in selective cholesteryl ester uptake by mouse granulosa cells even though SR-BI and SR-BII levels are greatly increased. The importance of the HDL receptor proteins to the selective pathway remains clear, however, since a significant portion of the selective process in both basal and stimulated granulosa cells is inhibitable by the use of blocking antibody. Another surface protein, caveolin, previously reported to co-localize with SR-BI in mouse cells shows no change in expression during periods when SR-BI/BII levels are undergoing major shifts.

Strong inclination toward transition mutation in nucleotide substitutions by poliovirus replicase.

A viable insertion mutant of the Sabin strain of type 1 poliovirus was constructed. The mutant carried an insertion sequence of 72 nucleotides at nucleotide position 702 in the 5' non-coding region (742 nucleotides long) of the genome of the Sabin strain. This mutant showed a small-plaque phenotype, as compared with the parental virus. Indeed, the final yield of the mutant in a single cycle of infection was tenfold fewer than that of the parental virus. Many large-plaque variants that are easily generated from the insertion mutant appeared to regain efficient viral replication and have single nucleotide changes. All nucleotide changes observed were limited to within three nucleotides of an AUG sequence in the insertion sequence. The result indicates strongly that the AUG sequence itself in this genome region functions in reducing the plaque size of the parental Sabin type 1 virus. The insertion mutant with a small-plaque phenotype may be the first in vitro mutant of poliovirus whose viability is lowered only by a primary sequence inserted into the 5' non-coding region of the genome. Base substitutions to alter the AUG sequence should largely be the result of errors of the virus-specific replicase, since variants with base substitutions must be subject to only minimum selection pressure. Accordingly, nucleotide sequence analysis of the genome region containing the AUG sequence was performed on a number of genomes of large-plaque variants to investigate types of nucleotide substitutions caused by characteristic errors in RNA replication. Only one transversion mutation was detected in the genomes of 44 independently isolated large-plaque variants with single base changes in the AUG sequence. This result suggests strongly that transition mutations occur predominantly as nucleotide substitutions caused by characteristic errors of poliovirus replicase.

Primary structure of poliovirus defective-interfering particle genomes and possible generation mechanisms of the particles.

The genomes of defective-interfering (DI) particles derived from the Sabin strain of type 1 poliovirus (PV1(Sab] were characterized by nuclease S1 mapping using complementary DNA (cDNA) copies of PV1(Sab) genome as probes. The results demonstrated variety in the size and location of the deletions, which were compatible with our previous prediction. The results further indicated that the locations of the deletions were limited within the internal genome region encoding viral capsid proteins and that the deletion sites were clustered in certain areas on the genome. Sequence analysis of a number of cloned cDNAs to the DI genomes revealed that every DI genome retained the correct reading frame for viral protein synthesis. These results strongly suggested that one or all of the viral non-structural proteins might be cis-acting at least at a certain stage in viral replication. A computer search for secondary structures with regard to the deletion sites provided a possible common structure from which, supported by sequences existing on the plus or minus RNA strand of PV1(Sab), deletion regions looped out from the remaining sequences. Replicase might, therefore, skip these transiently formed loop structures with certain frequencies, resulting in the generation of DI genomes. This model could also be considered as a model for genetic recombination in these RNA genomes. Possible "supporting sequences" were also found for every rearranged site on the RNAs of influenza virus and sindbis virus. Thus, we propose a new copy-choice model, designated the "supporting sequence-loop model", for the generation of rearrangements occurring on single-stranded RNA genomes.

Crystallization and preliminary X-ray diffraction studies of coxsackievirus B1.

Preparations of coxsackievirus B1 (CVB1) derived from an infectious cDNA clone have been crystallized in multiple crystal forms. Using high intensity synchrotron radiation, an orthorhombic form of the crystals was shown to diffract X-rays to at least 2.9 A resolution. The unit cell has a primitive lattice with dimensions a = 323 A, b = 450 A, and c = 522 A. A crystallographic asymmetric unit of these CVB1 crystals probably contains an entire virus particle, implying the presence of 60-fold non-crystallographic redundancy. This CVB1 crystal form appears to be suitable for high-resolution structure determination by X-ray crystallography.

Complete nucleotide sequences of all three poliovirus serotype genomes. Implication for genetic relationship, gene function and antigenic determinants.

The complete nucleotide sequences of the genomes of the type 2 ( P712 , Ch, 2ab ) and type 3 (Leon 12a1b ) poliovirus vaccine strains were determined. Comparison of the sequences with the previously established genome sequence of type 1 (LS-c, 2ab ) poliovirus vaccine strain revealed that 71% of the nucleotides in the genome RNAs were common, that the 5' and 3' termini of the genomes were highly homologous, and that more than 80% of the nucleotide differences in the coding region occurred in the third letter position of in-phase codons, resulting in a low frequency of amino acid difference. These results strongly suggested that the serotypes of poliovirus derived from a common prototype. A comparison of the amino acid sequences predicted from the genome sequences showed highest variation in the capsid protein region, whereas non-structural proteins are highly conserved. Initiation of polyprotein synthesis occurs in all three strains more than 740 nucleotides downstream from the 5' end. An analysis of the non-coding region suggests that small peptides that could potentially originate from this region are conserved. The amino acid sequences immediately surrounding the cleavage signals, however, show a higher than average degree of variation. The analysis of the amino acid sequences of the capsid protein VP1 of all serotypes has led to the prediction of potential antigenic sites on the virion involved in neutralization.

Expression and microvillar localization of scavenger receptor, class B, type I (a high density lipoprotein receptor) in luteinized and hormone-desensitized rat ovarian models.

Steroidogenic cells in rats and mice obtain most of their cholesterol for steroid production and cholesteryl ester (CE) storage via the selective uptake pathway in which high density lipoprotein CE (HDL-CE) is taken into the cell without the uptake and degradation of the HDL particle. A number of recent studies show that the scavenger receptor, class B, type I (SR-BI) can mediate HDL-CE selective uptake in cultured cells and suggest that this receptor may be responsible for HDL-CE selective uptake in steroidogenic cells in vivo. In the current study we examine the relationship between SR-BI expression and HDL-CE selective uptake in the gonadotropin-primed, luteinized rat ovary and in the ovary that is desensitized by multiple gonadotropin treatments. Results from this study demonstrate a tight association between expression of SR-BI and measurements of HDL-CE selective uptake regardless of the steroidogenic state of the ovary. Thus, in the luteinized ovary (which is actively producing progestins), HDL-CE selective uptake is high, as is the expression of SR-BI. In the desensitized ovary (where CE content is reduced by 90% and progestin production is virtually absent), HDL-CE selective uptake and SR-BI are induced 2- to 3-fold compared with those in the luteinized ovary. These data argue that SR-BI can be regulated by the cholesterol status of the luteal cell independently of gonadotropic stimulation. Immunostaining at the light microscopic level showed strong expression of SR-BI specifically on the surface of luteal cells in the luteinized and desensitized ovary. Immunolocalization at the electron microscopic level showed that SR-BI was associated with microvilli and microvillar channels of the luteal cell surface. This result supports the hypothesis that microvilli and microvillar channels represent a cell surface compartment that is specialized for the selective uptake of lipoprotein cholesterol into steroidogenic cells.

Cloned infectious complementary DNA of the poliovirus Sabin 1 genome: biochemical and biological properties of the recovered virus.

A complete cDNA copy of the genome of the attenuated type 1 poliovirus vaccine (Sabin 1) strain was constructed and inserted into the EcoRI site of the plasmid pBR325. When cultured mammalian cells were transfected with this recombinant plasmid, 20 to 50 poliovirus plaques per 10 micrograms plasmid DNA were observed. Fingerprints of the RNA of the recovered virus showed no changes when compared with those of the parental virus genome, an observation indicating that the primary structure of the cloned cDNA is a reflection of authentic poliovirus RNA. The recovered virus had the same properties as those of the Sabin 1 strain in regard to antigenicity, sensitivity to temperature, and dependency on bicarbonate concentration. These results suggest that the virus obtained by DNA transfection is indistinguishable from the Sabin 1 strain. The recombinant plasmid could therefore be used as a stable repository of the virus and as inoculum for the oral polio live vaccine.

Purification of enzymatically active poliovirus proteinase 3C produced in Escherichia coli.

A viral protein 3C of the poliovirus (PV) Sabin 2 strain, a possible core region of the viral proteinase, was expressed in Escherichia coli using a recombinant DNA technology. The protein was recovered as a soluble protein from the insoluble protein fraction of the bacterial lysate, and was purified by a simple procedure with column chromatography. The viral capsid precursor P1 (1ABCD) of the PV Sabin 3 strain, which had been similarly produced in E. coli, was mixed with the purified or crude recombinant 3C. Immunoblotting assay with monoclonal antibodies specific to capsid proteins 1C (VP3) and 1D (VP1) of the PV Sabin 3 strain revealed that the in vitro reaction products contained 1C (VP3), 1D (VP1) and 1ABC (VP0-VP3). The data indicated that processing of the polyprotein P1 by the recombinant 3C proceeded properly in vitro, although an undigested product, 1ABC, is always detected in the reaction mixture. The results strongly suggest that, in addition to a protein 3CD, the 3C protein itself is also catalytically active in the processing of the viral capsid precursor polyprotein P1.