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Query: UNIPROT:P06889 (Mol)
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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.
J Mol Biol 1992 Feb 20
PMID:Crystallization and preliminary X-ray diffraction studies of coxsackievirus B1. 131 88

The inadvertent carryover of amplified fragments of nucleic acids (amplicons) is a potential source of contamination in the polymerase chain reaction. Recently, a method has been developed to generate amplicons with deoxyuracil triphosphate (dUTP) and to specifically hydrolyze these amplicons with uracil-DNA glycosylase (UNG) following the completion of the assay. We evaluated this system for the specific amplification of RNA from coxsackievirus A3 and B3. We found that RNA from both viruses could be amplified with dUTP, although the use of this triphosphate in place of TTP resulted in some loss of assay sensitivity. We also found that the dUTP-containing amplicons could be efficiently hydrolyzed by UNG, resulting in a 10,000,000-fold reduction in amplicon concentration with little effect on the native nucleic acid. The dUTP-UNG method has a great deal of potential for reducing amplicon contamination during the routine performance of nucleic acid amplification reactions.
Mol Cell Probes 1992 Jun
PMID:Use of modified nucleotides and uracil-DNA glycosylase (UNG) for the control of contamination in the PCR-based amplification of RNA. 140 34

The presence of viral genomes in the murine heart in experimental coxsackievirus B3 myocarditis was investigated by Northern blotting analysis. Four-week-old C3H/He mice were inoculated with coxsackievirus B3. After sacrifice, the hearts were divided into 3 parts. Total RNA was extracted from one part of the heart. The other two parts were used for investigation of histopathology and of virus titer by plaque assay. The 32P-labeled cDNA probe was derived from the 5' end sequence of the coxsackievirus B3 genome. Northern blot autoradiograms of heart RNA were positive for viral RNA until day 7 and negative after day 10 and in control (uninfected) hearts. Positive autoradiograms always had the strongest signal at about 7.4 kilobase, corresponding to the size of the complete genome of the virus. The viral genomes were detected earlier than the appearance of the histopathologic changes in the murine heart. This type analysis of the viral genome in the murine myocardium may be useful in evaluating the effects of specific drugs on experimental viral myocarditis at the level of the viral genome.
J Mol Cell Cardiol 1990 Sep
PMID:The viral genome in experimental murine Coxsackievirus B3 myocarditis: a Northern blotting analysis. 217 95

Forty-eight different enterovirus serotypes were analysed by a nucleic-acid hybridization test using probes derived from the 3' end of coxsackievirus A21 (CA21) and B3 (CB3), poliovirus 3 (P3) and enterovirus 70 (E70). More than 90% of the serotypes could be detected with this collection of reagents. The CB3 probe reacted with all the coxsackie B viruses, with all three poliovirus serotypes, and with almost all of the 30 ECHO virus types tested. In addition some of the coxsackie A viruses and the BrDr 73 strain of enterovirus 71 gave a positive signal with this probe. The P3 probe detected all the poliovirus strains and also some coxsackievirus A isolates but no coxsackie B or ECHO viruses. A similar hybridization pattern as with the P3 probe was obtained when the CA 21 probe was used. The E70 probe appeared to be strain-specific. The results indicate that nucleic-acid hybridization is a useful method for rapid detection and subgrouping of enteroviruses during virus isolation, and that the test could be further developed for typing of the strains.
Mol Cell Probes 1987 Jun
PMID:Nucleic acid sequence relationships between enterovirus serotypes. 245 58

A non-isotopic hybridization assay is described for detection of enteroviral RNA in cell culture. Two biotin-labelled cDNA probes, corresponding to 1 kb from the 5' end and 3.5 kb from the 3' end of the coxsackievirus B3 genome, were hybridized in solution with protease and detergent-treated cell culture suspensions. Labelled DNA-RNA hybrids were captured on microtiter plates coated with anti-biotin antibody and bound hybrids were measured with a beta-galactosidase-labelled monoclonal antibody specific for DNA-RNA hybrids. Coxsackie B3 was detected at a concentration of 500 pfu ml-1. The limit of detection for other enteroviruses ranged from 10(3.3) to 10(5.8) pfu ml-1. The enteroviruses that could be detected included coxsackie B1 and 3, coxsackie A1-6 and 15, poliovirus types 1-3, and enteroviruses 7, 11, and 71. ECHO 22 was the only enterovirus, of those that were tested, that could not be detected. The solution hybridization reaction and enzyme immunoassay for DNA-RNA hybrids does not require the use of radiolabelled probes or extraction of RNA with phenol. The assay yields a quantitative endpoint, which avoids the subjectivity inherent in membrane-based methods. These features would make the assay more adaptable to clinical laboratories than other formats which have been devised for measurement of viral RNA.
Mol Cell Probes 1989 Dec
PMID:Solution hybridization and enzyme immunoassay for biotinylated DNA-RNA hybrids to detect enteroviral RNA in cell culture. 255 21

Molecular hybridization was used to measure poly(A)-containing mRNA and insulin mRNA, and to evaluate viral persistence, in pancreatic beta cells of coxsackievirus B4-induced diabetic mice. Cellular RNA was hybridized with [3H]poly(U) to measure poly(A)-containing total mRNA, 32P-labeled preproinsulin I and II probes to measure insulin mRNA, and a 32P-labeled virus-specific probe to evaluate persistence. The infected mice (80-90%) showed subnormal blood glucose at 72 h postinfection and were hyperglycemic at 6 and 8 weeks. Poly(A)-containing total mRNA decreased by about 26% at 72 h and 6 weeks and by 49% at 8 weeks, while preproinsulin I mRNA by 30% and preproinsulin II by 46% at 8 weeks postinfection compared to control. Viral sequences were abundant at 72 h and in fair amounts later. It appears that persistent viral infection produces a pathological state, which impairs beta cell function to reduce insulin mRNA and consequently insulin synthesis apparently leading to hyperglycemia.
Mol Cell Endocrinol 1988 Feb
PMID:Insulin mRNA content in pancreatic beta cells of coxsackievirus B4-induced diabetic mice. 283 17

In most patients with virus myocarditis, the diagnosis is still based on clinical data alone. Endomyocardial biopsies subjected to electron microscopy, immunofluorescence techniques and virus isolation procedures provide additional, but only occasionally conclusive information. In this communication we describe a new method which could possibly be used to improve the diagnostic possibilities in patients with suspected virus myocarditis. The method is based on the hybridization of radioactive complementary nucleotide sequences to virus-RNA. It is shown that in an experimental model (reovirus infected baby mice) this method can be used to demonstrate the virus infection of cardiac muscle. It is suggested that the method could be adapted to other viruses (e.g. coxsackie virus) and to endomyocardial biopsies derived from patients with suspected virus myocarditis.
J Mol Cell Cardiol 1985 Jan
PMID:Virus myocarditis: molecular hybridization allows the detection of virus-RNA in heart muscle after virus infection. 298 89

Defective-interfering (DI) particles of the Sabin strain of type 1 poliovirus were generated on serial high m.o.i. passaging. The deletions, measured by agarose gel electrophoresis, appeared to comprise approximately 10% of the total genome. Analysis of the RNAs, after digestion with RNase T1, by two-dimensional polyacrylamide gel electrophoresis revealed that the locations of the deleted genome regions were similar to those of the DI particles of the Mahoney strain of type 1 poliovirus (A. Nomoto, A. Jacobson, Y. F. Lee, J. Dunn, and E. Wimmer, (1979), J. Mol. Biol. 128, 179-196). Taking the known nucleotide sequences of the total genome and large RNase T1-resistant oligonucleotides into account, the deletions of almost all DI RNAs were found to exist between nucleotide positions 1307 and 2630, a genome region encoding capsid polypeptides VP2, VP3, and VP1. In cells coinfected with the purified DI particles and the Sabin strain of type 2 or type 3 poliovirus, particles containing the DI genomes were effectively produced. These results suggest that encapsidation signals are conserved in all three serotypes of polioviruses. However, only a very small amount of similar DI particles appeared to be produced in cells coinfected with coxsackie virus B1, although the genomes of polioviruses and coxsackie viruses have common sequences and therefore these viruses are considered to have arisen from a common ancestor. These data may suggest differences in encapsidation signals between polioviruses and coxsackie viruses.
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PMID:Isolation and characterization of defective-interfering particles of poliovirus Sabin 1 strain. 299 88

Single-stranded RNA probes were developed from cloned cDNA fragments derived from four picornaviruses; poliovirus type 1, coxsackievirus B3, ECHOvirus 9 and Theiler's murine encephalomyelitis virus. In comparative testing with a nick-translated cDNA probe, the RNA probe was found to be 10-100-fold more sensitive. Hybridization conditions were optimized for RNA probes to picornavirus targets. Longer hybridization times gave an improved signal, as did a temperature of 50 degrees C. Formamide concentration had little effect on signal strength. A crude transcription mix performed as well or better as a probe than did more purified preparations of RNA. Finally, a combination of the three probes derived from human picornaviruses detected all 16 serotypes of human enteroviruses tested. Used individually, the four probes detected different spectra of animal and human picornaviruses, shedding more light on the interrelationships among these pathogens.
Mol Cell Probes 1988 Mar
PMID:Development and application of RNA probes for the study of picornaviruses. 338 Jan 6

Translation extracts were prepared from various strains of Saccharomyces cerevisiae. The translation of mRNA molecules in these extracts were cooperatively enhanced by the presence of 5'-terminal cap structures and 3'-terminal poly(A) sequences. These cooperative effects could not be observed in other translation systems such as those prepared from rabbit reticulocytes, wheat germ, and human HeLa cells. Because the yeast translation system mimicked the effects of the cap structure and poly(A) tail on translational efficiency seen in vivo, this system was used to study cap-dependent and cap-independent translation of viral and cellular mRNA molecules. Both the 5' noncoding regions of hepatitis C virus and those of coxsackievirus B1 conferred cap-independent translation to a reporter coding region during translation in the yeast extracts; thus, the yeast translational apparatus is capable of initiating cap-independent translation. Although the translation of most yeast mRNAs was cap dependent, the unusually long 5' noncoding regions of mRNAs encoding cellular transcription factors TFIID and HAP4 were shown to mediate cap-independent translation in these extracts. Furthermore, both TFIID and HAP4 5' noncoding regions mediated translation of a second cistron when placed into the intercistronic spacer region of a dicistronic mRNA, indicating that these leader sequences can initiate translation by an internal ribosome binding mechanism in this in vitro translation system. This finding raises the possibility that an internal translation initiation mechanism exists in yeast cells for regulated translation of endogenous mRNAs.
Mol Cell Biol 1994 Nov
PMID:Cap-dependent and cap-independent translation by internal initiation of mRNAs in cell extracts prepared from Saccharomyces cerevisiae. 793 46


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