Bone and joint infections.
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Diagnostic Imaging↗
Biomedical subjects
J T Patton
Publications and source records attributed to J T Patton.
Structure and protein composition of the rotavirus replicase particle.
To understand the role of viral proteins in the replication of rotavirus RNA, we have characterized the structure of subviral particles (SVPs) that synthesize double-stranded RNA (DS RNA). Pulse-labeling of newly made RNA in infected cells showed that rotavirus DS RNA was synthesized either in single-shell (SS)-like particles or in precursor particles that rapidly mature into SS particles. Experiments using a cell-free system demonstrated that most replicase particles containing newly made DS RNA were of greater density in CsCl than single-shelled (SS) particles. However, this was partly due to the presence of single-stranded RNAs as the treatment of replicase particles with micrococcal nuclease reduced their density to between core particles and SS particles. Electrophoretic analysis indicated that replicase particles, purified by centrifugation on CsCl and glycerol gradients, were similar to SS particles, containing the structural proteins VP1, VP2, and VP6. Rotavirus replicase particles were also found to contain the nonstructural proteins NS34 and NS35 and possible host components. The presence of VP6 in enzymatically active replicase particles suggests that, like transcription, this protein may be required for rotavirus RNA replication.
Capsid↗
Synthesis of plus- and minus-strand RNA in rotavirus-infected cells.
The genomes of the rotaviruses consist of 11 segments of double-stranded RNA. During RNA replication, the viral plus-strand RNA serves as the template for minus-strand RNA synthesis. To characterize the kinetics of RNA replication, the synthesis and steady-state levels of viral plus- and minus-strand RNA and double-stranded RNA in simian rotavirus SA11-infected MA104 cells were analyzed by electrophoresis on 1.75% agarose gels containing 6 M urea (pH 3.0). Synthesis of viral plus-strand and minus-strand RNAs was detected initially at 3 h postinfection. The steady-state levels of plus- and minus-strand RNAs increased from this time until 9 to 12 h postinfection, at which time the levels were maximal. Pulse-labeling of infected cells with [3H]uridine showed that the ratio of plus- to minus-strand RNA synthesis changed during infection and that the maximal level of minus-strand RNA synthesis occurred several hours prior to the peak of plus-strand RNA synthesis. No direct correlation was found between the levels of plus-strand and minus-strand RNA synthesis in the infected cell. Pulse-labelling studies indicated that both newly synthesized and preexisting plus-strand RNA can act as templates for minus-strand RNA synthesis throughout infection. Studies also showed that less than 1 h was required between the synthesis of minus-strand RNA in vivo and its release from the cell within virions.
Animals↗
Characterization of subviral particles in cells infected with simian rotavirus SA11.
Subviral particles were isolated from lysates of simian rotavirus SA11-infected cells by sedimentation through sucrose gradients and separated by equilibrium centrifugation in CsCl gradients. A cell-free system that supports rotavirus RNA replication and transcription was used to identify particles in the CsCl gradients with associated polymerase activity. These data indicated that particles with densities of 1.34 and 1.38 g/cm3 were responsible for most of the transcriptase activity present in infected cells. Electrophoretic analysis showed that particles at 1.34 g/cm3 were analogous to double-shelled virus, consisting of the inner shell proteins VP1, VP2, and VP6, the outer shell proteins VP3 and VP7, and DS RNA. Particles of 1.38 g/cm3 were similar to single-shelled virus containing the inner shell proteins and DS RNA. The pellets of the CsCl gradients were enriched for subviral particles with replicase activity. Analysis of the pellets suggested that replicase particles contain a core of VP1 and VP2 that is similar to that found in single- and double-shelled virus but contain significantly less VP6 protein per particle than those with transcriptase activity. Two particles were detected in infected cells that contain no detectable polymerase activity; one consisted primarily of the structural proteins VP2, VP3, and VP6 and the other of the nonstructural protein NS35.
Animals↗
Electrophoretic separation of the plus and minus strands of rotavirus SA11 double-stranded RNAs.
The genome of the rotaviruses consists of eleven segments of completely double-stranded RNA (dsRNA). To provide a method for separating and identifying the complementary plus and minus strand RNAs within these segments, we have characterized their migration patterns under denaturing conditions on agarose-urea gels. Virion-derived 3H-labelled dsRNAs were resolved by electrophoresis on a polyacrylamide gel and the individual genome segments recovered by electroelution. Upon electrophoresis in a low pH 1.75% agarose gel containing 6 M urea, the dsRNAs were denatured with complementary plus and minus strands migrating at different rates. Our results showed that, like cytoplasmic polyhedrosis virus but unlike human reovirus (Smith et al., 1981), rotavirus plus strand RNA migrates faster than its complementary minus strand on agarose-urea gels.
Electrophoresis, Agar Gel↗
Synthesis of simian rotavirus SA11 double-stranded RNA in a cell-free system.
A cell-free system was developed to study the replication of simian rotavirus SA11. The components of the system included (i) subviral particles prepared from infected cells to template the synthesis of viral RNA and (ii) an mRNA-dependent rabbit reticulocyte lysate to support protein synthesis. Based upon nuclease-sensitivity, approximately 20% of the RNA made in vitro was double-stranded (dsRNA) and 80% single-stranded (ssRNA). Electrophoretic analysis of the RNA products on polyacrylamide and low pH agarose gels showed that the system supported the synthesis of 11 dsRNAs and 11 positive-sense ssRNAs that corresponded in size to authentic viral RNAs. The synthesis of dsRNA in vitro was determined to be an asymmetrical process in which a nuclease-sensitive positive-strand RNA acted as a template for the synthesis of negative-strand RNA. The system also supported the initiation of negative-strand RNA using exogenous viral positive-strand RNA as a template. Finally, analysis of subviral particles recovered from reactions suggested that viral proteins made in vitro assembled into nucleoprotein complexes which were similar to those present in infected cells. Together, these results indicate that the cell-free system supported rotavirus RNA replication, transcription and the assembly of subviral particles.
Animals↗
N protein alone satisfies the requirement for protein synthesis during RNA replication of vesicular stomatitis virus.
Genomic replication of the negative-strand RNA viruses is dependent upon protein synthesis. To examine the requirement for protein synthesis in replication, we developed an in vitro system that supports the genome replication of defective interfering particles of the negative-strand rhabdovirus vesicular stomatitis virus (VSV), as a function of protein synthesis (Wertz, J. Virol. 46:513-522, 1983). The system consists of defective interfering nucleocapsid templates and an mRNA-dependent reticulocyte lysate to support protein synthesis. We report here an analysis of the requirement for individual viral proteins in VSV replication. Viral mRNAs purified by hybridization to cDNA clones were used to direct the synthesis of individual proteins in the in vitro system. By this method, it was demonstrated that the synthesis of the VSV nucleocapsid protein, N, alone, resulted in the replication of genome-length RNA by both defective interfering intracellular nucleocapsids and virion-derived nucleocapsids. Neither the viral phosphoprotein, NS, nor the matrix protein, M, supported RNA replication. The amount of RNA replication for a given amount of N protein was the same in reactions in which either all of the VSV proteins or only N protein were synthesized. In addition, RNA replication products synthesized in reactions containing only newly made N protein assembled with the N protein to form nucleocapsids. These results demonstrate that the major nucleocapsid protein (N) can by itself fulfill the requirement for protein synthesis in RNA replication and allow complete replication, i.e., initiation and elongation, as well as encapsidation of genome-length progeny RNA.
Animals↗
Virally coded noncapsid protein associated with bovine parvovirus infection.
A phosphorylated protein (NP-1) with an Mr of 28,000 has been detected in nuclei of bovine parvovirus (BPV)-infected cells in association with chromatin. No protein in this size range was detected after infection of appropriate cells with several autonomous rodent parvoviruses although the BPV-specific protein is similar in size to noncapsid proteins associated with rabbit parvovirus or adeno-associated virus infection. Structural homology between NP-1 and a BPV capsid protein could be detected by electrophoretic analysis of the products of proteolysis with chymotrypsin. This protein can be detected after in vitro translation of RNA from BPV-infected cells and BPV-specific RNA. Homology between the in vivo- and in vitro-synthesized species was shown by the similarity of the chymotryptic products.
Animals↗
Inhibition of vesicular stomatitis virus RNA synthesis by 2',3'-dideoxycytidine 5'-triphosphate.
The ability of the compound 2',3'-dideoxycytidine 5'-triphosphate (ddCTP) to serve as an inhibitor of viral RNA synthesis was examined using an in vitro system that supports vesicular stomatitis virus (VSV) protein synthesis, transcription and replication. Viral RNA synthesis was inhibited by 87 and 98% of control, respectively, in reactions containing 1 mM- and 10 mM-ddCTP in place of CTP. VSV RNA replication and transcription were inhibited equally by ddCTP. At a concentration of 1 mM-ddCTP, there was no inhibitory effect on viral protein synthesis; at 10 mM-ddCTP, total protein synthesis was inhibited by 30% as compared to control reactions. The presence of ddCTP had no effect on the size or relative molar amounts of each protein synthesized as analysed by electrophoresis on polyacrylamide gels. This is the first report describing a compound that will inhibit VSV RNA synthesis in vitro without compromising the concurrent synthesis and modification of proteins.
Deoxycytosine Nucleotides↗
Cell-free synthesis and assembly of vesicular stomatitis virus nucleocapsids.
The association of newly synthesized vesicular stomatitis virus proteins into nucleocapsid structures was examined in a cell-free system that supports concurrent viral protein synthesis, transcription, and RNA replication. The vesicular stomatitis virus proteins synthesized by this system associated with the newly replicated RNA to form structures that banded in CsCl gradients with marker nucleocapsids. In reactions lacking nucleocapsid templates to program RNA synthesis, the newly synthesized proteins did not associate into nucleocapsid structures. The newly synthesized proteins associated with nucleocapsids were analyzed by electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate after separation from non-associated proteins by chromatography on Bio-Gel A15M agarose columns. The results of this analysis showed that newly synthesized L, NS, and N proteins associated into nucleocapsids in the in vitro system. In addition, a small amount of newly synthesized M protein was stably bound to the nucleocapsids. The molar ratio of the associated, newly synthesized proteins was 2:350:1,000:10 (L:NS:N:M). More than 90% of the newly synthesized NS protein that associated with nucleocapsids in vitro was of the NS2 subspecies, as assayed by DEAE-cellulose column chromatography. The stability of the association of the newly synthesized proteins with nucleocapsids in the system mimicked that of the association of viral proteins with nucleocapsids from infected cells as measured by salt sensitivity. These data indicate that nucleocapsids were assembled from newly synthesized proteins within our in vitro system and that the molar ratio of assembled proteins was similar to that observed for virion nucleocapsids.
Capsid↗
FRCR Part II.
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Education, Medical, Graduate↗
Transcription of the bovine parvovirus genome in isolated nuclei.
Transcription of the genome of the nondefective parvovirus BPV was examined in nuclei isolated from synchronized bovine fetal spleen cells. The relative levels of total RNA polymerase and RNA polymerase I, II, and III activities in nuclei isolated from BPV-infected and mock-infected cells were found to be similar throughout the course of infection. Hybridization of RNA synthesized in isolated nuceli indicated that BPV-specific RNA synthesis began during the period of 8 to 12 h postinfection and proceeded linearly until at least 20 h postinfection. By 20 h postinfection, 5% of the total RNA synthesized in nuclei from infected cells was virus specific. BPV-specific RNA synthesis was inhibited by 95% in the presence of 0.1 microgram of alpha-amanitin per ml, suggesting that the viral genome is transcribed by cellular RNA polymerase II.
Amanitins↗
Replication of nondefective parvoviruses: lack of a virion-associated DNA polymerase.
We have examined four of the nondefective parvoviruses for an associated DNA polymerase. Virions were purified from neuraminidase-treated infected-cell lysates by isopycnic centrifugation in CsCl or from infected cell material by CaCl(2) precipitation and centrifugation through sucrose into CsCl. Preparations of bovine parvovirus or Kilham rat virus obtained by the former procedure contained DNA polymerase activity but were not free of contaminating cellular proteins. The latter method produced viral preparations free of contaminating cellular proteins, and no DNA polymerase activity was detected in light infectious particles of H-1, LuIII, bovine parvovirus, or Kilham rat virus. Examination of levels of each cellular DNA polymerase in these preparations from each step of both purification procedures revealed that DNA polymerase beta had a greater tendency to copurify with bovine parvovirus and Kilham rat virus than did DNA polymerases alpha or gamma. Disruption of infectious virions obtained by the second purification method with detergents and sonic treatment did not result in the detection of a DNA polymerase activity. The biological activity and purity of each of the four different viruses obtained by the latter procedure were determined by hemagglutination and infectivity assays, polyacrylamide gel electrophoresis, and electron microscopy. In each case, the virions banding at a density of 1.39 to 1.41 g/cm(2) in CsCl were infectious and contained only the virion structural proteins. DNA polymerase activity was not detected in any of these preparations, and we have concluded that a virion-associated DNA polymerase is not required for productive infection with the nondefective parvoviruses.
Animals↗
Proceedings: Discography in assessment of lumbar disc disease.
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Humans↗
Paravertebral ossification in psoriasis and Reiter's disease.
In a retrospective study of psoriatic arthropathy and Reiter's disease, it is shown that paravertebral ossification (PVO), of the type described by Bywaters and Dixon (1965) and other types of non-marginal syndesmophytes (McEwan et al., 1971) may be the sole or major radiological abnormality. This sign is of particular value where the appearances of the sacro-iliac or other joints are normal or equivocal and is at least as significant as the peripheral I.P. joint involvement in the hands, where these conditions are suspected. This radiological sign may precede the typical psoriatic rash.
Arthritis, Reactive↗
Recent advances in the diagnosis of intervertebral disc disease.
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Contrast Media↗
Pseudo-achondrogenesis with fractures.
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Achondroplasia↗