Biophysical properties of the structural components of a granulosis virus isolated from the cabbage white butterfly (Pieris brassicae).
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Biomedical subjects
Publications and source records attributed to D C Kelly.
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The mol. wt. of the DNA from four nuclear polyhedrosis viruses isolated from Spodoptera littoralis, S. exempta, S. exigua and S. frugiperda were determined to be 84, 80, 68 and 74 X 10(6), respectively, by electron microscopy. The molecules were demonstrated to exist as double-stranded relaxed circular or supercoiled DNA, though linear forms of DNA were also observed.
The DNA contained by particles of densonucleosis viruses 1 and 2 were analyzed within the particle, and properties of DNA extracted from these particles were determined. The DNA appears to exist as a single-stranded molecule with limited secondary structure within particles, as assessed by spectral changes induced by formaldehyde, melting profiles, and circular dichroism studies. The single-stranded DNA had an apparent molecular weight of 1.9 X 10(6) to 2.2 X 10(6) as assessed by differences in the molecular weight of virus particles and top component and percentage of nucleic acid. DNA extracted from virus particles in low-salt buffers possessed properties typical of a single-stranded molecule. Double-stranded DNA could be extracted from virus particles under appropriate high salt and elevated temperature. The linear double-stranded DNA extracted from both viruses had a molecular weight of about 3.9 X 10(6) to 4.1 ZX 10(6) determined by neutral sedimentation and electron microscopy and an equivalent genome size determined by reassociation kinetics. About 87% of the DNA was homologous between the two viruses.
Densonucleosis viruses 1 and 2 both contain the three polyamines putrescine, spermidine, and spermine in complete virus particles but not in their respective top components. The polyamines, with spermidine predominant, comprise 1.41% of the virus particle by weight, which is sufficient to neutralize 26% of the single-stranded DNA contained within the particles.
The effects of inhibitors of nucleic acid and protein synthesis on the replication of Spodoptera frugiperda nuclear polyhedrosis virus have been determined. Two inhibitors of protein synthesis-cycloheximide and puromycin-were irreversible inhibitors of virus multiplication. Three inhibitors of nucleic acid synthesis-actinomycin D, cytosine arabinoside and camptothecin- prevented virus multiplication; only camptothecin was reversible. Rifampicin had no effect on virus multiplication.
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The replication of frog virus 3 in primary chick embryo fibroblasts has been studied by examination of thin sections with the electron microscope and the assay of infectious viurs. Uptake of frog virus 3 by the cells was observed to occur by pinocytosis and this may be the method of entry. Early in infection (i h p.i.) marked margination of the nuclear chromatin occurred and the chromatin remained in this condition throughtout the infection. Foci of infection were first detected in the cytoplasm of cells 24 h p.i. when production of infectious virus commenced. These foci appeared as electron translucent areas containing fine grains, surrounded by degenerate mitochondria. The foci usually contained virus particles. At this time budding of virus particles at the plasma membrane occurred. Later in infection at 36 and 48 h p.i. large numbers of virus particles were detected in the cytoplasm of cells either scattered loosely throughtout the cell, arranged as clusters or in paracrystalline arrays. Extensive budding at the plasma membrane then took place. Virus particles were detected in the nucleus of the cells at these late stages and it is possible that the virus may infect and replicate at this site. Throughout the productive stages of infection aberrant forms of the virus, namely particles devoid of cores, incompletely assembled particles and elongated bacilliform particles were noticed.
An examination of BHK, CEF, and FHM cells chronically infected with frog virus 3 has been made by scanning and transmission (thin section, freeze fracture, and surface replica) electron microscopy. With minor differences the pattern of virus development is similar in all three cell line. Virus particles were detected in cell nuclei which subsequently became degenerate very late in infection. Three inclusions were associated with frog virus 3 cytoplasmic foci of infection; lamella structures, extensive microtubule formation (in BHK and FHM cells), and linear crystalline structures. The last two structures may play a role in creating or maintaining the cell rounding c.p.e. revealed by scanning electron microscopy. Very late in infection most BHK and FHM, but not CEF, cells are stripped of the plasma membrane. Replicas of frozen fractured BHK cells featured cytoplasmic foci of infection, budding at the plasma membrane, and showed that at early times when virus is detected in the nucleus, the nuclear membranes are intact and morphologically unaltered. Budding at the plasma membrane was better resolved by scanning and as surface replicas. This demonstrated that sparse to profuse localized budding occurred. Frequently virus particles were located singly, or as multiples, at the end of, or along, cytoplasmic protrusions which occur both on the body of the cells and at the cytoplasmic/coverslip 'interface'.
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Studies employing indirect immunofluorescent staining, acrylamide gel electrophoresis of [(35)S]methionine-labeled cellular polypeptides, and RNA-RNA hybridization of [(3)H]uridine-labeled cellular RNA, failed to detect evidence of fowl plague virus infection of BHK cells enucleated with cytochalasin B, although virus-specific polypeptide and RNA synthesis was detected in nucleate BHK cells. The enucleate cells permitted the synthesis of Newcastle disease and vaccinia virus structural proteins. We conclude that influenza virus fails to initiate macromolecular synthesis in enucleate BHK cells, and the role of the nucleus in influenza virus replication is discussed.
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