Alan J.D. Bellett

A circular DNA—Protein complex from adenoviruses

Abstract DNA prepared from adenoviruses by treatment with sodium dodecyl sulphate (SDS) and Pronase is linear. However, several other methods gave DNA consisting of up to 70% relaxed circular molecules and up to 20% of oligomers. These forms were converted to linear duplex monomers by treatment with SDS, Pronase, trypsin, or α-chymotrypsin, and to linear single-stranded monomers by prolonged treatment with alkali. Viral DNA released directly into alkali sedimented fast in alkaline sucrose, but prolonged exposure to alkali or treatment with SDS converted it to a form that sedimented with linear single-stranded monomers. When a mixture of intact virus and Pronase-SDS treated linear DNA was extracted by a method that gave circular DNA from the virus, the linear DNA did not form circles. Partially disrupted virus spread by the protein film method often released apparently circular DNA, which sometimes appeared supercoiled. We propose that the adenovirus chromosome is a DNA-protein complex. It is probably a DNA molecule with a protein that joins the ends to form a circle, although a circular DNA with a tightly bound protein that can both cut and reform the circle is possible. We propose a hypothesis for the replication of the DNA based on these observations.

Semliki Forest virus temperature-sensitive mutants: Isolation and characterization

Abstract The normal replication of Semliki Forest virus involves RNA synthesis followed by the synthesis of two structural proteins (nucleocapsid and membrane). Temperature-sensitive mutants blocked at different steps of the growth cycle have been isolated from Semliki Forest virus by chemical mutagenesis. A group of the mutants defective in RNA synthesis (RNA − mutants) were prevented from producing viral antigen in cells infected at the restrictive temperature and also were unable to inhibit host cell RNA synthesis. The factor affecting viral RNA synthesis is expressed early in the growth cycle. Other mutants were defective in functions subsequent to viral RNA synthesis (RNA + mutants). Some were defective in the production of nucleocapsids only; others failed to make membrane only. A few RNA + mutants made both structural proteins, but not infectious virus, and may be defective in a maturation function.

Replication of the DNA of chick embryo lethal orphan virus

Abstract Replication of the DNA of chick embryo lethal orphan virus was semi-conservative. In CsCl density gradients a portion of pulse-labelled intracellular viral DNA was more dense than mature DNA and sometimes approached the density of denatured DNA. Chromatography on benzoylated naphthoylated DEAE-cellulose also suggested that replicating viral DNA had extensive single-stranded regions. In neutral sucrose, some pulse-labelled viral DNA sedimented faster than mature DNA. Short pulses of [3H]thymidine were incorporated into fragments that sedimented at about 12 s in alkaline sucrose. As the pulse length was increased, label was found in material that sedimented faster than 12 s fragments but more slowly than the strands of mature viral DNA, and finally in full length viral DNA strands. During a “chase” in unlabelled medium, pulse-labelled intracellular viral DNA was converted to a form with properties like those of mature DNA. No closed circular structures could be detected when pulse-labelled DNA was centrifuged in CsCl in the presence of ethidium bromide. Thus the replication of this DNA, which is linear and lacks terminal repetitions detectable by exonuclease digestion and annealing, does not involve circles or concatemers in which one or both strands are continuous. However, the 5′ ends of the daughter strands cannot be completed unless the nascent DNA forms a maturation intermediate, the most likely form of which is a concatemer with staggered nicks in both strands at one genome intervals. This implies an unusual structure of the ends of the DNA, or the existence of a protein that interacts with the ends.

Some properties of deoxyribonucleic acid preparations from Chilo, Sericesthis and Tipula iridescent viruses

Abstract The amount of DNA in the particles of three viruses of the iridescent virus group has been estimated, and some properties of DNA preparations from the viruses have been investigated. All the viruses contain double-stranded DNA. The DNA of Chilo iridescent virus contains 28 to 29% G + C base pairs, that of Sericesthis iridescent virus 31% G + C and that of Tipula iridescent virus 32% G + C. The differences in base ratios were confirmed by analysis of mixed DNA bands at equilibrium in CsCl. The contents of DNA per virus particle (expressed in molecular weight units) and the molecular weights of extracted DNA were estimated to be 135 million and 130 million for Chilo iridescent virus, 147 million and 134 million for Sericesthis iridescent virus and 155 million and 126 million for Tipula iridescent virus, respectively.

The multiplication of Serichesthis iridescent virus in cell cultures from Antheraea eucalypti Scott: II. An in vitro assay for the virus

Abstract The optimum temperature for production of the antigen of Sericesthis iridescent virus (SIV) by infected cell cultures was 20°. Virus titres in infective units (IU)/ml were estimated from the proportion of cells which stained with fluorescent antibody 6 1 2 days after inoculation with dilutions of SIV. The proportion of uninfected cells was close to that predicted by the Poisson distribution at multiplicities below 0.5 IU/cell. A plot of virus titre against virus concentration was linear and passed through the origin. The error of the method was estimated. The particle: infectivity ratio was about 80:1 for purified virus.

The Iridescent Virus Group

Publisher Summary In this chapter, the iridescent virus group will be considered to consist of three viruses of insects, Chilo iridescent virus (CIV), Sericesthis iridescent virus (SIV), and Tipula iridescent virus (TIV). The iridescent viruses contain deoxyribonucleic acid (DNA) but no detectable ribonucleic acid (RNA). The particles of SIV and TIV have lxen shown to be icosahedral by shadowing them in two directions, and comparing the shadows of the particles in the electron niicroscopc with those of models of regular polyhedral. The faces of TIV particles are approximately equilateral and triangular. Knowledge of the internal structure of iridescent viruses is even less certain. The core has been described as consisting of 42 “large knobs” forming an icosahedron but similar structures are found in uninfected insects, and there may be an internal component consisting of 12 subunits. Pellets or crystals of iridescent viruses appear blue-violet and green when viewed with oblique illumination. Crystals of these viruses have such large lattice constants that they diffract light of visible wavelengths just as ordinary crystals diffract X-rays, and light of selectively reflected wavelengths is seen as iridescence in the iridescent pellets. The optimum temperature for the replication of iridescent viruses is 20° to 25°, depending on the host and experimental conditions. Two diseases of mosquitoes appear to be caused by viruses with some resemblance to the iridescent viruses. One of these viruses, found in Aedes taeniorrhynchus , has been named “mosquito iridescent virus.”

The structural proteins of chick embryo lethal orphan virus (fowl adenovirus type 1)

Chick embryo lethal orphan (CELO) virus (fowl adenovirus type 1) contains at least 14 structural proteins with polypeptide molecular weights ranging from 100K to about 6K. A nomenclature of the CELO virion polypeptides is presented and the molar proportion of each polypeptide has been estimated. The CELO virus pentons were specifically released from the virion by dialysis against borate-based calcium-magnesium saline. The penton base (polypeptide III, mol. wt. 92K) and two fibres were separated, characterized and their polypeptides were correlated with their morphological positions in the virion. Peptide mapping suggested that the long fibre (polypeptide IV, mol. wt. 65K), and the short fibre (polypeptide VII, mol. wt. 44.5K) were not related in their primary sequences and are therefore probably encoded by separate genes. The time course of synthesis of the CELO virion polypeptides indicated that, like their mammalian adenovirus counterparts, they are synthesized late (after viral DNA replication).

An adenovirus protein associated with the ends of replicating DNA molecules.

Abstract The adenovirus DNA-protein complex contains the linear DNA molecule and a 55K protein covalently attached to each 5′ end. We describe a simple assay for adenovirus DNA-protein complex, in which DNA, covalently linked to the terminal protein, specifically binds to benzoylated naphthoylated DEAE-cellulose. The DNA-protein complex can be recovered intact from the column by elution with urea and SDS, or the DNA moiety can be eluted after incubation of the column with protease. Using this assay system, we have shown that protein is associated with pulse-labeled single strands of DNA from the terminal restriction enzyme fragments of replicating adenovirus DNA. We suggest that the terminal protein has a function in DNA replication.

Covalent integration of viral DNA into cell DNA in hamster cells transformed by an avian adenovirus.

Abstract A line of hamster skin cells transformed by an avian adenovirus was shown by DNA reassociation kinetics to contain about 16 ng of viral DNA per mg of cell DNA (2.5 copies per diploid amount of cell DNA). The viral DNA was covalently integrated into the cell DNA as judged by the network technique of H. E. Varmus, P. K. Vogt, and J. M. Bishop ((1973) Proc. Nat. Acad. Sci. USA 70, 3067–3071) and a more stringent modification of it. A quantitative method for the analysis of integration was developed and suggested that all of the viral DNA in transformed cells was in the integrated state, whereas all of the viral DNA in normal hamster cells 2 hr after inoculation with virus was free. This method could be used to determine the fraction of any given episomal DNA that is integrated, providing certain experimental conditions are met.

Relationships among the polyhedrosis and granulosis viruses of insects.

Abstract Insect viruses with inclusion bodies have been traditionally assigned to three “genera” according to the site of production and the morphology of the inclusion bodies and virus particles. These genera correspond to the nuclear polyhedroses, the cytoplasmic polyhedroses and the granuloses. Comparison of the viral nucleic acids does not support separation of the granuloses from the nuclear polyhedroses. Numerical analysis of data on the serological cross-reactions and amino acid composition of the viral proteins supports classification according to base composition of the nucleic acids. It is suggested that the granuloses and nuclear polyhedroses form a single complex of genetically related viruses, the subgroups of which do not correspond to the conventional “genera.” The cytoplasmic polyhedroses are completely unrelated to the nuclear polyhedroses and granuloses.