- Volume 18, Issue 2, 1973

Bacteria infected with bacteriophage T5 were disrupted with lysozyme and mild detergents and the intracellular phage-specific components resolved by sedimentation through neutral sucrose gradients. In pulse-chase experiments with [3H]-thymidine most of the radioactivity initially appeared in a fast-sedimenting form of DNA (fsf) which was very shear-sensitive and bound tightly to nitrocellulose. Label next appeared in a slower sedimenting form (ssf), then phage heads and finally virus particles. The ssf showed susceptibility to shear similar to that of DNA from intact virus, and sedimented with it on neutral gradients. The ssf DNA differed from virus DNA by binding to nitrocellulose and showing a different sedimentation profile on alkaline-sucrose gradients. The pulse-labelled replicating DNA was very heterogeneous in mol. wt. and appeared to contain many single-strand nicks which were extensively repaired while the DNA was still in the fast-sedimenting form. The conversion sequence fsf ―→ ssf ―→ heads → phage was supported by the accumulation of components in non-permissive host bacteria infected with certain amber mutants of T5. One of these, T5. B1, could not form the T5 phage-induced 5’ exonuclease and in these infections there was no conversion of replicating DNA to ssf, and net DNA synthesis stopped prematurely. It was concluded that maturation of T5 virus involved mature virus-size pieces of DNA of unusual structure as intermediates between replicating DNA and phage heads. The process appeared to require functional T5 induced exonuclease, but the role of this enzyme was unclear.

Poxvirus haemagglutinin has been separated into two components by ether/ethanol extraction and also by column chromatography after treatment with 2-chloroethanol. One component, lipid in nature, carried the haemagglutinating activity. The other, a protein termed the antibody blocking component, carried the virus specificity.

By the use of techniques applied by others to the study of lipoprotein membranes, poxvirus haemagglutinin of high specificity was reconstituted from the two components. The reconstituted material reacted with antibody to haemagglutinin but not with antibody to a non-haemagglutinating poxvirus. Reconstitution did not take place when either of the two components was replaced by fractions prepared from uninfected tissues or from tissues infected with a non-haemagglutinating poxvirus. Mixed haemagglutinins could be prepared from fractions prepared from different tissues or from different haemagglutinating poxviruses.

Some properties of a small RNA virus isolated from larvae of Gonometa podocarpi (Lepidoptera:Lasiocampidae) are described. The virus develops in the cytoplasm of gut and fat body and is 32 nm in diameter. The sedimentation coefficient of virus was 180S and the buoyant density was 1.35 g/cm3. The RNA:protein ratio was 37:63 and the RNA was single-stranded. Four principal polypeptides were found with mol. wt. in the range 12 100 to 36 500. It is proposed that this virus be included in the enterovirus group.

Mycoplasmatales virus-laidlawii 2 (MV-L2) was purified by ammonium sulphate precipitation and by sucrose density gradient fractionation. Electron micrographs of virus showed predominantly spherical enveloped particles with a mean diameter of about 80 nm (range 52 to 125 nm). The envelope had a ‘unit membrane’ structure and was probably serologically dissimilar to the ‘unit membrane’ of the host Acholeplasma laidlawii. No obvious isometric or helical capsid was observed within the envelope. The nucleic acid appeared to be DNA.

Selective changes were found in the constitutive enzymes of the host cell following infection of chick embryo fibroblast cells with Semliki Forest virus. The changes were limited to soluble dehydrogenases involved in pathways of glucose catabolism. Changes were also found in nucleotide levels which, along with the enzyme changes, suggested that glucose catabolism was stimulated in the virus infected cells. The enzyme changes were prevented by pretreatment of the cells with actinomycin D.

Infectious virus could not be recovered by disruption of human embryonic fibroblast cultures inoculated with cytomegalovirus and treated with cytosine-arabinoside (ara-C), either during treatment or 4 to 5 days after removing ara-C. However, when intact cells were used for infection, at least 1 in 70 cells was found harbouring virus. Examination by immunofluorescence revealed that 40 to 50% of the cells contained virus-specific antigens in the form of small granules diffusely distributed in the nuclei. The presence of infectious virus could be detected even in disrupted cells 4 to 5 days after removing ara-C; intracellular antigens of every kind were also found to develop. Experiments showed that the cultures continued to harbour latent infection indefinitely in the presence of ara-C. Cultures harbouring latent virus were susceptible to superinfection with cytomegalovirus.

Virus yield/cell from Balb-c 3T3 monolayer cultures infected with polyoma virus was found to decrease with increasing cell density. Close cell contact per se did not inhibit yield nor did moderate depletion of medium constituents by cell growth. Adsorption of input virus/cell decreased as the cultures aged.

Addition of fresh medium to resting cultures provoked a delayed increase in susceptibility. Treatment of resting cultures with trypsin initially enhanced their susceptibility, but a gradual decline to the original level followed; adsorption of input virus followed a similar course. These and other results described are consistent with the interpretation that only cells in a state of active division are susceptible and that resting cells acquire a trypsin-sensitive surface component which blocks infection.

Yields of virus from sparse cultures of several clones tested varied. Study of the clone of highest susceptibility revealed that cell DNA synthesis was not induced by virus infection and that the total amount of virus DNA made was small and closely equivalent to the amount found encapsidated.

The yield of polyoma virus from Balb-c 3T3 cells depends on the stage of the cell-cycle at which they are infected and is at a maximum at, or near, the beginning of G1.

Several of the physico-chemical properties of representative members of the Picornaviridae family have been examined. On the basis of their buoyant density in caesium chloride, stability at pH 3 to 7 and the base composition of the virus RNA, a division of this family of viruses into six subgroups is suggested.

Analysis of the X-ray diffraction patterns from oriented specimens of narcissus mosaic virus is described. This shows that there is a marked feature in the virus particle at a radial position of about 33 Å, and it is suggested that this corresponds to the sugar-phosphate backbone of the RNA. Electron microscope studies of stained transverse sections of the virus particles are not incompatible with this interpretation. Some aspects of the possible RNA conformation in the virus particle are discussed.