- Volume 16, Issue 1, 1972

The RNA polymerase activity of a cell-free particulate fraction of barley leaves infected with brome mosaic virus was investigated in the presence of actinomycin D and EDTA. The RNA products were fractionated according to their solubility in 2 m-LiCl. Both LiCl-soluble and LiCl-insoluble fractions of the RNA synthesized during a 2 min. pulse of [3H]-UTP were largely resistant to RNase in high salt concentration; the radioactive RNA of the insoluble fraction had sedimentation properties expected for replicative intermediates. After a 2 min. chase, LiCl-insoluble radioactivity was essentially RNase-sensitive in high salt, and sedimented in sucrose gradients in association with all three components of brome mosaic virus RNA, provided protective exogenous RNA was added throughout the experimental procedure. The overall results suggest that the in vitro synthesis of the RNAs found in virus particles occurs by a continuous process resulting possibly from recycling activity of the polymerase.

We have examined the effect of five local anaesthetics (dibucaine, tetracaine, cocaine, lignocaine and procaine) and five phenothiazine tranquillizers (trifluoperazine, fluphenazine, promethazine, chlorpromazine and diphenhydramine) on cell fusion induced by viruses. When used at physiologically relevant concentrations, all drugs significantly inhibited cell fusion without impairing virus replication. It is suggested that these drugs inhibit cell fusion by occupying sites within the plasma membrane which must be vacant in order for membrane fusion to occur. Inhibition of cell fusion by these drugs provides experimental support for the suggestions put forward elsewhere that events in membrane fusion and membrane depolarization are similar.

The basis for accumulation of capsid antigen in the cytoplasm of cells infected with a newly recognized variant of polyoma has been investigated. The virus progeny and the infectious cycle were compared with that of a normal large plaque strain (lp-s). No significant differences to account for the variation were observed. Expression of this phenotypic character was modified by reduced temperature of incubation combined with serum or arginine deprivation or independently by treatment of infected cells at specific times post-infection with 5-fluorodeoxyuridine actinomycin D or Actidione (cycloheximide).

These results are compatible with the hypothesis that accumulation of capsid antigen in the cytoplasm results from its synthesis at a rate greater than its diffusion into the nucleus. The molecular basis of the defect is discussed.

A microelectrophoresis technique has been used to study purified suspensions of buffalopox, vaccinia, variola and alastrim viruses. The electrophoretic behaviour of purified, but otherwise untreated buffalopox and vaccinia viruses was indistinguishable. Treatment with trypsin, lipase or p-toluene sulphonyl chloride altered the electrophoretic behaviour indicating that the virus surface is lipoprotein, but again, the viruses were indistinguishable. After treatment with 2-mercaptoethanol differences were found in the electrophoretic behaviour of buffalopox and vaccinia viruses. Preliminary experiments showed that cores, extracted from the viruses, also differed in electrophoretic behaviour.

The electrophoretic behaviour of variola virus was indistinguishable from that of alastrim virus even after treatment with 2-mercaptoethanol. However, further experiments indicated that the cores of these two viruses also differed in surface chemical composition.

Double stranded RNAs obtained from plants infected with alfalfa mosaic virus have been studied by electron microscopy using the Kleinschmidt technique. Two characteristic lengths were obtained. A change of length was observed when the two strands were separated with dimethylsulphoxide. This corresponded to a change of configuration from the A form in double-stranded RNA to the B form in single-stranded RNA.

Using dimethylsulphoxide it was possible to visualize single-stranded RNA molecules as elongated filaments.

The electron microscopic results are compared with reported mol. wt values for the different RNAs present in alfalfa mosaic virus.

The rates of synthesis and final yields of poliovirus RNA varied considerably during the four main phases (G1, S, G2 and m) of the life cycle of synchronized HeLa cells. The rate of RNA synthesis late in virus growth (as measured by uridine incorporation 2 to 4 hr after infection) and the final yield of RNA rose sharply if growth was initiated towards the end of phase S; that is, RNA was synthesized most rapidly if cells were infected during the period of most rapid DNA synthesis. In contrast, the initial rate of RNA synthesis (incorporation 0 to 2 hr after infection) was greatest if growth was initiated at the end of phase G2, just before mitosis. This differential effect on growth kinetics suggests that the balance between the two stages of virus RNA synthesis (production of complementary minus strands and of progeny plus strands) is dependent on unknown cellular factors.

The behaviour and properties of a temperature-sensitive mutant of cowpea chlorotic mottle virus are described. The mutant multiplied well at 21° but, unlike the wild-type, only slightly at 32° although considerable amounts of uncoated RNA accumulated in inoculated leaves at this temperature. The specific infectivity of the mutant was much lower than that of the wild-type virus because the largest species of encapsidated mutant RNA was almost completely degraded even in virus from plants grown at 21°. The temperature sensitivity and low specific infectivity of the mutant were related to properties of its coat protein, which was much less heat stable than that of the wild-type virus. Glutamic acid and alanine replaced lysine and valine respectively in the mutant coat protein and these replacements, in addition to affecting thermal stability, influenced the polymerization of isolated protein at pH 6.7.

Cheng (1958) reported that arboviruses of group B but not of group A were sensitive to proteases. Nicoli & Maydat (1965) apparently confirmed this although other reports suggested that protease sensitivity did not distinguish the groups. Merrill (1936) showed that equine encephalomyelitis virus (group A) was inactivated by chymotrypsin but not by trypsin. Takehara & Hotta (1961) found that Western equine encephalitis virus (group A) as well as the group B viruses of Japanese B encephalitis, dengue and yellow fever were sensitive to trypsin. Following the speculation by Bachrach (1963) on the taxonomic significance of the test, we decided to test 33 representative strains of arboviruses against trypsin, chymotrypsin and pronase.

Trypsin (Bovine type XI DCC treated No. T 1005) and α-chymotrypsin (Bovine pancreas type II No. C-4129) were purchased from the Sigma Chemical Company (St Louis, U.S.A.). Pronase (B Grade No. 53703) was purchased from Calbiochem (Los Angeles, U.S.A.).

We wish to present some evidence for the implication of an as yet uncharacterized member of the pox group of viruses in producing epidermal papillomata in a marsupial, Setonix brachyurus. Generally neoplasms in marsupials have not been investigated in any great detail (Barker, Calaby & Sharman, 1963), and it is interesting that in the population of S. brachyurus studied, animals with papillomata associated with poxvirus were frequently found. Apart from the lesions of molluscum contagiosum in humans, no other member of the poxvirus group is known to induce the formation of epidermal papillomata, although they may induce a few mesodermal neoplasms (Gross, 1970).

This particular population of S. brachyurus (or ‘quokka’) has been confined for some seven thousand years to Rottnest, a little island off the coast of Western Australia, and indeed, the species is found only in one or two other areas of this state (Main, 1963).

Heat stable (st) mutants have been isolated from phages T5 (Rubenstein, 1968), T1, T3, T7 (Ritchie & Malcolm, 1970) and λ (Parkinson & Huskey, 1971). In addition to their increased resistance to heat inactivation the st mutant phage particles also have a lower buoyant density and, with the possible exception of T1, contain less DNA than their respective wild-types. All these phages have DNA molecules with non-permuted base sequences (Olligs, 1967; Thomas & MacHattie, 1967) which have been suggested to arise from intracellular concatenated DNA forms (found with T5, T7 and λ) by a sequence specific cutting mechanism which recognizes sites marking the ends of mature DNA molecules (Thomas, Kelly & Rhoades, 1968). Therefore the deletion of non-essential DNA sequences would produce viable phage particles with less DNA than wild-type; this would reduce the buoyant density and apparently also increases the heat stability.

Strains of Newcastle disease virus (NDV) induce cell fusion by two different mechanisms. The first, called ‘fusion from without’ (Bratt & Gallaher, 1969), is independent of virus multiplication, can be induced equally well by infective and non-infective virus and does not require host-specific or virus-specific macromolecular synthesis. This type of cell fusion is completed within 1 to 3 hr of infection at high multiplicity and is caused by the direct interaction of the virus envelope with the plasma membrane of the cell. Fusion from without is a laboratory phenomenon and is similar to the rapid cell fusion induced by high multiplicities of Sendai virus, SV5, and measles virus (Poste, 1970). The second type of NDV-induced poly-karyocytosis, called ‘fusion from within’, is characterized by cell fusion beginning several hours after infection at moderate or low multiplicity. This type of cell fusion is related to the intracellular growth of virus and requires virus-specific macromolecular synthesis (Reeve & Poste, 1971a; Reeve et al. 1971).

Serotypes of human adenovirus have been classified by Huebner (1967) into three groups (A, B and C) based on their oncogenicity in hamsters. Rat cells transformed in vitro by viruses from both groups A and B have been reported to produce tumours in new-born rats (Freeman et al. 1967b, c). Some members of Huebner's non-oncogenic group C were subsequently shown to cause morphological transformation of primary rat embryo cells in vitro (Freeman et al. 1967a; McAllister et al. 1969). Clones derived from these epithelioid foci are similar in their in vitro characteristics to transformed clones induced by adenovirus in groups A and B. The cells grow to high density in medium with low serum, produce colonies in agar and possess adenovirus specific tumour antigen (T-antigen). No tumours were produced when cells transformed in vitro by the group C adenoviruses were inoculated into syngeneic neonate rats and it has been suggested that group C adenoviruses induce strong transplantation antigens in the cells they transform (McAllister et al. 1969).

Drosophila P virus was described by Plus & Duthoit (1969) as a small icosahedral virus, 25 nm. in diameter, endemic in many Drosophila populations. In naturally infected strains, this virus has little effect on flies. When injected, however, it causes female sterility and reduces the life span of both sexes by half (David & Plus, 1971). Just before death, injected flies become turgid with liquid, suggesting malfunction of the Malpighian tubules responsible for water exchanges in insects. An electron microscopic study was undertaken in order to characterize the virus and the response of the virus to certain chemicals was observed. The P virus exhibited properties consistent with those of the picornaviruses.

Heterozygous Drosophila melanogaster vg, free from known Drosophila viruses: P (Plus & Duthoit, 1969), σ (L'Héritier, 1970) and Iota (Jousset, 1970), were used as standard flies for injections.

Production of interferon from cultured cells has required the presence of serum in the cell culture medium although it may be omitted during the period of interferon induction. In the system reported here a fully defined medium, free of protein, is employed. This permits the culture of the cells and all phases of interferon induction and release to be studied under completely defined conditions. The final product can easily be separated from its inducers to yield a preparation of high specific activity.

The ls strain of mouse fibroblasts, adapted to grow in suspension, has been under regular culture in this laboratory (Griffiths & Pirt, 1967; Birch & Pirt, 1969, 1970, 1971; Blaker & Pirt, 1971) in the protein-free medium (Birch & Pirt, 1970). The only polymer used in the medium is the uncharged polysaccharide methylcellulose.