- Volume 29, Issue 2, 1975

High mol. wt. RNA (HMW-RNA) extracted from nuclei of HEp-2 cells 5 h post infection with herpes simplex 1 virus has been shown to have the following characteristics: (i) the amount of HMW-RNA hybridizing to viral DNA fixed on filters increased with input multiplicity as well as following heat denaturation prior to hybridization; (ii) purified self-annealed HMW-RNA was enriched for viral RNA sequences which hybridized to viral DNA following denaturation; (iii) hybridization of excess unlabelled HMW-RNA which labelled viral DNA fragments followed by isopycnic centrifuging in Cs2SO4 led to the partitioning of a fraction of DNA with HMW-RNA. This DNA self annealed at a faster rate than the parental DNA population from which it was derived indicating that the HMW-RNA contained transcripts derived from symmetric transcription of a fraction of viral DNA; (iv) excess unlabelled, heat-denatured, HMW viral RNA drove 50% of viral DNA into DNA-RNA hybrid in hybridization tests with trace amounts of labelled viral DNA. Analysis of the kinetics of hybridization indicated that HMW-RNA consisted of 2 classes arising from 24 to 26% of viral DNA and differing 5000-fold in molar concentration. Since HMW-RNA contains symmetric viral transcripts which self anneal during the hybridization this is probably a minimal estimate of the amount of viral DNA represented in HMW-RNA.

Several of the major structural polypeptides of herpes simplex virus were obtained in purified form by polyacrylamide gel electrophoresis of purified virus particle polypeptides. Antisera made by footpad inoculation of these polypeptides into rabbits were used to study the antigenic properties of two envelope glycoproteins and of the major capsid protein.

Adsorption of u.v.-inactivated Sendai virus on to NIL8 hamster cells causes fusion of the cells into polykaryocytes within 2 h. ‘Infected’ cells were incubated at 37 °C for periods of 10 min to 8 h and their surface proteins iodinated with [125I] catalysed by peroxidase. Structural components of the viral envelope, such as haemagglutinin-neuraminidase (HN) and probably also the fusion protein (F) were detected in the cell membrane for periods up to 4 h post infection.

The effects of trypsin and chymotrypsin on the infectivity, morphology and antigenic properties of the Indiana and Brazil strains of vesicular stomatitis virus have been studied. Each enzyme reduced the infectivity of the Indiana strain by about 10000-fold but the infectivity of the Brazil strain was unaffected. Electron microscopy of the treated particles and polyacrylamide gel electrophoresis of the viral polypeptides showed that each enzyme removed all the surface projections of the Indiana virus but about one-third of the projections of the Brazil virus were resistant. Complement fixation tests showed that, in contrast to the complete removal of the antigenic activity from the surface of the Indiana virus, about one-third of the surface antigenic activity of the Brazil strain was retained. The enzymeresistant projections of the Brazil virus absorbed neutralizing antibody from the homologous antiserum but were much less active in stimulating the production of neutralizing antibody in guinea pigs although the level of complement fixing antibody produced was similar to that produced by the intact virus. Amino acid analysis also gave differences between the total and enzyme-resistant surface projections. These results suggest that, in contrast to the Indiana virus, the Brazil virus possesses two surface projections which differ in their resistance to proteolytic enzymes.

The pattern of viral polypeptide synthesis and cleavage in poliovirus-infected cells was shown by autoradiography to be considerably more complex than previously thought. In normal growth, at least 26 distinct polypeptides were found, and various modifications of the cleavage process revealed a total of at least 34. Most of the new polypeptides were minor components that were unstable during a chase. Different cultural modifications led to different polypeptide ratios, and it appeared likely that several cleavage activities were involved. Minor differences were found in the polypeptide contents of cytoplasmic extracts and whole infected cells. The complexity of the cleavage pattern necessitated a new nomenclature based on mol. wt. (e.g. Pp110, ‘poliovirus protein’ of 110000). Particular attention was paid to mol. wt. determinations, notably in the use of internal protein standards and more fully denaturing gel conditions. The size of the ‘primary translation product’ of poliovirus RNA was found to be 210000 daltons, so that either 20% of the viral genome is not translated in vivo, or some is read as a smaller independent translation unit.

Poliovirus proteins were labelled in vivo with [35S]-methionine, and the major products of translation and cleavage were separated by electrophoresis and compared in terms of two-dimensional tryptic peptide maps visualized by autoradiography. The main intermediates p110 and p90 had few or no methionine-labelled sequences in common, but were both contained in, and therefore almost fully account for, the presumed primary translation product p210. The sequences of p79, a major stable product of cleavage and a non-structural protein, were almost completely contained in p90, which in turn is the major component of the larger intermediates p168 and p155. P110 is confirmed as the precursor of virus particle protein, and VPo as the precursor of VP2. However, the sequences of p31, the other major product of translation that is not a structural protein, were not contained in any of the viral polypeptides mentioned above.

In addition to the major infective component, which bands at a density of 1.34 g/ml in caesium chloride (‘light component’), a component with a density of 1.44 g/ml (‘heavy component’) has been found in harvests of poliovirus (type 1), Coxsackie B5 virus, a bovine enterovirus (VG-5-27) and swine vesicular disease virus (SVDV). With SVDV about 98% of the infectivity equilibrated at 1.34 g/ml but approx. 2% was present as a peak at 1.44 g/ml. The morphology of the two forms was similar but the heavy component had a smaller diameter (28 nm) than the light component (30 nm). No inter-conversion of the two forms was observed on re-cycling in fresh caesium chloride gradients and the two components had the same proportions of RNA and protein and the same polypeptide composition. Each component gave a similar proportion of the light and heavy forms on replication, but the light component had a specific infectivity about fourfold higher than that of the heavy component and was also much more efficient in eliciting the formation of neutralizing antibodies in guinea pigs. Although these results suggest that the two particles are alternative stable configurations of the virus, iodination failed to reveal any differences in the extent or pattern of labelling of the polypeptides in the two forms.

Biophysical characterization of two mycobacteriophages (Phlei phage and Butyricum phage) was carried out. Biophysical parameters obtained were: (i) buoyant densities in CsCl of 1.51 g/ml for both phages; (ii) sedimentation coefficient of 490 S and 410S; (iii) DNA content of 42 and 34% and (iv) mol. wt. calculated by electron microscopic dimensions to be 123 × 106 and 116 × 106 for Butyricum and Phlei phage, respectively.

Temperature-sensitive mutants of human adenovirus types 12 and 5, defective in viral DNA synthesis, were able to support growth of adeno-associated virus type 1 at the non-permissive temperature.

Synthesis of rabbitpox DNA was inhibited in mouse L cells deprived of isoleucine. Time-course patterns of incorporation of radiolabelled precursors into viral DNA revealed that synthesis of viral DNA began about 6 h after reversal of the isoleucine-deficient state.