- Volume 24, Issue 3, 1974

The properties and function of parental nucleocapsid-like particles (NLP) in the cytoplasm of Sendai virus-infected Ehrlich tumour cells were studied. The formation of NLP stems from partial uncoating of the virus particles leading to the loss of about 60% of the mol. mass of virus protein, the genome in the NLP being fully conserved. Uncoating proceeds at 37 °C, but not at 0 °C, and does not require the synthesis of protein and host RNA. NLP sedimentation rate (∼ 200S) and buoyant density in caesium chloride (1.34 g/ml) are fairly close to those of nucleocapsids produced in vitro by treatment of the virus with sodium deoxycholate. However, the following differences between the two types of structures are found: (i) NLP are sensitive to ribonuclease; (ii) NLP contain the largest virus particle protein in addition to the nucleocapsid protein; (iii) most molecules of parental RNA in NLP sediment slower than RNA of native virus particles (50S), this phenomenon apparently not being due to genome RNA degradation.

The involvement of NLP in the parental genome transcription is deduced from the following observations: (i) NLP possess the RNA polymerase activity in vitro; (ii) virus-induced RNA synthesis can be detected in the infected cells at a stage when all cytoplasmic parental RNA seems to be in NLP; (iii) a part of newly synthesized virus-specific RNA is associated with NLP; the kinetics of labelling suggest that the RNA is synthesized in NLP and released after synthesis.

SV40 (small-plaque type) generates heterogeneous defective virus particles during serial undiluted passages. DNA synthesis in resting African green monkey kidney cells infected with such defective particles was studied by autoradiography and band-sedimentation. The defective particles were found to have capacities to induce synthesis of cell DNA and to direct synthesis of virus DNA. The progeny virus DNA was more heterogeneous than the standard DNA.

The phosphorylation of structural proteins of ten rhabdoviruses has been studied. Each virus preparation has been found to possess at least one phosphoprotein which, in the case of Chandipura, Cocal, Piry viruses or vesicular stomatitis virus (VSV), Indiana and New Jersey serotypes, is the minor NS protein. In rabies virus the sole phosphoprotein appears to be the N protein of the nucleocapsid. For Mokola and Lagos bat viruses, two or three phosphoproteins have been observed, one of which is the N protein. Spring viraemia of carp virus (SVCV) preparations contain two phosphoproteins one of which is, or has a mobility close to, that of the N protein. Kern Canyon virus preparations also possess two phosphoproteins, one with the same electrophoretic mobility as (and possible identity to), the virus glycoprotein while the other has a mobility slightly slower than the N protein. Antigenically the core proteins of rabies, Mokola and Lagos bat viruses have been shown to be related to each other but distinct from those of SVCV or VSV.

Incubating tobacco leaf tissue, from which the lower epidermis was peeled, overnight with 0.3 to 0.4% Macerozyme and 0.6 to 1.2% cellulase, depending on leaf condition, produced a good yield of protoplasts that were susceptible to infection by TMV. Fluorescent antibody staining showed that 20 to 80% of protoplasts became infected, and infectivity tests indicated that an average of about 4 × 106 virus particles/infected protoplast, or 100 to 500 µg virus/106 protoplasts were produced by 2 days after inoculation. The production and infection of protoplasts depended less on the season when the plants were grown than with the ‘two-step’ method. Also, plentiful stable protoplasts were obtained without adding potassium dextran sulphate to the macerating medium. Calcium is required in the incubating medium for virus infection but can be partially replaced by Mg, which is not essential in the presence of Ca. Virus attained the greatest concentration when the protoplasts were inoculated as soon as they were washed free from the enzymes.

Semliki Forest virus (SFV) established a persistent, non-cytopathic infection in cultured Aedes albopictus cells with no effect on cumulative cell number as compared with control cultures. All cells were initially infected by SFV as judged by infective centre and immunofluorescence assay and released approx. 50 to 70 p.f.u./cell in the initial 24 h after infection. At 12 h after infection there was a ‘shut-down’ of virus-specific RNA synthesis followed by a sharp decline in the percentage of cells infected, stabilizing at about 2% by 48 h. Significant amounts of virus were released from about 2% of cells in persistently infected cultures. At 8 to 10 days a small-plaque, attenuated variant of SFV appeared in infected cultures; it replaced ‘wild-type’ virus completely by day 16.

The establishment of persistence could not be related to (i) the release of interferon-like activity from infected cells, (ii) the presence of a directly antiviral agent in infected cells or medium, or (iii) changes in the total levels or subcellular distribution of acid phosphatase.

Subculturing persistently infected cells stimulated cell division and promoted a (reduced) burst of virus production; simultaneous superinfection with SFV gave no increase in virus yields.

Genetical and biochemical evidence indicates that herpes simplex virus (types 1 and 2) specifies a unique kinase activity which is able to phosphorylate both thymidine and deoxycytidine. Pseudorabies virus specifies an enzyme which has only thymidine kinase activity and so does vaccinia virus, while equine abortion virus does not induce either activity.

It is shown that the HSV specified deoxypyrimidine kinase is indispensable for virus growth in serum starved cells but not important for virus replication in actively growing culture cells.

The response to various stimuli of herpes simplex virus-specified deoxypyrimidine kinase activity differs significantly from that of the thymidine kinase and deoxycytidine kinase of mammalian cells. However, the two phosphorylating activities of the HSV deoxypyrimidine kinase are themselves quite distinct in their biochemical behaviour.

It has been impossible to separate the HSV-coded thymidine and deoxycytidine phosphorylating activities by their sedimentation, electrophoretic or size characteristics. The evidence suggests that both deoxypyrimidine nucleosides are phosphorylated at the same active site.

Both l-canavanine, the oxyguanidinium analogue of arginine, and l-canaline, the equivalent oxyamine analogue of ornithine, inhibit the replication of vaccinia virus in HeLa cells. Inhibition by canavanine is reversed progressively by the simultaneous addition of increasing amounts of arginine but inhibition by canaline is not reversed by addition of ornithine. However, the inhibitory effect of canaline is reversed progressively by the simultaneous addition of increasing amounts of pyridoxal phosphate. The effect of canavanine on virus production is suppressed completely by the presence of a tenfold greater concentration of arginine: the effect of canaline is suppressed completely by the presence of an equimolar concentration of pyridoxal phosphate. Additionally, the inhibition of virus growth in canalinetreated cells is reversed partially by the subsequent addition of pyridoxal phosphate. These observations suggest that canavanine inhibits competitively as a result of its structural analogy to arginine but that canaline does not act as a structural analogue of ornithine. It is proposed that canaline inhibits by its interaction with pyridoxal phosphate which results in a deficiency of this cofactor. The effect of each inhibitor on the incorporation of precursors of DNA, RNA and protein into both infected and control cells is reported. These results are compatible with the mode of action proposed for each inhibitor.

Mild treatment of chick embryo and L cells with proteases resulted in decreased induction by poly(rI).poly(rC) of interferon and of antiviral resistance, as well as in reduced uptake of [3H]-poly(rI).poly(rC). The enzyme treatment of the cells neither significantly affected their sensitivity to exogenous interferon nor suppressed interferon induction by a virus inducer. The supernatant fluid from the trypsintreated cells contained a substance which complexed with poly(rI).poly(rC), resulting in inhibition, but not in irreversible inactivation, of its interferon-inducing ability. The induction was also inhibited when the cells were first incubated with the inducer and then treated with trypsin. Since trypsin presumably acts primarily on the cell surface, these results are interpreted to indicate that the binding of poly(rI).poly(rC) to cell surface sites and its persistence at the surface for a certain period of time plays an essential role in interferon induction.

A bovine enterovirus (serotype VG-5-27) was grown in BHK 21 cells and purified using gel filtration and sedimentation. A minor component which sedimented at 145S, relative to 165S virus particles, was purified by repeated sedimentation on sucrose density gradients. The 145S particles contained RNA of the same size as virus RNA. Five types of polypeptides were present corresponding to 30 VP0, 60 VP1, 30 VP2, 60 VP3 and 30 VP4 chains, in contrast to 165S virus particles which contain 60 VP1, 60 VP2, 60 VP3 and 30 VP4 chains. The particles appeared to be non-infective but could be isolated from early passages of plaque purified clones of the virus. Iodination of the polypeptides of the 145S component differed considerably from that for mature virus particles. We suggest that the particle may be an intermediate in the maturation of the virus particle.

A bovine enterovirus (serotype VG-5-27) was iodinated with [125I] by treatment with either chloramine T or lactoperoxidase. Virus particles were degraded in steps by either procedure. Initially, RNA and VP4 were released to give an unstable component which sedimented at 75S. This was degraded eventually to slowly sedimenting mateial. When treated for less than 2 min, virus particles were iodinated in only VP1, whereas all three polypeptides were labeled in the artificial top components produced by prolonged iodination. The patternof labelling of the polypeptides in procapsids (natural empty particles) was different from that in mature virus particles.With lactoperoxidase, in particular, VP0 and VP3 were predominantly labelled in contrast to the virus particles, in which only VP1 was labelled. The observations suggest that during maturationof virus particles a substantial conformational change occurs among the proteins present in the procapsid.

The site of influenza virus-induced RNA synthesis in infected chick embryo cells has been determined by autoradiography. Following 5 min pulses of [3H]-uridine, two distinguishable phases of induced RNA synthesis could be detected by grain counting in the nucleus, both of which occurred predominantly in the nucleoplasm. Cytoplasmic RNA synthesis could not be detected in fowl plague virus (FPV) infected cells; a significant increase in cytoplasmic grain count was detected in NDV-infected cells from 4 to 8 h after infection.

Cordycepin (′3-deoxyadenosine) inhibited nucleolar RNA synthesis in chicken embryo fibroblasts (CEF) to a greater extent than nucleoplasmic RNA synthesis; FPV-induced RNA synthesis in cordycepin-treated cells occurred in the nucleoplasm. α-amanitin treatment of FPV-infected cells inhibited the first peak of virus-induced nucleoplasmic RNA synthesis.

Fixed preparations of whole FPV-infected cells were incubated with an RNA-dependent RNA polymerase reaction mixture and examined by autoradiography. A peak of enzyme activity was detected at 3 h after infection in the nucleoplasm; a second peak of activity was detected at 6 h after infection and was wholly cytoplasmic.

We conclude that RNA synthesis in vivo in cells infected with influenza viruses occurs in the cell nucleus and that the increased level of nucleoplasmic RNA synthesis at approx. 1 h after infection signifies increased transcription of cell DNA. The evidence suggests that the microsomal RNA-dependent RNA polymerase found in FPV-infected cells does not function in vivo.

Surface changes were demonstrated in duck embryo cells following infection with both Marek’s disease virus (MDV) and a herpes virus of turkeys (HVT) by agglutination with concanavalin A (con A). Materials labelled with fucose were extracted from the surface of cells with Nonidet. Antigens and con A receptors were isolated from these extracts by affinity chromatography and were compared using double labelling and electrophoresis in polyacrylamide gels. Eight or nine glycoproteins containing fucose were detected in cell surface extracts. Major con A receptors (average mol. wt. 95000 and 130000) detected in MDV and in HVT infected preparations were also present in uninfected cells. Antigens isolated from MDV and HVT infected preparations by immunoadsorption using MDV antiserum were different from the major con A receptors. The relationship between con A binding glycoproteins and antigenic glycoproteins is discussed. Two antigens (average mol. wt. 30000 and 43000) associated with the surface of MDV infected cells were MDV specific but the antigens precipitated from culture fluids of MDV and HVT infected cells with MDV antiserum appear to be identical.

Maize cells infected with maize rough dwarf virus were exposed to [3H]-uridine for various times and later examined by autoradiography. The results suggest that virus RNA is synthesized and the virus particles are assembled in the viroplasms, from which the particles slowly pass into other parts of the cytoplasm.

Mouse L cell interferon that had been either partially or completely inactivated by heating at 56 °C, boiling at 100 °C, shaking, repeated freezing and thawing, or treatment with mercaptoethanol and urea could be partly reactivated by addition of the anionic detergents, decyl-, dodecyl-, or tetradecyl sulphate. For complete reactivation it was necessary to disrupt disulphide bonds of interferon in solutions containing detergents.

The tryptophan content of hepatitis B surface antigen (HBsAg) was studied by spectrophotometric titration with N-bromosuccinimide (NBS) at pH 4.0 in 8.0 m-urea. A tryptophan value of 13.9 % or 22.4 residues/mol (mol. wt. = 30000) was obtained by this method. This value coupled with 2.3 mol per cent tyrosine content obtained by amino acid composition studies accounts for the unusually high extinction coefficient of HBsAg (at 280 nm = 37.26). The modification of tryptophan residues with NBS did not cause cleavage of the protein or loss of biologic activity as determined by radioimmunoassay.